Patent Application
20220127586
Second messenger signaling molecules allow cells to amplify stimuli, and rapidly control downstream responses. This concept is illustrated in human cells where viral double-stranded DNA stimulates the cytosolic enzyme cyclic GMP-AMP synthase (cGAS) to synthesize the cyclic dinucleotide (CDN) 2′-5′/3′-5′ cyclic GMP-AMP (2′3′ cGAMP) (Sun et al. (2013) Science 339:786-791; Wu et al. (2013) Science 339:826-830). 2′3′ cGAMP diffuses throughout the cell, activates the receptor Stimulator of Interferon Genes (STING), and induces type I interferon and NF-κB responses to elicit protective anti-viral immune responses (Wu & Chen (2014) Annu. Rev. Immunol 32:461-488). Most recently, synthetic CDN analogues have emerged as promising lead compounds for immune modulation and cancer immunotherapy (Minn & Wherry (2016) Cell 165:272-275). Enzymatic synthesis of 2′3′ cGAMP transforms local detection of limited stimuli (i.e., cytoplasmic dsDNA) into a spatially-disseminated response. Nucleotide triphosphates like the ATP and GTP used for 2′3′ cGAMP synthesis are ideal building blocks for second messengers due to their abundance and high-energy bonds (Nelson & Breaker (2017) Sci. Signal 10.eaam8812). CDNs were first identified in bacteria (Ross et al (1987) Nature 325:279-281), and established the foundation for later recognition of the importance of CDN signaling in mammalian cells (Danilchanka and& Mekalanos (2013) Cell 154:962-970). Nearly all bacterial phyla encode CDN signaling pathways, yet enigmatically, all known CDN signals are constructed only using purine nucleotides. CDNs control diverse responses in bacterial cells. For example, cyclic di-GMP coordinates the transition between planktonic and sessile growth, cyclic di-AMP controls osmoregulation, cell wall homeostasis, and DNA-damage responses, and 3′-5′/3′-5′ cGAMP (3′3′ cGAMP) modulates chemotaxis, virulence, and exoelectrogenesis (Krasteva & Sondermann (2017) Nat. Chem. Biol. 13:350-359). The human receptor STING also senses these bacterial CDNs as pathogen (or microbe) associated molecular patterns (PAMPs), revealing a direct, functional connection between bacterial and human second messenger signaling (Burdette et al. (2011) Nature 478:515-518). However, the understanding of the true scope of immune responses to bacterial second messenger products is limited and restricted to cyclic dipurine molecules.
Accordingly, there remains a great need in the art to understand the diversity of the bacterial second messenger products and their functions in modulating immune responses in order to design better therapeutics.
The present invention is based, at least in part, on the elucidation of the diversity of products synthesized by a family of microbial synthases related to the Vibrio cholerae enzyme dinucleotide cyclase in Vibrio (DncV) and its metazoan ortholog cGAS.
For example, in one aspect, a modified polypeptide that catalyzes production of nucleotides, wherein said polypeptide comprises an amino acid sequence having at least 70% identity to any one of CD-NTase amino acid sequences listed in Table 1, or a biologically active fragment thereof, and further comprises a nucleotidyltransferase protein fold and an active site, wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]XnA1Y1B1, optionally wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]XnA1Y1B1Z1Z2[ . . . ]ZmC1, wherein: A1, B1, and C1 independently represent amino acid residue D or E; X1, X2, . . . , Xn, Y1, Z1, Z2, . . . , and Zn independently represent any amino acid residue; and n and/or m is any integer, optionally wherein n is 5-40 residues and m is 10-200 residues, is provided.
Numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the polypeptide comprises an amino acid sequence having at least 90% identity to to any one of CD-NTase amino acid sequences listed in Table 1, or a biologically active fragment thereof, and further comprises a nucleotidyltransferase protein fold and an active site, wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]XnA1Y1B1, optionally wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]XnA1Y1B1Z1Z2[ . . . ]ZmC1, wherein: A1, B1, and C1 independently represent amino acid residue D or E; X1, X2, . . . , Xn, Y1, Z1, Z2, . . . , and Zn independently represent any amino acid residue; and n or m is any integer, optionally wherein n is 5-40 residues and m is 10-200 residues. In another embodiment, the polypeptide functions as a monomer. In still another embodiment, the active site of the polypeptide comprises at least two magnesium ions. In yet another embodiment, the magnesium ions are coordinated by a triad of acidic amino acid residues. In another embodiment the GS motif in the active site interacts with the terminal phosphate of a nucleotide and participates in magnesium ion coordination. In still another embodiment, the polypeptide comprises one or more domains selected from the group consisting of Mab-21 protein domain. PAP_central domain, CCA domain, and transcription factor NFAT domain. In yet another embodiment, the polypeptide comprises an N-terminal Pol-β-like nucleotidyltransferase core domain. In another embodiment, the polypeptide comprises a C-terminal OAS1_C domain or a C-terminal tRNA_NucTransf2 domain, optionally wherein the C-terminal OAS1_C domain or a C-terminal tRNA_NucTransf2 domain are contiguous with an N-terminal Pol-β-like nucleotidyltransferase core domain. In still another embodiment, the polypeptide comprises an alpha helix that braces the N-terminal Pol-β-like nucleotidyltransferase core domain and the C-terminal domain. In yet another embodiment, the polypeptide catalyzes production of nucleotides, optionally wherein the nucleotides are cyclic or linear nucleotides. In another embodiment, the polypeptide catalyzes production of nucleotides in the absence of a ligand, such as a double-stranded DNA ligand. In still another embodiment, the nucleotides are cyclic nucleotides, optionally wherein the cyclic nucleotides are selected from the group consisting of cyclic dipurines, cyclic dipynmidines, cyclic purine-pyrimidine hybrids, and cyclic tri-nucleotide molecules in yet another embodiment, the cyclic dipurine is c-di-AMP, cGAMP, or c-di-GMP. In another embodiment, the cyclic dipyrimidine is c-di-UMP or cUMP-CMP. In still another embodiment, the cyclic purine-pyrimidine hybrid is cUMP-AMP or cUMP-GMP. In yet another embodiment, the cyclic tri-nucleotide molecule is cAMP-AMP-GMP. In another embodiment, the active site of the polypeptide comprises an amino acid sequence of GSYX10DVD, wherein X is any amino acid. In still another embodiment, the active site of the polypeptide comprises an amino acid sequence of GSYX10DVDX72D, wherein X is any amino acid.
In some embodiments, the polypeptide comprises amino acid residue N at the position corresponding to N166 of Em-CdnE shown in
In another aspect, a composition comprising a modified polypeptide described herein, and a pharmaceutically acceptable agent selected from the group consisting of excipients, diluents, and carriers, is provided.
In still another aspect, an isolated nucleic acid molecule encoding a polypeptide described herein, is provided.
In yet another aspect, an isolated nucleic acid molecule comprising a nucleotide sequence, which is complementary to a nucleic acid sequence described herein, is provided.
In another aspect, a vector, such as an expression vector, comprising a nucleic acid molecule described herein, is provided.
In still another aspect, a host cell transfected with an expression vector described herein, is provided.
In yet another aspect, a method of producing a polypeptide described herein, comprising culturing a host cell described herein in an appropriate culture medium to, thereby, produce the polypeptide, is provided.
As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the host cell is a bacterial cell or a eukaryotic cell. In another embodiment, the host cell is genetically engineered to express a selectable marker. In still another embodiment, the method further comprises isolating the polypeptide from the medium or host cell.
In another aspect, a method for detecting the presence of a polypeptide described herein in a sample comprising: a) contacting the sample with a compound which selectively binds to the polypeptide; and b) determining whether the compound binds to the polypeptide in the sample to thereby detect the presence of the polypeptide in the sample, is provided. In one embodiment, the compound which binds to the polypeptide is an antibody.
In still another aspect, a non-human animal model engineered to express a polypeptide described herein, is provided. In one embodiment, the polypeptide is overexpressed. In another embodiment, the animal is a knock-in or a transgenic animal. In still another embodiment, thee animal is a rodent.
In yet another aspect, a method of synthesizing nucleotides comprising contacting a polypeptide described herein, or biologically active fragment thereof, with nucleotide substrates.
As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the method further comprises adding a ligand, such as a double-stranded DNA, to the mixture. In another embodiment, the method further comprises purifying the synthesized nucleotides. In still another embodiment, the nucleotide substrates are selected from ATP, CTP, GTP, UTP, and any combination thereof. In yet another embodiment, the nucleotide substrate is modified or unnatural nucleoside triphosphates. In another embodiment, the nucleotide-based second messenger is a cyclic or linear nucleotide-based second messenger. In still another embodiment, the synthesized nucleotides are selected from the group consisting of cyclic dipurine, cyclic dipyrimidine, cyclic purine-pyrimidine hybrid, and cyclic tri-nucleotide. In yet another embodiment, the cyclic dipurine is c-di-AMP, cGAMP, or c-di-GMP. In another embodiment, the cyclic dipyrimidine is c-di-UMP or cUMP-CMP. In still another embodiment, the cyclic purine-pyrimidine hybrid is cUMP-AMP or cUMP-GMP. In yet another embodiment, the cyclic tri-nucleotide molecule is cAMP-AMP-GMP. In another embodiment, the synthesized nucleotides comprise modified or unnatural nucleoside triphosphates. In still another embodiment, the step of contacting occurs in vivo, ex vivo, or in vitro.
In another aspect, a method for identifying an agent which modulates the expression and/or activity of a polypeptide described herein, or biologically active fragment thereof, comprising: a) contacting the polypeptide or biologically active fragment thereof, or a cell expressing the polypeptide or biologically active fragment thereof, with a test agent; and b) determining the effect of the test agent on the expression and/or activity of the polypeptide or biologically active fragment thereof to thereby identify an agent which modulates the expression and/or activity of the polypeptide or biologically active fragment thereof, is provided.
As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the activity is selected from the group consisting of: a) nucleotide-based second messenger synthesis; b) enzyme kinetics; c) nucleotide coordination; d) protein stability; e) interactions with DNA; f) enzyme conformation; and g) STING and/or RECON pathway regulation. In another embodiment, the step of contacting occurs in vivo, ex vivo, or in vitro. In still another embodiment, the agent increases the expression and/or activity of the polypeptide, or biologically active fragment thereof. In yet another embodiment, the agent is selected from the group consisting of a nucleic acid molecule described herein, polypeptide described herein, and a small molecule that binds to a polypeptide described herein. In another embodiment, the agent decreases the expression and/or activity of the polypeptide, or biologically active fragment thereof. In still another embodiment, the agent is a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interfering agent, nucleotide-based second messenger, peptide or peptidomimetic inhibitor, aptamer, antibody, or intrabody. In yet another embodiment, the RNA interfering agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), CRISPR guide RNA (gRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA). In another embodiment, the agent comprises an antibody and/or intrabody, or an antigen binding fragment thereof, which specifically binds to the polypeptide or biologically active fragment thereof. In still another embodiment, the antibody and/or intrabody, or antigen binding fragment thereof, is chimeric, humanized, composite, or human. In yet another embodiment, the antibody and/or intrabody, or antigen binding fragment thereof, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, and diabodies fragments.
In still another aspect, a crystal of a polypeptide described herein, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the polypeptide to a resolution of greater than 5.0 Angstroms, is provided.
As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the polypeptide is crystallized in apo form. In another embodiment, the polypeptide is crystallized in complex with nucleotide substrates. In still another embodiment, the crystal has a space group P 21 21 21. In yet another embodiment, the crystal has a unit cell of dimensions of α=β=γ=90.0°. In another embodiment, the crystal has a set of structural coordinates listed in Table 3+/− the root mean square deviation from the backbone atoms of the the polypeptide of less than 2 Angstroms. In still another embodiment, the crystal is obtained by hanging drop vapor diffusion. In yet another embodiment, the crystal is obtained by incubating hanging drops at a ratio of 1:1 to 1.2:0.8 (protein:reservoir) at 18° C. In another embodiment, the conformation of the complex is the conformation shown in
In yet another aspect, a method for identifying an agent which modulates activity of a polypeptide described herein, comprising the steps of: a) using a three-dimensional structure of the polypeptide as defined by atomic coordinates according to Table 3; b) employing the three-dimensional structure to design or select an agent; c) synthesizing the agent; and d) contacting the agent with the polypeptide, or biologically active fragment thereof, to determine the ability of the agent to modulate activity of the polypeptide, is provided.
As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the step of employing the three-dimensional structure to design or select an agent comprises the steps of: a) identifying chemical entities or fragments capable of associating with the polypeptide; and b) assembling the identified chemical entities or fragments into a single molecule to provide the structure of the agent. In another embodiment, the agent is designed de novo. In still another embodiment, the agent is designed from a known agonist or antagonist of the polypeptide. In yet another embodiment, the activity of the polypeptide is selected from the group consisting of: a) nucleotide-based second messenger synthesis; b) enzyme kinetics; c) nucleotide coordination; d) protein stability; e) interactions with DNA; f) enzyme conformation; and g) STING and/or RECON pathway regulation.
In another aspect, a method of using the three-dimensional structure coordinates of Table 3, comprising: a) determining structure factors from the coordinates; b) applying said structure factor information to a set of X-ray diffraction data obtained from a crystal of a CD-NTase family enzyme; and c) solving the three-dimensional structure of the CD-NTase family enzyme, is provided.
For any figure showing a bar histogram, curve, or other data associated with a legend, the bars, curve, or other data presented from left to right for each indication correspond directly and in order to the boxes from top to bottom of the legend.
The present invention is based, at least in part, on the elucidation of the diversity of products synthesized by a family of microbial synthases related to the Vibrio cholerae enzyme dinucleotide cyclase in Vibrio (DncV) (Davies et al. (2012) Cell 149, 358-370) and its metazoan ortholog cGAS (Sun et al. (2013) Science 339:786-791). Using a systematic biochemical screen for bacterial nucleotide second messengers, a broad family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize an exceptionally diverse range of CDNs was discovered. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analysis of these novel nucleotide second messengers demonstrated that these signals active distinct host receptors and modulate the interaction of both pathogenic and commensal microbiota with their animal and plant hosts. Accordingly, compositions based on the CD-NTase polypeptides, and methods of use thereof, such as methods of producing nucleotide-based second messengers and methods of screening for modulators of CD-NTase, are provided.
The articles “a” and “an” are used herein to refer to one or to more than one (i e to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “administering” is intended to include routes of administration which allow an agent to perform its intended function. Examples of routes of administration for treatment of a body which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.
Unless otherwise specified here within, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
In addition, intrabodies are well-known antigen-binding molecules having the characteristic of antibodies, but that are capable of being expressed within cells in order to bind and/or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods are well-known in the art for adapting antibodies to target (e.g., inhibit) intracellular moieties, such as the use of single-chain antibodies (scFvs), modification of immunoglobulin VL domains for hyperstability, modification of antibodies to resist the reducing intracellular environment, generating fusion proteins that increase intracellular stability and/or modulate intracellular localization, and the like. Intracellular antibodies can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g., as a gene therapy) (see, at least PCT Publs. WO 08/020079, WO 94/02610, WO 95/22618, and WO 03/014960; U.S. Pat. No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.); Kontermann (2004)Methods 34:163-170. Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412. Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).
The term “antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a CD-NTase polypeptide encompassed by the present invention, or a complex thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain, and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak el al. (1994) Structure 2:1121-1123).
Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, protein subunit peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.
Antibodies may be polyclonal or monoclonal; xenogenic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the invention bind specifically or substantially specifically to a modified CD-NTase polypeptide. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
Antibodies may also be “humanized,” which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, have been grafted onto human framework sequences.
A “blocking” antibody or an antibody “antagonist” is one which inhibits or reduces at least one biological activity of the antigen(s) it binds. In certain embodiments, the blocking antibodies or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological activity of the antigen(s).
As used herein, the term “isotype” refers to the antibody class (e.g., IgM, IgG1, IgG2C, and the like) that is encoded by heavy chain constant region genes.
The terms “cancer” or “tumor” or “hyperproliferative” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term “cancer” includes premalignant as well as malignant cancers. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenström's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma. Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.
The terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
The term “coding region” refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term “noncoding region” refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5′ and 3′ untranslated regions).
The term “complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
As used herein, the term “inhibiting” and grammatical equivalents thereof refer decrease, limiting, and/or blocking a particular action, function, or interaction A reduced level of a given output or parameter need not, although it may, mean an absolute absence of the output or parameter. The invention does not require, and is not limited to, methods that wholly eliminate the output or parameter. The given output or parameter can be determined using methods well-known in the art, including, without limitation, immunohistochemical, molecular biological, cell biological, clinical, and biochemical assays, as discussed herein and in the examples. The opposite terms “promoting,” “increasing,” and grammatical equivalents thereof refer to the increase in the level of a given output or parameter that is the reverse of that described for inhibition or decrease.
As used herein, the term “interacting” or “interaction” means that two molecules (e.g., protein, nucleic acid), or fragments thereof, exhibit sufficient physical affinity to each other so as to bring the two interacting molecules, or fragments thereof, physically close to each other. An extreme case of interaction is the formation of a chemical bond that results in continual and stable proximity of the two entities. Interactions that are based solely on physical affinities, although usually more dynamic than chemically bonded interactions, can be equally effective in co-localizing two molecules Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds. Van der Waals force, ionic force, covalent linkages, and combinations thereof. The state of proximity between the interaction domains, fragments, proteins or entities may be transient or permanent, reversible or irreversible. In any event, it is in contrast to and distinguishable from contact caused by natural random movement of two entities. Typically, although not necessarily, an “interaction” is exhibited by the binding between the interaction domains, fragments, proteins, or entities. Examples of interactions include specific interactions between antigen and antibody, ligand and receptor, enzyme and substrate, and the like.
Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The activity may be a direct activity of one or both of the molecules, (e.g., signal transduction). Alternatively, one or both molecules in the interaction may be prevented from binding their ligand, and thus be held inactive with respect to ligand binding activity (e.g., binding its ligand and triggering or inhibiting an immune response) To inhibit such an interaction results in the disruption of the activity of one or more molecules involved in the interaction. To enhance such an interaction is to prolong or increase the likelihood of said physical contact, and prolong or increase the likelihood of said activity.
An “interaction” between two molecules, or fragments thereof, can be determined by a number of methods. For example, an interaction can be determined by functional assays. Such as the two-hybrid Systems. Protein-protein interactions can also be determined by various biophysical and biochemical approaches based on the affinity binding between the two interacting partners. Such biochemical methods generally known in the art include, but are not limited to, protein affinity chromatography, affinity blotting, immunoprecipitation, and the like. The binding constant for two interacting proteins, which reflects the strength or quality of the interaction, can also be determined using methods known in the art. See Phizicky and Fields, (1995) Microbiol. Rev., 59:94-123.
As used herein, a “kit” is any manufacture (e.g. a package or container) comprising at least one reagent. e.g. a probe, for specifically detecting or modulating the expression of a modified CD-NTase polypeptide encompassed by the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods encompassed by the present invention.
As used herein, the term “modulate” includes up-regulation and down-regulation. e.g., enhancing or inhibiting the expression and/or activity of the modified CD-NTase polypeptide encompassed by the present invention.
An “isolated protein” refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of a polypeptide or fragment thereof, in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of a modified CD-NTase polypeptide or fragment thereof, having less than about 30% (by dry weight) of non-CD-NTase protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-CD-NTase protein, still more preferably less than about 10% of non-CD-NTase protein, and most preferably less than about 5% non-CD-NTase protein. When antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
As used herein, the term “nucleic acid molecule” is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. As used herein, the term “isolated nucleic acid molecule” is intended to refer to a nucleic acid molecule in which the nucleotide sequences are free of other nucleotide sequences, which other sequences may naturally flank the nucleic acid in human genomic DNA.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.
For nucleic acids, the term “substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more of the nucleotides, and more preferably at least about 97%, 98%, 99% or more of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available on the world wide web at the GCG company website), using a NWSgapdna. CMP matrix and a gap w eight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11 17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444 453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at the GCG company website), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The nucleic acid and protein sequences encompassed by the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules encompassed by the present invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules encompassed by the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (available on the world wide web at the NCBI website).
The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well-known in the art (see, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).
A “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a modified CD-NTase nucleic acid and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
An “RNA interfering agent” as used herein, is defined as any agent which interferes with or inhibits expression of a target gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to a modified CD-NTase nucleic acid encompassed by the present invention, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target modified CD-NTase nucleic acid by RNA interference (RNAi).
“RNA interference (RNAi)” is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target modified CD-NTase nucleic acid results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G, and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting expression of the target modified CD-NTase nucleic acid. In one embodiment, the RNA is double stranded RNA (dsRNA) This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs, shRNAs, or other RNA interfering agents, to inhibit or silence the expression of target modified CD-NTase nucleic acids. As used herein, “inhibition of a modified CD-NTase nucleic acid expression” or “inhibition of modified CD-NTase gene expression” includes any decrease in expression or protein activity or level of the modified CD-NTase nucleic acid or protein encoded by the modified CD-NTase nucleic acid. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a modified CD-NTase nucleic acid or the activity or level of the protein encoded by a modified CD-NTase nucleic acid which has not been targeted by an RNA interfering agent.
In addition to RNAi, genome editing can be used to modulate the copy number or genetic sequence of a protein of interest, such as constitutive or induced knockout or mutation of a protein of interest, such as a modified CD-NTase polypeptide encompassed by the present invention. For example, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g., for creating non-functional or null mutations). In such embodiments, the CRISPR guide RNA and/or the Cas enzyme may be expressed. For example, a vector containing only the guide RNA can be administered to an animal or cells transgenic for the Cas9 enzyme. Similar strategies may be used (e.g., designer zinc finger, transcription activator-like effectors (TALEs) or homing meganucleases). Such systems are well-known in the art (see, for example, U.S. Pat. No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Pat. Publ. 2014/0087426 and 2012/0178169; Boch et al. (2011) Nat. Biotech. 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One 6:e19722, Li et al. (2011) Nucl. Acids Res. 39:6315-6325; Zhang et al. (2011) Nat Biotech. 29:149-153; Miller et al. (2011) Nat. Biotech. 29:143-148; Lin et al. (2014) Nucl. Acids Res. 42:e47). Such genetic strategies can use constitutive expression systems or inducible expression systems according to well-known methods in the art.
“Piwi-interacting RNA (piRNA)” is the largest class of small non-coding RNA molecules, piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ lime cells, particularly those in spermatogenesis. They are distinct from microRNA (miRNA) in size (26-31 nt rather than 21-24 nt), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, specifically the silencing of transposons. The majority of piRNAs arc antisense to transposon sequences, indicating that transposons are the piRNA target. In mammals it appears that the activity of piRNAs in transposon silencing is most important during the development of the embryo, and in both C. elegans and humans, piRNAs are necessary for spermatogenesis. piRNA has a role in RNA silencing via the formation of an RNA-induced silencing complex (RISC).
“Aptamers” are oligonucleotide or peptide molecules that bind to a specific target molecule. “Nucleic acid aptamers” arc nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently. SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. “Peptide aptamers” are artificial proteins selected or engineered to bind specific target molecules. These proteins consist of one or more peptide loops of variable sequence displayed by a protein scaffold. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. The “Affimer protein”, an evolution of peptide aptamers, is a small, highly stable protein engineered to display peptide loops which provides a high affinity binding surface for a specific target protein. It is a protein of low molecular weight, 12-14 kDa, derived from the cysteine protease inhibitor family of cystatins. Aptamers are useful in biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
“Short interfering RNA” (siRNA), also referred to herein as “small interfering RNA” is defined as an agent which functions to inhibit expression of a modified CD-NTase nucleic acid, e.g., by RNAi. A siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell. In one embodiment, siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands. i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
In another embodiment, a siRNA is a small hairpin (also called stem loop) RNA (shRNA). In one embodiment, these shRNAs are composed of a short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loosoop structure and the antisense strand may follow. These shRNAs may be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA April; 9(4):493-501 incorporated by reference herein).
RNA interfering agents, e.g., siRNA molecules, may be administered to a host cell or organism, to inhibit expression of a modified hsGAS polypeptide encompassed by the present invention and thereby inhibit the expression and/or activity of hsGAS.
The term “small molecule” is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282-63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.
The term “specific binding” refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity (KD) of approximately less than 10−7 M, such as approximately less than 10−8 M, 10−9 M or 10−10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using an antigen of interest as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.” Selective binding is a relative term referring to the ability of an antibody to discriminate the binding of one antigen over another.
As used herein, the term “molecular complex” means a composite unit that is a combination of two or more molecular components (e.g., protein, nucleic acid, nucleotide, compound) formed by interaction between the molecular components. Typically, but not necessarily, a “molecular complex” is formed by the binding of two or more molecular components together through specific non-covalent binding interactions. However, covalent bonds may also be present between the interacting partners. For instance, the two interacting partners can be covalently crosslinked so that the molecular complex becomes more stable. The molecular complex may or may not include and/or be associated with other molecules such as nucleic acid, such as RNA or DNA, or lipids or further cofactors or moieties selected from a metal ions, hormones, second messengers, phosphate, sugars. A “molecular complex” of the invention may also be part of or a unit of a larger physiological molecular complex assembly.
The term “isolated molecular complex” means a molecular complex present in a composition or environment that is different from that found in nature, in its native or original cellular or body environment. Preferably, an “isolated molecular complex” is separated from at least 50° %, more preferably at least 75%, most preferably at least 90% of other naturally co-existing cellular or tissue components. Thus, an “isolated molecular complex” may also be a naturally existing molecular complex in an artificial preparation or anon-native host cell. An “isolated molecular complex” may also be a “purified molecular complex”, that is, a substantially purified form in a substantially homogenous preparation substantially free of other cellular components, other polypeptides, viral materials, or culture medium, or, when the components in the molecular complex are chemically synthesized, free of chemical precursors or by-products associated with the chemical synthesis. A “purified molecular complex” typically means a preparation containing preferably at least 75%, more preferably at least 85%, and most preferably at least 95% of a particular molecular complex. A “purified molecular complex” may be obtained from natural or recombinant host cells or other body samples by standard purification techniques, or by chemical synthesis.
The term “CD-NTase” refers to cGAS/DncV-like nucleotidyltransferase family of proteins. CD-NTases are nucleotidyltransferase identified from bacteria which typically function as monomers and capable of nucleotide second messenger synthesis. It is a highly diverse family of proteins that share a common nucleotidyltransferase protein fold and an active site with a consensus sequence of GSX1X2[ . . . ]XnA1Y1B1, optionally wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]Xn A1Y1B1Z1Z2[ . . . ]ZmC1, wherein A1, B1, and C1 independently represent amino acid residue D or E; X1, X2, . . . . Xn, Y1, Z1, Z2, . . . , and Zn independently represent any amino acid residue; and n or m is any integer. In some embodiments, n is 5-40 residues and m is 10-200 residues, or any range in between, inclusive, such as n is 6-15 residues and m is 50-100 residues.
In some embodiments, the nucleotidyltransferase protein fold is a protein structure having a core of an alpha-beta-trun-beta-X-beta-(alpha); mixed beta-sheet, order of core strands: 123, as defined according to d.218: nucleotidyltransferase [81302] (1 superfamily) of the SCOPe database, release 2.07 (updated 2018 Aug. 3, stable release March 2018). The active site may have two or more magnesium ions, which are typically coordinated by a triad of acidic amino acid residues. The “GS” motif in the active site interacts with the terminal phosphates of a nucleotide and participates in magnesium ion coordination. In one embodiment, CD-NTase contains conserved domains which include Mab-21 protein domain (PFAM database PF03281 and/or EuKaryotic Orthologous Groups (KOG) database KOG3963, PAP_central domain (PFAM database PF04928, Clusters of Orthologous Groups (COG) database COG5186, NCBI conserved domain database CD05402, and/or KOG database KOG2245), CCA domain (COG database COG1746), and transcription factor NFAT domain (KOG database KOG3792/37933). In another embodiment, CD-NTase is a bipartite protein having a N-terminal Pol-β-like nucleotidyltransferase core domain (such as defined according to PFAM database PF14792/PF01909, COG database COG1665/1669, and/or NCBI conserved domain database CD05400/CD5397) contiguous with either a C-terminal OAS1_C domain (PFAM database PF10421) or a C-terminal tRNA-NucTransf2 domain (PFAM database PF9249). The following database references apply for the referenced databases herein: Pfam database v31.0, updated March 2017. KOG and COG databases v1.0, updated 2014; and NCBI conserved domain database v3.16, updated 2017. CD-NTase may further contain an alpha helix that braces the N-terminal nucleotidyltransferase core domain and C-terminal domain. Representative sequences of CD-NTase family proteins are listed in Table 1 and Table 2. The classification, crystal structures, and functional characterizations of the representative CD-NTase family proteins are described in the Examples below.
The term “modified CD-NTase polypeptide” refers to CD-NTase polypeptide that is different from that found in nature, in its native or original cellular or body environment. The term “modification” as used herein refers to all modifications of a protein, DNA, or protein-DNA complex of the invention including cleavage and addition or removal of a group. The “modified CD-NTase polypeptide” of this invention may be, e.g., homolog, derivative, or fragment of native CD-NTase polypeptide having an amino acid sequence listed in Table 1. Preferably, the “modified CD-NTase polypeptide” has one or more following biologically activities a) circular or linear nucleotide-based second messenger synthesis; b) active enzyme conformation; and c) STING or RECON pathway regulation. The term “modified CD-NTase nucleic acid” refers to nucleic acid (e.g., DNA, mRNA) that encodes the modified CD-NTase polypeptide of described herein.
As used herein, the term “nucleotide-based second messenger” refers to a second messenger having a relatively small number (e.g., one, two, or three) of nucleotides or derivatives thereof that transduces signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. It can be circular or linear. In one embodiment, the nucleotide-based second messenger is a cyclic dinucleotide which includes but is not limited to a cyclic di-purine (e.g. cyclic di-AMP, cyclic di-GMP, cyclic AMP-CMP), acyclic pyrimidine (e.g., cyclic di-UMP or cyclic UMP-CMP), or a cyclic pruine-pynmidine hybrid (e.g., cyclic UMP-AMP or cyclic UMP-GMP). In another embodiment, the nucleotide-based second messenger is a cyclic trinucleotide (e.g., cyclic AMP-AMP-GMP). Several bona fide nucleotide signaling pathways, (p)ppGpp, cAMP, cGMP, c-di-AMP, c-di-GMP and cGAMP, have been characterized with respect to basic pathway modules and phenotypic and physiological output (Martin-Rodriguez et al. (2017) Curr Top Med Chem 17:1928-1944). In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions (Pesavento et al. (2009) Curr Opin Microbiol 12:170-176).
The nucleotide-based second messenger may contain modified or unnatural nucleotides. The modified nucleotides can be naturally occurring modified RNA base analogs (Limbach et al. (1994) Nucleic Acids Res 22:2183-2196; Cantara et al. (2011) Nucleic Acids Res 39:D195-D201; Czerwoniec et al. (2009) Nucleic Acids Res 37:D118-D121; Grosjean et al. (1998) Modification and Editing of RNA. ASM Press, Washington D.C.), including but not limited to N6-Methyladenosine-5′-Triphosphate, 5-Methylcytidine-5′-Triphosphate, 2′-O-Methyladenosine-5′-Triphosphate, 2′-O-Methylcytidine-5′-Triphosphate, 2′-O-Methylguanosine-5′-Triphosphate, 2′-O-Methyluridine-5′-Triphosphate, Pseudouridine-5′-Triphosphate, Inosine-5′-Triphosphate, 2′-O-Methylinosine-5′-Triphosphate, 5-Methyluridine-5′-Triphosphate, 4-Thiouridine-5′-Triphosphate, 2-Thiouridine-5′-Triphosphate, 5,6-Dihydrouridine-5′-Triphosphate, 2-Thiocytidine-5′-Triphosphate, 2′-O-Methylpseudouridine-5′-Triphosphate, N1-Methyladenosine-5′-Triphosphate, 2′-O-Methyl-5-methyluridine-5′-Triphosphate, N4-Methylcytidine-5′-Triphosphate, N1-Methylpseudouridine-5′-Triphosphate, 5,6-Dihydro-5-Methyluridine-5′-Triphosphate, 5-Formylcytidine-5′-Triphosphate, 5-Hydroxymethylcytidine-5′-Triphosphate, 5-Hydroxycytidine-5′-Triphosphate, 5-Hydroxyuridine-5′-Triphosphate, 5-Methoxyuridine-5′-Triphosphate, and 5-Carboxymethylesteruridine-5′-Triphosphate.
Unnatural nucleotides include but are not limited to 2′ Fluoro and 2′ O-Methyl NTPs, for example, 2′-Amino-2′-deoxyadenosine-5′-Triphosphate, 2′-Amino-2′-deoxycytidine-5′-Triphosphate, 2′-Amino-2′-deoxyuridine-5′-Triphosphate, 2′-Azido-2′-deoxyadenosine-5′-Triphosphate, 2′-Azido-2′-deoxycytidine-5′-Triphosphate, 2′-Azido-2′-deoxyguanosine-5′-Triphosphate, 2′-Azido-2′-deoxyuridine-5′-Triphosphate, 2′-Fluoro-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxycytidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyguanosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyuridine-5′-Triphosphate, 2′-Fluorothymidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxycytidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyguanosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyuridine-5′-Triphosphate, 2′-Fluorothymidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxycytidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyguanosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyuridine-5′-Triphosphate, 2′-O-Methyladenosine-5′-Triphosphate, 2′-O-Methylcytidine-5′-Triphosphate, 2′-O-Methylguanosine-5′-Triphosphate, 2′-O-Methyluridine-5′-Triphosphate, Pseudouridine-5′-Triphosphate, 2′-O-Methylinosine-5′-Triphosphate, 2′-Amino-2′-deoxycytidine-5′-Triphosphate, 2′-Amino-2′-deoxyuridine-5′-Triphosphate, 2′-Azido-2′-deoxycytidine-5′-Triphosphate, 2′-Azido-2′-deoxyuridine-5′-Triphosphate, 2′-O-Methylpseudouridine-5′-Triphosphate, 2′-O-Methyl-5-methyluridine-5′-Triphosphate, 2′-Azido-2′-deoxyadenosine-5′-Triphosphate, 2′-Amino-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-thymidine-5′-Triphosphate, 2′-Azido-2′-deoxyguanosine-5′-Triphosphate, N4-Methylcytidine-5′-Triphosphate, 2′-O-Methyladenosine-5′-Triphosphate, 2′-O-Methylcytidine-5′-Triphosphate, 2′-O-Methylguanosine-5′-Triphosphate, 2′-O-Methyluridine-5′-Triphosphate, 2′-Amino-2′-deoxyadenosine-5′-Triphosphate, 2′-Amino-2′-deoxycytidine-5′-Triphosphate, 2′-Amino-2′-deoxyuridine-5′-Triphosphate, Araadenosine-5′-Triphosphate, Aracytidine-5′-Triphosphate, Araguanosine-5′-Triphosphate, Arauridine-5′-Triphosphate, 2′-Azido-2′-deoxyadenosine-5′-Triphosphate, 2′-Azido-2′-deoxycytidine-5′-Triphosphate, 2′-Azido-2′-deoxyguanosine-5′-Triphosphate, 2′-Azido-2′-deoxyuridine-5′-Triphosphate, 2′-Fluoro-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxycytidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyguanosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyuridine-5′-Triphosphate, 2′-Fluorothymidine-5′-Triphosphate, 2′-O-Methyladenosine-5′-Triphosphate, 2′-O-Methylcytidine-5′-Triphosphate, 2′-O-Methylguanosine-5′-Triphosphate, 2′-O-Methyluridine-5′-Triphosphate, 2′-Fluoro-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxycytidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyguanosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyuridine-5′-Triphosphate, 2′-Fluorothymidine-5′-Triphosphate, 2′-O-Methyladenosine-5′-Triphosphate, 2′-O-Methylcytidine-5′-Triphosphate, 2′-O-Methylguanosine-5′-Triphosphate, and 2′-O-Methyluridine-5′-Triphosphate.
As used herein, the term “domain” means a functional portion, segment or region of a protein, or polypeptide. “Interaction domain” refers specifically to a portion, segment or region of a protein, polypeptide or protein fragment that is responsible for the physical affinity of that protein, protein fragment or isolated domain for another protein, protein fragment or isolated domain.
If not stated otherwise, the term “compound” as used herein are include but are not limited to peptides, nucleic acids, carbohydrates, natural product extract libraries, organic molecules, preferentially small organic molecules, inorganic molecules, including but not limited to chemicals, metals and organometallic molecules.
The terms “derivatives”. “analogs” or “variants” as used herein include, but are not limited, to molecules comprising regions that are substantially homologous to the modified CD-NTase polypeptide, in various embodiments, by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to a sequence encoding the component protein under stringent, moderately stringent, or nonstringent conditions. It means a protein which is the outcome of a modification of the naturally occurring protein, by amino acid substitutions, deletions and additions, respectively, which derivatives still exhibit the biological function of the naturally occurring protein although not necessarily to the same degree. The biological function of such proteins can e.g. be examined by suitable available in vitro assays as provided in the invention.
The term “functionally active” as used herein refers to a polypeptide, namely a fragment or derivative, having structural, regulatory, or biochemical functions of the protein according to the embodiment of which this polypeptide, namely fragment or derivative is related to.
“Function-conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (e.g., polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A “function-conservative variant” also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.
The terms “polypeptide fragment” or “fragment”, when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino-terminus, internally, or at the carboxyl-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500 or more amino acids long. They can be, for example, at least and/or including 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1020, 1040, 1060, 1080, 1100, 1120, 1140, 1160), 1180, 1200, 1220, 1240, 1260, 1280, 1300, 1320, 1340 or more long so long as they are less than the length of the full-length polypeptide. Alternatively, they can be no longer than and/or excluding such a range so long as they are less than the length of the full-length polypeptide.
“Homologous” as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotide sequence 5′-TATGGC-3′ share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90° %, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
The term “probe” refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
As used herein, the term “host cell” is intended to refer to a cell into which a nucleic acid encompassed by the present invention, such as a recombinant expression vector encompassed by the present invention, has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
As used herein, the term “vector” refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and ‘vector’ may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals.
The term “activity” when used in connection with proteins or molecular complexes means any physiological or biochemical activities displayed by or associated with a particular protein or molecular complex including but not limited to activities exhibited in biological processes and cellular functions, ability to interact with or bind another molecule or a moiety thereof, binding affinity or specificity to certain molecules, in vitro or in vivo stability (e.g., protein degradation rate, or in the case of molecular complexes ability to maintain the form of molecular complex), antigenicity and immunogenecity, enzymatic activities, etc. Such activities may be detected or assayed by any of a variety of suitable methods as will be apparent to skilled artisans.
As used herein, the term “interaction antagonist” means a compound that interferes with, blocks, disrupts or destabilizes a protein-protein interaction or a protein-DNA interaction; blocks or interferes with the formation of a molecular complex, or destabilizes, disrupts or dissociates an existing molecular complex.
The term “interaction agonist” as used herein means a compound that triggers, initiates, propagates, nucleates, or otherwise enhances the formation of a protein-protein interaction or a protein-DNA interaction; triggers, initiates, propagates, nucleates, or otherwise enhances the formation of a molecular complex; or stabilizes an existing molecular complex.
The terms “polypeptides” and “proteins” are, where applicable, used interchangeably herein. They may be chemically modified, e.g. post-translationally modified. For example, they may be glycosylated or comprise modified amino acid residues They may also be modified by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. They may be tagged with a tag. They may be tagged with different labels which may assists in identification of the proteins in a molecular complex. Polypeptides/proteins for use in the invention may be in a substantially isolated form. It will be understood that the polypeptide/protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide/protein for use in the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.
The terms “hybrid protein”, “hybrid polypeptide.” “hybrid peptide”, “fusion protein”, “fusion polypeptide”, and “fusion peptide” are used herein interchangeably to mean a non-naturally occurring protein having a specified polypeptide molecule covalently linked to one or more polypeptide molecules that do not naturally link to the specified polypeptide. Thus, a “hybrid protein” may be two naturally occurring proteins or fragments thereof linked together by a covalent linkage. A “hybrid protein” may also be a protein formed by covalently linking two artificial polypeptides together. Typically but not necessarily, the two or more polypeptide molecules are linked or fused together by a peptide bond forming a single non-branched polypeptide chain.
The term “tag” as used herein is meant to be understood in its broadest sense and to include, but is not limited to any suitable enzymatic, fluorescent, or radioactive labels and suitable epitopes, including but not limited to HA-tag, Myc-tag, T7, His-tag, FLAG-tag, Calmodulin binding proteins, glutathione-S-transferase, strep-tag, KT3-epitope, EEF-epitopes, green-fluorescent protein and variants thereof.
The term “structure coordinates” refers to mathematical detercoordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a molecule or molecule complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the backbone of CD-NTase or a binding pocket portion thereof, as defined by the structure coordinates of CD-NTase described herein.
The term “binding pocket,” as used herein, refers to a region of a molecule or molecular complex, which, as a result of its shape, favorably associates with another chemical entity. Thus, a binding pocket may include or consist of features such as cavities, surfaces, or interfaces between domains. Chemical entities that may associate with a binding pocket include, but are not limited to, cofactors, substrates, modifiers, agonists, and antagonists.
The term “unit cell” refers to a basic parallelepiped shaped block. The entire volume of a crystal may be constructed by regular assembly of such blocks. Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal.
The term “space group” refers to the arrangement of symmetry elements of a crystal.
The term “molecular replacement” refers to a method that involves generating a preliminary model of a CD-NTase crystal whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known (e.g., CD-NTase coordinates from Table 3) within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal (Lattman et al. (1985) Methods in Enzymology 115:55-77; M. G. Rossmann, ed., “The Molecular Replacement Method”, Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York, (1972)). Using the structure coordinates of CD-NTase provided herein, molecular replacement may be used to determine the structure coordinates of a crystalline mutant or homologue of CD-NTase or of a different crystal form of CD-NTase.
In the context of this invention, the term “crystal” refers to a regular assemblage of a modified CD-NTase polypeptide or a complex of a modified CD-NTase polypeptide for X-ray crystallography. That is, the assemblage produces an X-ray diffraction pattern when illuminated with abeam of X-rays Thus, a crystal is distinguished from an agglomeration or other complex of CD-NTase that does not give a diffraction pattern.
The term “RECON” refers to CDN sensor reductase controlling NF-κB. RECON is a mammalian host receptor for bacterial cdNs. The oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs) CDN binding inhibits RECON's enzymatic activity and subsequently promotes inflammation. High-affinity cdN binding inhibited RECON enzyme activity by simultaneously blocking the substrate and cosubstrate sites, as revealed by structural analyses. CDN inhibition of RECON promotes a proinflammatory, antibacterial state that is distinct from the antiviral state associated with STING activation. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for cdNs. RECON also negatively regulates NF-κB activation (McFarland et al. (2017) Immunity 46:433-445; McFarland et al. (2018) mBio 9:e00526-18).
The term “STING” or “stimulator of interferon genes”, also known as transmembrane protein 173 (TMEM173), refers to a five transmembrane protein that functions as a major regulator of the innate immune response to viral and bacterial infections STING is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activating TBK1/1RF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. The term “STING” is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof. Representative human STING cDNA and human STING protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human STING isoforms include the longer isoform 1 (NM_198282.3 and NP_938023.1), and the shorter isoform 2 (NM_001301738.1 and NP_001288667.1; which has a shorter 5′ UTR and lacks an exon in the 3′ coding region which results in a shorter and distinct C-terminus compared to variant 1). Nucleic acid and polypeptide sequences of STING orthologs in organisms other than humans are well-known and include, for example, chimpanzee STING (XM_016953921.1 and XP_016809410.1. XM_009449784.2 and XP_009448059.1; XM_001135484.3 and XP_001135484.1), monkey STING (XM_015141010.1 and XP_014996496.1), dog STING (XM_022408269.1 and XP_022263977.1; XM_005617260.3 and XP_005617317.1; XM_022408249.1 and XP_022263957.1; XM_005617262.3 and XP_005617319.1, XM_005617258.3 and XP_005617315.1; XM_022408253.1 and XP_022263961.1; XM_005617257.3 and XP_005617314.1; XM_022408240.1 and XP_022263948.1; XM_005617259.3 and XP_005617316.1, XM_022408259.1 and XP_022263967.1; XM_022408265.1 and XP_022263973.1), cattle STING (NM_001046357.2 and NP_001039822.1), mouse STING (NM_001289591.1 and NP_001276520.1; NM_001289592.1 and NP_001276521.1; NM_028261.1 and NP_082537.1), and rat STING (NM_001109122.1 and NP_001102592.1).
STING agonists have been shown as useful therapies to treat cancer. Agonists of STING well-known in the art and include, for example, MK-1454, STING agonist-1 (MedChem Express Cat No. HY-19711), cyclic dinucleotides (CDNs) such as cyclic di-AMP (c-di-AMP), cyclic-di-GMP (c-di-GMP), cGMP-AMP (2′3′cGAMP or 3′3′cGAMP), or 10-carboxymethyl-9-acridanone (CMA) (Ohkuri et al. (2015) Oncoimnunology 4(4):e999523), rationally designed synthetic CDN derivative molecules (Fu et al. (2015) Sci Transl Med. 7(283):283ra52. doi. 10.1126/scitranslmed.aaa4306), and 5,6-dimethyl-xanthenone-4-acetic acid (DMXAA) (Corrales et al. (2015) Cell Rep. 11(7):1018-1030). These agonists bind to and activate STING, leading to a potent type I IFN response. On the other hand, targeting the cGAS-STING pathway with small molecule inhibitors would benefit for the treatment of severe debilitating diseases such as inflammatory and autoimmune diseases associated with excessive type I IFNs production due to aberrant DNA sensing and signaling STING inhibitors are also known and include, for example, CCCP (MedChem Express, Cat No. HY-100941) and 2-bromopalmitate (Tao et al. (2016) IUBMB Life. 68(11):858-870). It is to be noted that the term can further be used to refer to any combination of features described herein regarding STING molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe a STING molecule encompassed by the present invention.
The term “STING pathway” or “cGAS-STING pathway” refers to a STING-regulated innate immune pathway, which mediates cytosolic DNA-induced signalling events. Cytosolic DNA binds to and activates cGAS, which catalyzes the synthesis of 2′3′-cGAMP from ATP and GTP. 2′3′-cGAMP binds to the ER adaptor STING, which traffics to the ER-Golgi intermediate compartment (ERGIC) and the Golgi apparatus. STING then activates IKK and TBK1. TBK1 phosphorylates STING, which in turn recruits IRF3 for phosphorylation by TBK1. Phosphorylated IRF3 dimerizes and then enters the nucleus, where it functions with NF-kB to turn on the expression of type I interferons and other immunomodulatory molecules. The cGAS-STING pathway not only mediates protective immune defense against infection by a large variety of DNA-containing pathogens but also detects tumor-derived DNA and generates intrinsic antitumor immunity. However, aberrant activation of the cGAS-STING pathway by self DNA can also lead to autoimmune and inflammatory disease.
The term “cGAS” or “Cyclic GMP-AMP Synthase”, also known as Mab-21 Doman-Containing Protein 1, refers to nucleotidyltransferase that catalyzes the formation of cyclic GMP-AMP (cGAMP) from ATP and GTP (Sun et al. (2013) Science 339:786-791; Krazusch et al. (2013) Cell Rep 3:1362-1368; Civril et al. (2013) Nature 498:332-227; Ablasser et al. (2013) Nature 503:530-534; Kranzusch et al. (2014) Cell 158:1011-1021). cGAS involves both the formation of a 2.5 phosphodiester linkage at the GpA step and the formation of a 3,5 phosphodiester linkage at the ApG step, producing c[G(2,5)pA(3,5)p] (Tao et al. (2017) J Immunol 198:3627-3636, Lee et al. (2017) FEBS Lett. 591:954-961). cGAS acts as a key cytosolic DNA sensor, the presence of double-stranded DNA (dsDNA) in the cytoplasm being a danger signal that triggers the immune responses (Tao et al (2017) J Immunol 198:3627-3636) cGAS binds cytosolic DNA directly, leading to activation and synthesis of cGAMP, a second messenger that binds to and activates TMEM173/STING, thereby triggering type-I interferon production (Tao et al. (2017) J Immunol 198:3627-3636; Wang et al (2017) Immunity 46:393-404). cGAS has antiviral activity by sensing the presence of dsDNA from DNA viruses in the cytoplasm (Tao et al. (2017) J Immunol 198:3627-3636). cGAS also acts as an innate immune sensor of infection by retroviruses, such as HIV-1, by detecting the presence of reverse-transcribed DNA in the cytosol (Gao et al. (2013) Science 341:903-906). The detection of retroviral reverse-transcribed DNA in the cytosol may be indirect and be mediated via interaction with PQBP1, which directly binds reverse-transcribed retroviral DNA (Yoh el al. (2015) Cell 161:1293-1305). cGAS also detects the presence of DNA from bacteria, such as M. tuberculosis (Wassermann et al. (2015) Cell Host Microbe 17:799-810). cGAMP can be transferred from producing cells to neighboring cells through gap junctions, leading to promote TMEM173/STING activation and convey immune response to connecting cells (Ablasser et al. (2013)Nature 503:530-534). cGAMP can also be transferred between cells by virtue of packaging within viral particles contributing to IFN-induction in newly infected cells in a cGAS-independent but TMEM173/STING-dependent manner (Gentili et al. (2015) Science 349:1232-1236). In addition to antiviral activity, cGAS is also involved in the response to cellular stresses, such as senescence. DNA damage or genome instability (Mackenzie et al. (2017) Nature 548:461-465; Harding et al. (2017) Nature 548:466470). cGAS acts as a regulator of cellular senescence by binding to cytosolic chromatin fragments that are present m senescent cells, leading to trigger type-I interferon production via TMEM173/STING and promote cellular senescence. cGAS is also involved in the inflammatory response to genome instability and double-stranded DNA breaks. cGAS acts by localizing to micronuclei arising from genome instability (PubMed:28738408; Harding et al. (2017) Nature 548:466-470). Micronuclei, which is frequently found in cancer cells, is consist of chromatin surrounded by its own nuclear membrane Following breakdown of the micronuclear envelope, a process associated with chromothripsis, MB21D1/cGAS binds self-DNA exposed to the cytosol, leading to cGAMP synthesis and subsequent activation of TMEM173/STING and type-I interferon production (Mackenzie et al. (2017) Nature 548.461-465; Harding et al. (2017) Nature 548:466-470). In one embodiment, human cGAS has 522 amino acids with a molecular mass of 58814 Da. cGAS is a monomer in the absence of DNA and when bound to dsDNA (Tao et al. (2017) J Immunol 198:3627-3636). cGAS interacts with PQBP1 (via WW domain) (Yoh et al. (2015) Cell 161:1293-1305). cGAS also interacts with TRIM14 and this interaction stabilizes cGAS/MB21D1 and promotes type 1 interferon production (Chen et al. (2016) Mol Cell 64:105-119). cGAS also interacts with herpes virus 8/HHV-8 protein ORF52, and this interaction inhibits cGAS enzymatic activity.
The term “cGAS” is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof. Representative human cGAS cDNA and human cGAS protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human cGAS isoforms include the protein (NP_612450.2) encoded by the transcript (NM_138441.2). Nucleic acid and polypeptide sequences of cGAS orthologs in organisms other than humans are well-known and include, for example, chimpanzee cGAS (XM_009451553.3 and XP_009449828.1; and XM_009451552.3 and XP_009449827.1), Monkey cGAS (NM_001318175.1 and NP_001305104.1), cattle cGAS (XM_024996918.1 and XP_024852686.1, XM_005210662.4 and XP_005210719.2, and XM_002690020.6 and XP_002690066.3), mouse cGAS (NM_173386.5 and NP_775562.2), rat cGAS (XM_006243439.3 and XP_006243501.2), and chicken cGAS (XM_419881.6 and XP_419881.4).
Anti-cGAS antibodies suitable for detecting cGAS protein are well-known in the art and include, for example, antibody TA340293 (Origene), antibodies NBP1-86761 and NBP1-70755 (Novus Biologicals. Littleton, Colo.), antibodies ab224144 and ab176177 (AbCam, Cambridge, Mass.), antibody 26-664 (ProSci), etc. In addition, reagents are well-known for detecting cGAS. Multiple clinical tests of cGAS are available in NIH Genetic Testing Registry (GTR®) (e.g., GTR Test ID: GTR000540854.2, offered by Fulgent Clinical Diagnostics Lab (Temple City, Calif.). Moreover, multiple siRNA, shRNA, CRISPR constructs for reducing cGAS expression can be found in the commercial product lists of the above-referenced companies, such as siRNA product #sc-95512 from Santa Cruz Biotechnology, RNAi products SR314484 and TL305813V, and CRISPR product KN212386 (Origene), and multiple CRISPR products from GenScript (Piscataway, N.J.). It is to be noted that the term can further be used to refer to any combination of features described herein regarding cGAS molecules. For example, any combination of sequence composition, percentage identify, sequence length, domain structure, functional activity, etc. can be used to describe a cGAS molecule encompassed by the present invention.
There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.
An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
In view of the foregoing, the nucleotide sequence of a DNA or RNA encoding a modified CD-NTase polypeptide nucleic acid (or any portion thereof) can be used to derive the modified CD-NTase polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
Finally, nucleic acid and amino acid sequence information for the CD-NTase polypeptide encompassed by tbc present invention are well-known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided in Table 1 below.
* Included in Table 1 are orthologs of the proteins, as well as polypeptide molecules comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence of any SEQ ID NO listed in Table 1, or a portion thereof. Such polypeptides can have a function of the full-length polypeptide as described further herein.
* Included in Table 2 are RNA nucleic acid molecules (e.g., thymines replaced with uredines), nucleic acid molecules encoding orthologs of the encoded proteins, as well as DNA or RNA nucleic acid sequences comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the nucleic acid sequence of any SEQ ID NO listed in Table 2, or a portion thereof. Such nucleic acid molecules can have a function of the full-length nucleic acid as described further herein.
a. Isolated Nucleic Acids
One aspect encompassed by the present invention pertains to isolated nucleic acid molecules that encode a modified polypeptide that catalyzes production of nucleotide-based second messengers, wherein said polypeptide comprises an amino acid sequence having at least 70% identity to any one of CD-NTase amino acid sequences listed in Table 1 and further comprises a nucleotidyltransferase protein fold and an active site, wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]Xn A1Y1B1, optionally wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]XnA1Y1B1Z1Z2[ . . . ]ZmC1, wherein:
A1, B1, and C1 independently represent amino acid residue D or E;
X1, X2, . . . , Xn, Y1, Z1, Z2, . . . , and Zn independently represent any amino acid residue; and
n or m is any integer. As described above, in some embodiments, n is 5-40 residues and m is 10-200 residues, or any range in between, inclusive, such as n is 6-15 residues and in is 50-100 residues.
Another way to express this amino acid sequence motif is by the following: GSXx(D/E)X(D/E)Xx(D/E), wherein X is any amino acid residue and Xx is any number of any amino acid residues. As described above, in some embodiments, Xx is 5-40 residues, 10-200, residues, or any range in between, inclusive, such as 6-100 residues, 6-15 residues, 50-100) residues, etc.
As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (i.e., cDNA or genomic DNA) and RNA molecules (i.e., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule that encodes a modified CD-NTase polypeptide, or biologically active portions thereof, can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule that encodes a modified CD-NTase polypeptide, or biologically active portions thereof, encompassed by the present invention, e.g., a nucleic acid molecule having the nucleotide sequence shown in Table 2, or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more (e.g., about 98%) homologous to the nucleotide sequence shown in Table 2, or a portion thereof (i.e., 100, 200, 300, 400, 450, 500, or more nucleotides), wherein the polypeptide encoded by the nucleic acid molecule further comprises a nucleotidyltransferase protein fold and an active site described herein, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a modified CD-NTase polypeptide cDNA can be isolated from a bacterium using all or portion of the nucleotide sequence shown in Table 2, or fragment thereof, as a hybridization probe and standard hybridization techniques (i.e., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or a portion of the nucleotide sequence shown in Table 2, or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence shown in Table 2, or fragment thereof, wherein the polypeptide encoded by the nucleic acid molecule further comprises a nucleotidyltransferase protein fold and an active site described herein, can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of the nucleotide sequence shown in Table 2, or fragment thereof, or the homologous nucleotide sequence. For example, mRNA can be isolated from human cancer cells (i.e., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in Table 2, or fragment thereof, or to the homologous nucleotide sequence. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. In addition, a nucleic acid of the invention can be generated by site-directed mutagenesis technique using cDNA, or genomic DNA of wild-type CD-NTase as a template and specific oligonucleotide primers that contain the intended mutation. The nucleic acid so amplified or generated can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a modified CD-NTase polypeptide nucleotide sequence can be prepared by standard synthetic techniques, i.e., using an automated DNA synthesizer.
Probes based on the modified CD-NTase polypeptide nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, i.e., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which express a modified CD-NTase polypeptide, such as by measuring a level of a modified CD-NTase polypeptide-encoding nucleic acid in a sample of cells from a subject, i.e., detecting mRNA levels of modified CD-NTase polypeptides.
Nucleic acid molecules encoding other modified CD-NTase polypeptides and thus having a nucleotide sequence which differs from the nucleotide sequences shown in Table 2, or fragment thereof are contemplated. Moreover, nucleic acid molecules encoding modified CD-NTase polypeptides from different species, and thus which have a nucleotide sequence which differs from the nucleotide sequences shown in Table 2 are also intended to be within the scope encompassed by the present invention.
In one embodiment, the nucleic acid molecule(s) of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence shown in Table 1 and further comprises a nucleotidyltransferase protein fold and an active site described herein, or fragment thereof, such that the protein or portion thereof catalyzes production of cyclic or linear nucleotide-based second messengers. Methods and assays for measuring each such biological activity are well-known in the art and representative, non-limiting embodiments are described in the Examples below and Definitions above.
As used herein, the language “sufficiently homologous” refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in an amino acid sequence shown in Table 1, or fragment thereof) amino acid residues to an amino acid sequence shown in Table 1, or fragment thereof, such that the protein or portion thereof catalyzes production of cyclic or linear nucleotide-based second messengers.
In another embodiment, the protein is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the entire amino acid sequence of an amino acid sequence shown in Table 1, or a fragment thereof.
Portions of proteins encoded by the modified CD-NTase nucleic acid molecule encompassed by the present invention are preferably biologically active portions of the modified CD-NTase polypeptide. As used herein, the term “biologically active portion of the modified CD-NTase polypeptide” is intended to include a portion, e.g., a domain/motif, of the modified CD-NTase polypeptide that has one or more of the biological activities of the full-length modified CD-NTase polypeptide, respectively.
Standard binding assays, e.g., immunoprecipitations and yeast two-hybrid assays, as described herein, or functional assays, e.g., RNAi or overexpression experiments, can be performed to determine the ability of a modified CD-NTase polypeptide or a biologically active fragment thereof to maintain a biological activity of the full-length modified CD-NTase polypeptide.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in Table 2, or fragment thereof due to degeneracy of the genetic code and thus encode the same modified CD-NTase polypeptide, or fragment thereof. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in Table 1, or fragment thereof, or a protein having an amino acid sequence which is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to an amino acid sequence shown in Table 1, or fragment thereof, or differs by at least 1, 2, 3, 5 or 10 amino acids but not more than 30, 20, 15 amino acids from an amino acid sequence shown in Table 1, wherein the protein further comprises a nucleotidyltransferase protein fold and an active site described herein. In another embodiment, a nucleic acid encoding a modified CD-NTase polypeptide consists of nucleic acid sequence encoding a portion of a full-length modified CD-NTase polypeptide of interest that is less than 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 amino acids in length.
It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the modified CD-NTase polypeptides may exist within a population (e.g., a human population). Such genetic polymorphism in the modified CD-NTase gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a modified CD-NTase protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the modified CD-NTase gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in the modified CD-NTase polypeptide that are the result of natural allelic variation and that do not alter the functional activity of the modified CD-NTase polypeptide are intended to be within the scope of the invention Nucleic acid molecules corresponding to natural allelic variants and homologues of the modified CD-NTase cDNAs encompassed by the present invention can be isolated based on their homology to the modified CD-NTase nucleic acid sequences disclosed herein using the becterium cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions (as described herein).
In addition to naturally-occurring allelic variants of the modified CD-NTase polypeptide sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences shown in Table 2, or fragment thereof, thereby leading to changes in the amino acid sequence of the encoded modified CD-NTase polypeptide, without altering the functional ability of the modified CD-NTase polypeptide. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence shown in Table 2, or fragment thereof. A “non-essential” amino acid residue is a residue that can be altered from the sequence of the modified CD-NTase polypeptide (e.g., the sequence shown in Table 1, or fragment thereof) without significantly altering the activity of the modified CD-NTase polypeptide, whereas an “essential” amino acid residue is required for the modified CD-NTase polypeptide activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved between mouse and human) may not be essential for activity and thus are likely to be amenable to alteration without altering the modified CD-NTase polypeptide activity.
Accordingly, another aspect encompassed by the present invention pertains to nucleic acid molecules encoding modified CD-NTase polypeptides that contain changes in amino acid residues that are not essential for the modified CD-NTase polypeptide activity. Such modified CD-NTase polypeptides differ in amino acid sequence from an amino acid sequence shown in Table 1, or fragment thereof, yet retain at least one of the modified CD-NTase polypeptide activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein lacks one or more modified CD-NTase polypeptide domains. As stated in the Definitions section, the structure-function relationship of CD-NTase polypeptide is known or disclosed in the present disclosure, such that the ordinarily skilled artisan readily understands the regions that may be mutated or otherwise altered while preserving at least one biological activity of the modified CD-NTase polypeptide.
“Sequence identity or homology”, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared ×100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified “loop out regions”, e.g., those arising from deletions or insertions in one of the sequences are counted as mismatches.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. Preferably, the alignment can be performed using the Clustal Method. Multiple alignment parameters include GAP Penalty=10, Gap Length Penalty=10, For DNA alignments, the pairwise alignment parameters can be Htuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For protein alignments, the pairwise alignment parameters can be Ktuple=1, Gap penalty=3, Window=5, and Diagonals Saved=5.
In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a NWSgapdna CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0) (available online), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
An isolated nucleic acid molecule encoding a modified CD-NTase polypeptide homologous to the protein show in Table 1 and further comprising a nucleotidyltransferase protein fold and an active site described herein, or fragment thereof, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequences shown in Table 2, or fragment thereof, or a homologous nucleotide sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into a nucleotide sequence shown in Table 2, or fragment thereof, or the homologous nucleotide sequence by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in the modified CD-NTase polypeptide is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a modified CD-NTase polypeptide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for the modified CD-NTase polypeptide activity described herein to identify mutants that retain the modified CD-NTase polypeptide activity. Following mutagenesis of a nucleotide sequence shown in Table 2, or fragment thereof, the encoded protein can be expressed recombinantly (as described herein) and the activity of the protein can be determined using, for example, assays described herein.
The levels of the modified CD-NTase polypeptides may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
In preferred embodiments, the levels of the modified CD-NTase polypeptides are ascertained by measuring gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Expression levels can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g. genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
In a particular embodiment, the modified CD-NTase polypeptide mRNA expression level can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g. Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well-known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).
The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array, e.g., an Affymetrix™ gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of the modified CD-NTase mRNA expression levels.
An alternative method for determining the modified CD-NTase mRNA expression level in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to the modified CD-NTase polypeptide mRNA.
As an alternative to making determinations based on the absolute the modified CD-NTase polypeptide expression level, determinations may be based on the normalized modified CD-NTase polypeptide expression level. Expression levels are normalized by correcting the absolute modified CD-NTase polypeptide expression level by comparing its expression to the expression of a non-CD-NTase polypeptide gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, e.g., a normal sample, or between samples from different sources.
The level or activity of a modified CD-NTase polypeptide can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The modified CD-NTase polypeptide can be detected and quantified by any of a number of means well-known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express the modified CD-NTase polypeptide.
b. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to the use of vectors, preferably expression vectors, containing a nucleic acid encoding a modified CD-NTase polypeptide (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. In one embodiment, adenoviral vectors comprising a modified CD-NTase nucleic acid molecule are used.
The recombinant expression vectors encompassed by the present invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
The recombinant expression vectors of the invention can be designed for expression of the modified CD-NTase polypeptide in prokaryotic or eukaryotic cells. For example, the modified CD-NTase polypeptide can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the modified CD-NTase polypeptide is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-modified CD-NTase polypeptide. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant modified CD-NTase polypeptide unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Rev. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the modified CD-NTase polypeptide expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
Alternatively, the modified CD-NTase polypeptide can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40 For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) (Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
Another aspect encompassed by the present invention pertains to host cells into which a recombinant expression vector or nucleic acid encompassed by the present invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, the modified CD-NTase polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Fao hepatoma cells, primary hepatocytes, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g. DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989), and other laboratory manuals.
A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well-known in the art. A modified CD-NTase polypeptide or fragment thereof, may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, a modified CD-NTase polypeptide or fragment thereof, may be retained cytoplasmically and the cells harvested, lysed and the protein or molecular complex isolated. A modified CD-NTase polypeptide or fragment thereof, may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the modified CD-NTase polypeptide or a fragment thereof.
In some embodiments, the modified CD-NTase polypeptide, or biologically active fragment thereof, and may be fused to a heterologous polypeptide. In certain embodiments, the fused polypeptide has greater half-life and/or cell permeability than the corresponding unfused modified CD-NTase polypeptide, or biologically active fragment thereof. For example, the modified CD-NTase polypeptide may be fused to a cell permeable peptide to facilitate the delivery of the modified CD-NTase polypeptide into the intact cells. Cell permeable peptides, also known as protein transduction domains (PTDs), are carriers with small peptide domains that can freely cross cell membranes. Several PTDs have been identified that allow a fused protein to efficiently cross cell membranes in a process known as protein transduction. Studies have demonstrated that a TAT peptide derived from the HIV TAT protein has the ability to transduce peptides or proteins into various cells PTDs have been utilized in anticancer strategy, for example, a cell permeable Bcl-2 binding peptide, cpm1285, shows activity in slowing human mycloid leukemia growth in mice. Cell-permeable phosphopeptides, such as FGFR730pY, which mimics receptor binding sites for specific SH2 domain-containing proteins are potential tools for cancer research and cell signaling mechanism studies. In other embodiments, heterologous tags can be used for purification purposes (e.g., epitope tags and Fc fusion tags), according to standards methods known in the art.
Thus, a nucleotide sequence encoding all or a selected portion of the modified CD-NTase polypeptide may be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect, or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant modified CD-NTase polypeptides, or fragments thereof, by microbial means or tissue-culture technology in accordance with the subject invention.
In another variation, protein production may be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are well-known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis., Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used. Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors in vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.
In certain embodiments, the modified CD-NTase polypeptide, or fragment thereof, may be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell. Chemical synthesis may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules. (see e.g., U.S. Pat. Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., (1993) Curr. Opin Biotech.: vol. 4, p 420; M. Miller, et al., (1989) Science: vol. 246, p 1149; A. Wlodawer, et al. (1989) Science: vol. 245, p 616; L. H. Huang, et al., (1991) Biochemistry: vol. 30, p 7402; M. Sclmolzer, et al., (1992) Int J. Pept. Prot. Res: vol. 40, p 180-193; K. Rajarathnam, et al., (1994) Science: vol. 264, p 90; R. E. Offord, “Chemical Approaches to Protein Engineering”, in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., (1992) J. Biol. Chem.: vol. 267, p 3852. L. Abrahamsen, et al., (1991) Biochemistry: vol. 30, p 4151; T. K. Chang, et al., (1994) Proc. Natl. Acad. Sci USA 91: 12544-12548; M. Schnlzer, et al., (1992) Science: vol., 3256, p 221; and K. Akaji, et al., (1985) Chem. Pharm. Bull. (Tokyo) 33: 184).
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the modified CD-NTase polypeptide or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell encompassed by the present invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) the modified CD-NTase polypeptide. Accordingly, the invention further provides methods for producing the modified CD-NTase polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding the modified CD-NTase polypeptide has been introduced) in a suitable medium until the modified CD-NTase polypeptide is produced. In another embodiment, the method further comprises isolating the modified CD-NTase polypeptide from the medium or the host cell.
The host cells of the invention can also be used to produce human or non-human transgenic animals and/or cells that, for example, overexpress the modified CD-NTase polypeptide or oversecrete the modified CD-NTase polypeptide. The non-human transgenic animals can be used in screening assays designed to identify agents or compounds, e.g., drugs, pharmaceuticals, etc., which are capable of ameliorating detrimental symptoms of selected disorders such as diffuse gastric cancer (DGC), lobular breast cancer, or other types of EMT cancers. For example, in one embodiment, a host cell encompassed by the present invention is a fertilized oocyte or an embryonic stem cell into which the modified CD-NTase polypeptide-encoding sequences, or fragments thereof, have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous modified CD-NTase polypeptide sequences have been introduced into their genome or homologous recombinant animals in which endogenous CD-NTase sequences have been altered. Such animals are useful for studying the function and/or activity of the modified CD-NTase polypeptide, or fragments thereof, and for identifying and/or evaluating modulators of the modified CD-NTase polypeptide activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include nonhuman primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a nonhuman animal, preferably a mammal, more preferably a mouse, in which an endogenous CD-NTase gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal encompassed by the present invention can be created by introducing nucleic acids encoding the modified CD-NTase polypeptide, or a fragment thereof, into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The modified CD-NTase cDNA sequence can be introduced as a transgene into the genome of a nonhuman animal. Alternatively, a nonhuman homologue of the modified CD-NTase gene can be used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the modified CD-NTase transgene to direct expression of the modified CD-NTase polypeptide to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the modified CD-NTase transgene in its genome and/or expression of the modified CD-NTase mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding the modified CD-NTase polypeptide can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a modified CD-NTase gene. For example, a modified CD-NTase gene can be used to construct a homologous recombination vector suitable for altering an endogenous CD-NTase gene, m the mouse genome. In the homologous recombination vector, the modified CD-NTase gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the CD-NTase gene to allow for homologous recombination to occur between the exogenous modified CD-NTase gene carried by the vector and an endogenous CD-NTase gene in an embryonic stem cell. The additional flanking CD-NTase nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the modified CD-NTase gene has homologously recombined with the endogenous CD-NTase gene are selected (see e.g., Li, E. et al. (1992) Cell 69.915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Current Opinion in Biotechnology, 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.
In another embodiment, transgenic nonhuman animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the nonhuman transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter GO phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte front an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
c. Modified CD-NTase Polypeptides
The present invention also provides soluble, purified and/or isolated forms of modified CD-NTase polypeptides that catalyzes production of circular or linear nucleotide-based second messengers, wherein said polypeptide comprises an amino acid sequence having at least 70% identity to any one of CD-NTase amino acid sequences listed in Table 1 and further comprises a nucleotidyltransferase protein fold and an active site, wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]Xn A1Y1B1, optionally wherein the active site comprises the amino acid sequence GSX1X2[ . . . ]XnA1Y1B1Z1Z2[ . . . ]ZmC1, wherein:
A1, B1, and C1 independently represent amino acid residue D or E;
X1, X2, . . . , Xn, Y1, Z1, Z2, . . . , and Zn independently represent any amino acid residue; and
n or m is any integer, for use according to methods described herein.
In one aspect, a modified CD-NTase polypeptide may comprise a CD-NTase amino acid sequence of any one of CD-NTase amino acid sequences listed in Table 1 and further comprising a nucleotidyltransferase protein fold and an active site described herein, or a CD-NTase amino acid sequence of any one of CD-NTase amino acid sequences listed in Table 1 and further comprising a nucleotidyltransferase protein fold and an active site described herein with 1 to about 20 additional conservative amino acid substitutions. Amino acid sequence of any modified CD-NTase polypeptide described herein can also be at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to a CD-NTase amino acid sequence of any one of CD-NTase amino acid sequences listed in Table 1 and further comprises a nucleotidyltransferase protein fold and an active site described herein, or a fragment thereof.
In another aspect, the present invention contemplates a composition comprising an isolated modified CD-NTase polypeptide described herein and less than about 25%, or alternatively 15%, or alternatively 5%, contaminating biological macromolecules or polypeptides.
The present invention further provides compositions related to producing, detecting, or characterizing a modified CD-NTase polypeptide, or fragment thereof, such as nucleic acids, vectors, host cells, and the like. Such compositions may serve as compounds that modulate a modified CD-NTase polypeptide's expression and/or activity, such as antisense nucleic acids.
In certain embodiments, a modified CD-NTase polypeptide of the invention may be a fusion protein containing a domain which increases its solubility and bioavailability and/or facilitates its purification, identification, detection, and/or structural characterization. In some embodiments, it may be useful to express a modified CD-NTase polypeptide in which the fusion partner enhances fusion protein stability in blood plasma and/or enhances systemic bioavailability. Exemplary domains, include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His-Asp or FLAG fusion proteins and tags. Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type 21 secretion system-targeting peptides, transcytosis domains, nuclear localization signals, etc. In various embodiments, a modified CD-NTase polypeptide of the invention may comprise one or more heterologous fusions. Polypeptides may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains. The fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. It is also within the scope of the invention to include linker sequences between a polypeptide of the invention and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein. In another embodiment, the polypeptide may be constructed so as to contain protease cleavage sites between the fusion polypeptide and polypeptide of the invention in order to remove the tag after protein expression or thereafter. Examples of suitable endoproteases, include, for example, Factor Xa and TEV proteases.
In some embodiments, the modified CD-NTase polypeptides, or fragments thereof, are fused to an antibody (e.g., IgG1, IgG2, IgG3, IgG4) fragment (e.g., Fc polypeptides). Techniques for preparing these fusion proteins are known, and are described, for example, in WO 99/31241 and in Cosman et. al. (2001) Immunity 14:123-133. Fusion to an Fc polypeptide offers the additional advantage of facilitating purification by affinity chromatography over Protein A or Protein G columns.
In still another embodiment, a modified CD-NTase polypeptide may be labeled with a fluorescent label to facilitate their detection, purification, or structural characterization. In an exemplary embodiment, a modified CD-NTase polypeptide of the invention may be fused to a heterologous polypeptide sequence which produces a detectable fluorescent signal, including, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).
In preferred embodiments, the modified CD-NTase polypeptide or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence shown in Table 1 or fragment thereof and further comprises a nucleotidyltransferase protein fold and an active site described herein, such that the modified CD-NTase polypeptide or portion thereof catalyzes production of circular or linear nucleotide-based second messengers. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, the modified CD-NTase polypeptides has an amino acid sequence shown in Table 1, or fragment thereof, and further comprises a nucleotidyltransferase protein fold and an active site described herein, or an amino acid sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in Table 1, or fragment thereof, and further comprises comprises a nucleotidyltransferase protein fold and an active site described herein. In yet another preferred embodiment, the modified CD-NTase polypeptide has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence shown in Table 2, or fragment thereof, or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence shown in Table 2, or fragment thereof. The preferred modified CD-NTase polypeptides encompassed by the present invention also preferably possess at least one of the modified CD-NTase polypeptide biological activities described herein.
Biologically active portions of a modified CD-NTase polypeptide include peptides comprising amino acid sequences derived from the amino acid sequence of the modified CD-NTase protein, or the amino acid sequence of a protein homologous to the modified CD-NTase protein, which include fewer amino acids than the full-length modified CD-NTase protein or the full-length polypeptide which is homologous to the modified CD-NTase protein, and exhibit at least one activity of the modified CD-NTase protein. Typically, biologically active portions (peptides, e.g., peptides which are, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length) comprise a domain or motif, (e.g., the full-length protein minus the signal peptide). In a preferred embodiment, the biologically active portion of the protein which includes one or more the domains/motifs described herein catalyzes production of circular or linear nucleotide-based second messengers. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of the modified CD-NTase protein include one or more selected domains/motifs or portions thereof having biological activity. In one embodiment, a modified CD-NTase polypeptide fragment of interest consists of a portion of a full-length modified CD-NTase polypeptide that is less than 240, 230, 220, 210, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 amino acids in length.
The modified CD-NTase polypeptides of the present invention can be produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the modified CD-NTase polypeptide is expressed in the host cell. The modified CD-NTase polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a modified CD-NTase protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, modified CD-NTase protein can be isolated from cells (e.g., engineered cells that harboring modified CD-NTase), for example using an anti-CD-NTase antibody.
The invention also provides modified CD-NTase chimeric or fusion proteins. As used herein, a modified CD-NTase “chimeric protein” or “fusion protein” comprises a modified CD-NTase polypeptide operatively linked to a non-CD-NTase polypeptide. A “modified CD-NTase polypeptide” refers to a polypeptide having an amino acid sequence having at least 70% identity to CD-NTase with a nucleotidyltransferase protein fold and an active site described herein, whereas a “non-CD-NTase polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the modified CD-NTase protein, e.g., a protein which is different from the modified CD-NTase protein and which is derived from the same or a different organism. Within the fusion protein, the term “operatively linked” is intended to indicate that the modified CD-NTase polypeptide and the non-CD-NTase polypeptide are fused in-frame to each other. The non-CD-NTase polypeptide can be fused to the N-terminus or C-terminus of the modified CD-NTase polypeptide. For example, in one embodiment the fusion protein is a modified CD-NTase-GST and/or modified CD-NTase-Fc fusion protein in which the modified CD-NTase sequences, respectively, are fused to the N-terminus of the GST or Fc sequences. Such fusion proteins can be made using the modified CD-NTase polypeptides. Such fusion proteins can also facilitate the purification, expression, and/or bioavailability of recombinant modified CD-NTase polypeptides. In another embodiment, the fusion protein is a modified CD-NTase protein containing a heterologous signal sequence at its C-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of the modified CD-NTase polypeptides can be increased through use of a heterologous signal sequence.
Preferably, a modified CD-NTase chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A modified CD-NTase-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the modified CD-NTase protein.
The present invention also pertains to homologues of the modified CD-NTase proteins. Homologues of the modified CD-NTase protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the modified CD-NTase protein, respectively. As used herein, the term “homologue” refers to a variant form of the modified CD-NTase protein. In one embodiment, treatment of a subject with a homologue having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the modified CD-NTase protein.
In an alternative embodiment, homologues of the modified CD-NTase protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the modified CD-NTase protein. In one embodiment, a variegated library of the modified CD-NTase variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of the modified CD-NTase variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential modified CD-NTase sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of the modified CD-NTase sequences therein. There are a variety of methods which can be used to produce libraries of potential modified CD-NTase homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential modified CD-NTase sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev Biochem 53-323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the modified CD-NTase protein coding can be used to generate a variegated population of the modified CD-NTase fragments for screening and subsequent selection of homologues of a modified CD-NTase protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a modified CD-NTase coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the modified CD-NTase protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of the modified CD-NTase homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinational genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify modified CD-NTase homologues (Arkin and Youvan (1992) Proc. Natl. Acad Sci. USA 89:7811-7815).
The modified CD-NTase nucleic acid and polypeptide molecules described herein may be used to produce nucleotide-based second messengers. For example, the modified CD-NTase nucleic acid or polypeptide molecules may be delivered into a cell or an organism cultured at an optimal condition so that the modified CD-NTase nucleic acid or polypeptide molecules catalyze nucleotide-based second messenger synthesis. The delivery method is known in the art and also described herein. For example, the modified CD-NTase nucleic acid or polypeptide molecules may be delivered using chemical vehicles like liposomes or through viral delivery. In other embodiments, the modified CD-NTase nucleic acid or polypeptide molecules may be contacted with nucleotide substrates in a cell-free condition where buffers, ions, and/or ligands required for the catalytic activity of the modified CD-NTase are supplied.
Second messenger synthesis by the CD-NTases can be modulated further in addition to expressing the CD-NTases. For example, the nucleotide substrates may be modified or unnatural nucleotides as described in the definitions, so that the nucleotide-based second messengers synthesized may include modified or unnatural nucleotides. Methods for identifying, purifying, and/or characterizing the produced nucleotide-based second messengers are known in the art and described in the examples below. The nucleotide-based second messengers may be further modified for better properties. For example nonhydrolyzable sulfate analogs or lapidated versions of the nucleotide-based second messengers may be synthesized. In some embodiments, making non-natural linear or cyclic oligonucleotides available as substrates for the CD-NTases can modulate the second messengers synthesized (e.g., feeding the CD-NTases non-natural linear or cyclic oligonucleotides of interest, such as by ingestion in vivo or contact in vitro).
The CD-NTases themselves and/or nucleotide-based second messengers produced using the modified CD-NTase nucleic acid and polypeptide molecules described herein, can be used as therapeutics.
The modified CD-NTase nucleic acid and polypeptide molecules described herein may be used to design and/or screen for modulators of one or more of biological activities of CD-NTase polypeptides or complexes. In particular, information useful for the design of therapeutic and diagnostic molecules, including, for example, the protein domain, structural information, and the like for modified CD-NTase polypeptides of the invention is now available or attainable as a result of the ability to prepare, purify and characterize the modified CD-NTase polypeptides and complexes, and domains, fragments, variants and derivatives thereof.
Therefore, one aspect encompassed by the present invention pertains to methods of screening for modulators of the modified CD-NTase nucleic acid and polypeptide molecules. For example, in one such method, a modified CD-NTase nucleic acid and/or polypeptide, is contacted with a test compound, and the activity of the modified CD-NTase nucleic acid and/or polypeptide is determined in the presence of the test compound, wherein a change in the activity of the modified CD-NTase nucleic acid and/or polypeptide in the presence of the compound as compared to the activity in the absence of the compound (or in the presence of a control compound) indicates that the test compound modulates the activity of the modified CD-NTase nucleic acid and/or polypeptide. The modulators of the invention may elicit a change in one or more of the following activities: (a) a change in the level and/or rate of formation of a CD-NTase-nucleotide complex and/or a CD-NTase-DNA-nucleotide complex, (b) a change in the activity of a CD-NTase nucleic acid and/or polypeptide, including, e.g., circular or linear nucleotide-based second messenger synthesis, enzyme kinetics, STING pathway activity, RECON pathway activity, etc., (c) a change in the stability of a CD-NTase nucleic acid and/or polypeptide, (d) a change in the conformation of a CD-NTase nucleic acid and/or polypeptide, or (e) a change in the activity of at least one component contained in a CD-NTase-nucleotide complex and/or a CD-NTase-DNA-nucleotide complex.
Compounds to be tested for their ability to act as modulators of CD-NTase nucleic acids and/or polypeptides, can be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly. Compounds for use with the above-described methods may be selected from the group of compounds consisting of lipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleic acids (PNAs), small molecules, natural products, aptamers and polynucleotides. In certain embodiments, the compound is a polynucleotide. In some embodiments, said polynucleotide is an antisense nucleic acid. In other embodiments, said polynucleotide is a siRNA. In certain embodiments, the compound comprises a biologically active fragment of a CD-NTase polypeptide (e.g., a dominant negative form that binds to DNA and/or nucleotide substrates, but does not activate, nucleotide-based second messenger synthesis).
A variety of assay formats will suffice and, in light encompassed by the present disclosure, those not expressly described herein may nevertheless be comprehended by one of ordinary skill in the art based on the teachings herein. Assay formats for analyzing activity of a modified CD-NTase nucleic acid and/or polypeptide, may be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Simple binding assays can also be used to detect agents which modulate a modified CD-NTase, for example, by enhancing the binding of a modified CD-NTase polypeptide to DNA, and/or by enhancing the binding of the modified CD-NTase-DNA complex to a substrate. Another example of an assay useful for identifying a modulator of CD-NTase is a competitive assay that combines one or more modified CD-NTase polypeptides with a potential modulator, such as, for example, polypeptides, nucleic acids, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. The modified CD-NTase polypeptides can be labeled, such as by radioactivity or a colorimetric compound, such that CD-NTase-DNA complex formation and/or activity can be determined accurately to assess the effectiveness of the potential modulator Assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof. Assays may also employ any of the methods for isolating, preparing and detecting the modified CD-NTase polypeptide, or complexes thereof, as described above.
Complex formation between a modified CD-NTase polypeptide, or fragment thereof, and a binding partner (e.g., DNA or nucleotides) may be detected by a variety of methods. Modulation of the complex's formation may be quantified using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners, by immunoassay, or by chromatographic detection. Methods of isolating and identifying CD-NTase-DNA complexes described above may be incorporated into the detection methods.
In certain embodiments, it may be desirable to immobilize a modified CD-NTase polypeptide to facilitate separation of modified CD-NTase complexes from uncomplexed forms of modified CD-NTase polypeptides, DNA fragments, and/or nucleotide substrates, as well as to accommodate automation of the assay. Binding of a modified CD-NTase polypeptide to a binding partner may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein may be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis. Mo.) or glutathione derivatized microtitre plates, which are then combined with the binding partner, e.g. an 35S-labeled binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes may be dissociated from the matrix, separated by SDS-PAGE, and the level of the modified CD-NTase polypeptides found in the bead fraction quantified from the gel using standard electrophoretic techniques such as described in the appended examples.
Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, a modified CD-NTase polypeptide may be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated polypeptide molecules may be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well-known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide may be derivatized to the wells of the plate, and polypeptide trapped in the wells by antibody conjugation. As above, preparations of a binding partner and a test compound are incubated in the polypeptide presenting wells of the plate, and the amount of complex trapped in the well may be quantified. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with the modified CD-NTase polypeptide and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme may be chemically conjugated or provided as a fusion protein with the binding partner. To illustrate, the binding partner may be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of the modified CD-NTase polypeptide trapped in the modified CD-NTase-DNA complex and/or CD-NTase-DNA-nucleotide complex may be assessed with a chromogenic substrate of the enzyme, e.g. 3,3′-diamino-benzadine terahydrochloride or 4-chloro-1-napthol. Likewise, a fusion protein comprising the modified CD-NTase polypeptide and glutathione-S-transferase may be provided, and the modified CD-NTase-DNA complex and/or CD-NTase-DNA-nucleotide complex formation may be quantified by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
Antibodies against the modified CD-NTase polypeptide can be used for immunodetection purposes. Alternatively, the modified CD-NTase polypeptide to be detected may be “epitope-tagged” in the form of a fusion protein that includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST fusion proteins described above may also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N.J.).
In certain in vitro embodiments encompassed by the present assay, the protein or the set of proteins engaged in a protein-protein, protein-substrate, or protein-nucleic acid interaction comprises a reconstituted protein mixture of at least semi-purified proteins By semi-purified, it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins. For instance, in contrast to cell lysates, the proteins involved in a protein-substrate, protein-protein or nucleic acid-protein interaction are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and more preferably are present at 90-95% purity. In certain embodiments of the subject method, the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-substrate, protein-protein interaction, or nucleic acid-protein interaction.
In one embodiment, the use of reconstituted protein mixtures allows more careful control of the protein-substrate, protein-protein, or nucleic acid-protein interaction conditions. Moreover, the system may be derived to favor discovery of modulators of particular intermediate states of the protein-protein interaction. For instance, a reconstituted protein assay may be carried out both in the presence and absence of a candidate agent, thereby allowing detection of a modulator of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.
Assaying biological activity resulting from a given protein-substrate, protein-protein or nucleic acid-protein interaction, in the presence and absence of a candidate modulator, may be accomplished in any vessel suitable for containing the reactants Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
In another embodiment, the modified CD-NTase polypeptide, or complexes thereof, of interest may be generated in whole cells, taking advantage of cell culture techniques to support the subject assay. For example, the modified CD-NTase polypeptide, or complexes thereof, may be constituted in a prokaryotic or eukaryotic cell culture system. Advantages to generating the modified CD-NTase polypeptide, or complexes thereof, in an intact cell includes the ability to screen for modulators of the level and/or activity of the modified CD-NTase polypeptide, or complexes thereof, which are functional in an environment more closely approximating that which therapeutic use of the modulator would require, including the ability of the agent to gain entry into the cell. Furthermore, certain of the in vivo embodiments of the assay are amenable to high through-put analysis of candidate agents.
The modified CD-NTase nucleic acids and/or polypeptide can be endogenous to the cell selected to support the assay. Alternatively, some or all of the components can be derived from exogenous sources. For instance, fusion proteins can be introduced into the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the fusion protein itself or mRNA encoding the fusion protein. Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of the protein-protein interaction.
The amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain, western blots or an intrinsic activity. In certain embodiments, the product of the reporter gene is detected by an intrinsic activity associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays encompassed by the present invention which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target. Accordingly, potential modulators of a modified CD-NTase may be detected in a cell-free assay generated by constitution of a functional modified CD-NTase in a cell lysate. In an alternate format, the assay can be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.
The activity of a modified CD-NTase nucleic acid and/or polypeptide may be identified and/or assayed using a variety of methods well-known to the skilled artisan. For example, the activity of a modified CD-NTase nucleic acid and/or polypeptide may be determined by assaying for the level of expression of RNA and/or protein molecules. Transcription levels may be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product. Translation levels may be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product (e.g., fluorescence, luminescence, enzymatic activity, etc.). Depending on the particular situation, it may be desirable to detect the level of transcription and/or translation of a single gene or of multiple genes. In another embodiment, the biological activity of a modified CD-NTase nucleic acid and/or polypeptide may be assessed by monitoring the modification of the substrate. For example, the synthesis of nucleotide-based second messengers may be monitored as described in the examples herein.
In yet another embodiment, the biological activity of a modified CD-NTase nucleic acid and/or polypeptide may be assessed by monitoring changes in the phenotype of a targeted cell. For example, the repression of V. cholera chemotaxis may be detected as described in the examples herein. The detection means can also include a reporter gene construct which includes a transcriptional regulatory element that is dependent in some form on the level and/or activity of a modified CD-NTase nucleic acid and/or polypeptide. The modified CD-NTase nucleic acid and/or polypeptide may be provided as a fusion protein with a domain that binds to a DNA element of a reporter gene construct. The added domain of the fusion protein can be one which, through its DNA-binding ability, increases or decreases transcription of the reporter gene. Whichever the case may be, its presence in the fusion protein renders it responsive to a modified CD-NTase nucleic acid and/or polypeptide. Accordingly, the level of expression of the reporter gene will vary with the level of expression of a modified CD-NTase nucleic acid and/or polypeptide.
Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. A reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable. The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. For instance, the product of the reporter gene can be an enzyme which confers resistance to an antibiotic or other drug, or an enzyme which complements a deficiency in the host cell (i.e. thymidine kinase or dihydrofolate reductase). To illustrate, the ammoglycoside phosphotransferase encoded by the bacterial transposon gene Tn5 neo can be placed tinder transcriptional control of a promoter element responsive to the level of a modified CD-NTase nucleic acid and/or polypeptide present in the cell. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of inhibition of the modified CD-NTase nucleic acid and/or polypeptide.
The present invention provides, crystals of CD-NTase polypeptides, as well as structures determined therefrom. In one aspect, the invention relates to a crystal of a CD-NTase polypeptide, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the CD-NTase polypeptide to a resolution of greater than 5.0 Angstroms, alternatively greater than 3.0 Angstroms, or alternatively greater than 2.0 Angstroms. In one embodiment, the crystal of a CD-NTase polypeptide has a space group P 212121. In another embodiment, the crystal of a CD-NTase polypeptide has a unit cell of dimensions of α=β=γ=90.0°. In yet another embodiment, the crystal has the set of structural coordinates as given in Table 3+/−the root mean square deviation from the backbone atoms of the CD-NTase polypeptide of less than 2 Angstroms, e.g., less than 1.5 Angstroms, less than 1.25 Angstroms, less than 1.0 Angstroms, less than 0.75 Angstroms, less than 0.5 Angstroms, less than 0.45 Angstroms, less than 0.4 Angstroms, less than 0.35 Angstroms, less than 0.3 Angstroms, less than 0.25 Angstroms, or less than 0.2 Angstroms.
In one embodiment, CD-NTase in the crystals encompassed by the present invention is a modified CD-NTase polypeptide having at least 70% identity to the CD-NTase amino acid sequence of any one listed in Table 1 and further comprising a nucleotidyltransferase protein fold and an active site described herein. In another embodiment, the modified CD-NTase is a fragment of CD-NTase, e.g., a biologically active fragment of CD-NTase. The CD-NTase polypeptide may be in an Apo form or nucleotide-bound form in the crystal. In still another embodiment, the conformation of the CD-NTase polypeptide is the conformation shown in
X-ray structure coordinates define a unique configuration of points in space. Those of skill in the art understand that a set of structure coordinates for protein or an protein/ligand complex, or a portion thereof, define a relative set of points that, in turn, define a configuration in three dimensions A similar or identical configuration can be defined by an entirely different set of coordinates, provided the distances and angles between coordinates remain essentially the same. In addition, a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same.
The present invention thus includes the scalable three-dimensional configuration of points derived from the structure coordinates of at least a portion of a CD-NTase molecule or molecular complex, as listed in Table 3, as well as structurally equivalent configurations, as described below. Preferably, the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of a plurality of the amino acids defining a CD-NTase binding pocket.
In certain embodiments, the structure coordinates of CD-NTase, as determined by X-ray crystallography, are listed in Table 3 Slight variations in structure coordinates can be generated by mathematically manipulating the CD-NTase structure coordinates. For example, the structure coordinates set forth in Table 3 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above. Alternatively, modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal, could also yield variations in structure coordinates. Such slight variations in the individual coordinates will have little effect on overall shape. If such variations are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to be structurally equivalent.
It should be noted that slight variations in individual structure coordinates of the CD-NTase polypeptide would not be expected to significantly alter the nature of chemical entities such as modulators that could associate with the binding pockets. In this context, the phrase “associating with” refers to a condition of proximity between a chemical entity, or portions thereof, and a CD-NTase molecule or portions thereof. The association may be non-covalent, wherein the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or it may be covalent. Thus, for example, a modulator that bound to a binding pocket of CD-NTase would also be expected to bind to or interfere with a structurally equivalent binding pocket.
For the purpose of this invention, any molecule or molecular complex or binding pocket thereof, or any portion thereof, that has a root mean square deviation of conserved residue backbone atoms (N, Ca, C, O) of less than about 0.75 Å, when superimposed on the relevant backbone atoms described by the reference structure coordinates listed in Table 3, is considered “structurally equivalent” to the reference molecule. That is to say, the crystal structures of those portions of the two molecules are substantially identical, within acceptable error. As used herein, “residue” refers to one or more atoms. Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structure coordinates listed in Table 3±a root mean square deviation from the conserved backbone atoms of those amino acids of less than about 0.45 Å. More preferably, the root mean square deviation is at most about 0.35 Å, and most preferably at most about 0.2 Å.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the backbone of a CD-NTase polypeptide or a binding pocket portion thereof, as defined by the structure coordinates of the CD-NTase polypeptides described hemin.
Likewise, the invention also includes the scalable three-dimensional configuration of points derived from structure coordinates of molecules or molecular complexes that are structurally homologous to CD-NTase, as well as structurally equivalent configurations. Structurally homologous molecules or molecular complexes are defined below. Advantageously, structurally homologous molecules can be identified using the structure coordinates of CD-NTase according to a method of the invention.
Various computational analyses can be used to determine whether a molecule or a binding pocket portion thereof is “structurally equivalent,” defined in terms of its three-dimensional structure, to all or part of CD-NTase or its binding pockets. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, Calif.) version 4.1, and as described in the accompanying User's Guide.
The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps: (1) load the structures to be compared; (2) define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results.
Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure), all remaining structures are working structures (i.e., moving structures). Since atom equivalency within QUANTA is defined by user input, for the purpose of this invention equivalent atoms are defined as protein backbone atoms (N, Ca, C, and O) for all conserved residues between the two structures being compared. A conserved residue is defined as a residue which is structurally or functionally equivalent. Only rigid fitting operations are considered.
When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
The configurations of points in space derived from structure coordinates according to the invention can be visualized as, for example, a holographic image, a stereodiagram, a model, or a computer-displayed image, and the invention thus includes such images, diagrams or models.
In one aspect, the invention relates to methods of producing crystals of a CD-Ntase polypeptide. Crystals of the CD-Ntase polypeptide can be produced or grown by a number of techniques including batch crystallization, vapor diffusion (either by sitting drop or hanging drop), soaking, and by microdialysis. Seeding of the crystals in some instances is required to obtain X-ray quality crystals. Standard micro and/or macro seeding of crystals may therefore be used. Preferably, the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein-ligand complex to a resolution greater than 5.0 Angstroms, alternatively greater than 3.0 Angstroms, or alternatively greater than 2.0 Angstroms. Exemplified in the Examples section below is the hanging-drop vapor diffusion procedure.
Once a crystal encompassed by the present invention is produced, X-ray diffraction data can be collected. The example below used standard cryogenic conditions for such X-ray diffraction data collection though alternative methods may also be used. For example, diffraction data can be collected by using X-rays produced in a conventional source (such as a sealed tube or rotating anode) or using a synchrotron source. Methods of X-ray data collection include, but are not limited to, precession photography, oscillation photography and diffractometer data collection. Data can be processed using packages including, for example, DENZO and SCALPACK (Z. Otwinowski and W. Minor) and the like.
The three-dimensional structure of the CD-NTase polypeptide constituting the crystal may be determined by conventional means as described herein. Where appropriate, the structure factors from the three-dimensional structure coordinates of a related CD-NTase polypeptide may be utilized to aid the structure determination of the CD-NTase polypeptide. Structure factors are mathematical expressions derived from three-dimensional structure coordinates of a molecule. These mathematical expressions include, for example, amplitude and phase information. The term “structure factors” is known to those of ordinary skill in the art. Alternatively, the three-dimensional structure of the protein-ligand complex may be determined using molecular replacement analysis. This analysis utilizes a known three-dimensional structure as a search model to determine the structure of a closely related protein-ligand complex. The measured X-ray diffraction intensities of the crystal are compared with the computed structure factors of the search model to determine the position and orientation of the CD-NTase polypeptide crystal. Computer programs that can be used in such analyses include, for example, X-PLOR and AmoRe (J. Navaza, Acta Crystallographics ASO, 157-163 (1994)). Once the position and orientation are known, an electron density map may be calculated using the search model to provide X-ray phases. The electron density can be inspected for structural differences and the search model may be modified to conform to the new structure. Using this approach, one may use the structure of the CD-NTase polypeptide described herein to solve other CD-NTase polypeptide crystal structures, or other polypeptide crystal structures, particularly where the polypeptide is homologous to CD-NTase. Computer programs that can be used in such analyses include, for example, QUANTA and the like.
The present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including agonist and antagonist, capable of binding to CD-NTases and/or modulating CD-NTases.
One approach enabled herein, is to use the structure coordinates of CD-NTases to design compounds that bind to the CD-NTases and alter the physical properties of the compounds in different ways, e.g., solubility. For example, this invention enables the design of compounds that act as inhibitors of the CD-NTase protein by binding to, all or a portion of, the inhibitor packet above the nucleotide donor site in the active enzyme conformation of CD-NTase. In certain embodiments, this invention also enables the design of compounds that act as modulators of CD-NTases by binding to, all or a portion of, residues involved in DNA-binding, nucleotide coordination, and/or overall protein stability.
Another design approach is to probe a crystal of a CD-NTase polypeptide with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate CD-NTase modulators and the enzyme. For example, high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of where each type of solvent molecule sticks. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their effects on modulating activity of the CD-NTase polypeptide (see, e.g., Travis et al. (1993) Science 262:1374).
This invention also enables the development of compounds that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that binds to CD-NTase. Thus, the time-dependent analysis of structural changes in CD-NTase during its interaction with other molecules is enabled. The reaction intermediates of CD-NTase can also be deduced from the reaction product in co-complex with CD-NTase. Such information is useful to design improved analogues of known CD-NTase modulators or to design novel classes of modulators based on the reaction intermediates of the CD-NTase enzyme and CD-NTase-modulator co-complex. This provides a novel route for designing CD-NTase modulators with both high specificity and stability.
Another approach made possible and enabled herein, is to screen computationally small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to the CD-NTase enzyme in this screening, the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity or by estimated interaction energy (see, e.g., Meng et al. (1992) J. Camp. Chem 13.505-524). Because CD-NTase may crystallize in more than one crystal form, the structure coordinates, or portions thereof, as provided herein are particularly useful to solve the structure of those other crystal forms of CD-NTases. They may also be used to solve the structure of CD-NTase mutants. CD-NTase co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of CD-NTase.
One method that may be employed for this purpose is molecular replacement. In this method, the unknown crystal structure, whether it is another crystal form of CD-NTase, an CD-NTase mutant, or an CD-NTase co-complex, or the crystal of some other protein with significant amino acid sequence homology to any functional domain of CD-NTase, may be determined using the CD-NTase structure coordinates of this invention as provided in Table 3. This method may provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
In addition, in accordance with this invention. CD-NTase mutants may be crystallized in co-complex with known CD-NTase modulators. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of wild-type CD-NTase. Potential sites for modification within the various binding sites of the enzyme may thus be identified. This information may provide an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between CD-NTase and a chemical entity or compound.
All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 2-3 Å resolution X-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR (Yale University, ©1992, distributed by Molecular Simulations, Inc.). See, e.g., Blundel & Johnson, supra; Methods in Enzymology, vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985). This information may thus be used to optimize known classes of CD-NTase modulators, and more importantly, to design and synthesize novel classes of CD-NTase modulators.
The structure coordinates of CD-NTase mutants provided in this invention also facilitate the identification of related proteins or enzymes analogous to CD-NTase in function, structure or both, thereby further leading to novel therapeutic modes for treating or preventing CD-NTase-mediated diseases, such as cancer and autoimmune diseases.
The design of compounds that bind to or modulate CD-NTase according to this invention may involve consideration of two factors. First, the compound may be capable of physically and structurally associating with CD-NTase. Noncovalent molecular interactions important in the association of CD-NTase with its substrate include hydrogen bonding, van der Waals and hydrophobic interactions. Second, the compound may be able to assume a conformation that allows it to associate with CD-NTase. Although certain portions of the compound will not directly participate in this association with CD-NTase, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site of ICE, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with CD-NTase.
The potential modulatory or binding effect of a chemical compound on CD-NTase may be analyzed prior to its actual synthesis and testing by the use of computer modelling techniques. If the theoretical structure of the given compound indicates insufficient interaction and association between it and CD-NTase, synthesis and testing of the compound may be obviated. However, if computer modelling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to CD-NTase and modulate activity of CD-NTase, e.g., by measuring nucleotide-based second messenger synthesis. In this manner, synthesis of inoperative compounds may be avoided.
A modulatory or other binding compound of CD-NTase may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding pockets or other areas of CD-NTase. One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with CD-NTase and more particularly with the individual binding pockets of the CD-NTase S active site. This process may begin by visual inspection of, for example, the active site on the computer screen based on the coordinates of the CD-NTase polypeptides in Table 3. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within an individual binding pocket of CD-NTase as defined supra. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.
Specialized computer programs may also assist in the process of selecting fragments or chemical entities. For example, these may include:
Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or inhibitor. Assembly may be proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of the CD-NTase polypeptides. This would be followed by manual model building using software such as Quanta or Sybyl.
For example, useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments may include:
Instead of proceeding to build a CD-NTase modulator in a step-wise fashion one fragment or chemical entity at a time as described above, modulatory or other CD-NTase binding compounds may be designed as a whole or “de novo” using either an empty active site or optionally including some portion(s) of a known modulator(s). For example, these methods may include:
Other molecular modelling techniques may also be employed in accordance with this invention. See, e.g., Cohen, N. C. et al., “Molecular Modeling Software and Methods for Medicinal Chemistry”, J. Med. Chem., 33, pp. 883-894 (1990). See also, Navia, M. A and M. A Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in Structural Biology, 2, pp. 202-210 (1992).
Once a compound has been designed or selected by the above methods, the efficiency with which that compound may bind to CD-NTase may be tested and optimized by computational evaluation. For example, a compound that has been designed or selected to function as a CD-NTase-modulator may also preferably traverse a volume not overlapping that occupied by the active site when it is bound to the native substrate. An effective CD-NTase modulator may preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient CD-NTase modulators may preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, preferably, not greater than 7 kcal/mole. CD-NTase modulators may interact with the enzyme in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the modulator binds to the enzyme.
A compound designed or selected as binding to CD-NTase may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the modulator and the enzyme when the modulator is bound to CD-NTase, preferably make a neutral or favorable contribution to the enthalpy of binding.
Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction Examples of programs designed for such uses may include: Gaussian 92, revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. ©1992); AMBER, version 4.0 (P. A Kollman, University of California at San Francisco, ©1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington. Mass. 51994); and Insight 11/Discover (Biosysm Technologies Inc., San Diego, Calif. ©1994). These programs may be implemented, for instance, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art.
Once an CD-NTase-binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to CD-NTase by the same computer methods described in detail, above.
The present invention also enables mutants of ICE and the solving of their crystal structure. More particularly, by virtue encompassed by the present invention, the location of the active site and interface of CD-NTase based on its crystal structure permits the identification of desirable sites for mutation.
For example, mutation may be directed to a particular site or combination of sites of wild-type CD-NTase, i.e., the active site, or a location on the interface site may be chosen for mutagenesis. Similarly, only a location on, at or near the enzyme surface may be replaced, resulting m an altered surface charge of one or more charge units, as compared to the wild-type enzyme. Alternatively, an amino acid residue in CD-NTase may be chosen for replacement based on its hydrophilic or hydrophobic characteristics.
Such mutants may be characterized by any one of several different properties as compared with wild-type CD-NTase. For example, such mutants may have altered surface charge of one or more charge units, or have an increased stability to component dissociation. Or such mutants may have an altered substrate specificity in comparison with, or a higher specific activity than, wild-type CD-NTase.
The mutants of CD-NTase prepared herein may be prepared in a number of ways as discussed above. Once the CD-NTase mutants have been generated in the desired location, i.e., active site or DNA binding interface, the mutants may be tested for any one of several properties of interest. For example, one or more of the following activities may be tested: a) nucleotide-based second messenger synthesis; b) enzyme kinetics; c) nucleotide coordination; d) protein stability; e) interactions with the ligand; f) enzyme conformation; g) STING pathway regulation and h) RECON pathway regulation.
In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a modified CD-NTase polypeptide comprising an amino acid sequence that has at least 70% identity to any one of the amino acid sequences listed in Table 1 and further comprising a nucleotidyltransferase protein fold and an active site described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
As described in detail below, the pharmaceutical compositions encompassed by the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
The phrase “therapeutically-effective amount” as used herein means that amount of an agent that modulates (e.g., inhibits or enhances) expression and/or activity of the modified CD-NTase which is effective for producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit/risk ratio.
The phrase “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution, (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
The term “pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., enhances or inhibits) modified CD-NTase polypeptide expression and/or activity. These salts can be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).
In other cases, the agents useful in the methods encompassed by the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of a modified CD-NTase polypeptide encompassed by the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting the purified respiration uncoupling agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations useful in the methods encompassed by the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association a modified CD-NTase polypeptide encompassed by the present invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a respiration uncoupling agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a respiration uncoupling agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.
In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pirrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.
Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in the pharmaceutical-formulating art They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more respiration uncoupling agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a modified CD-NTase polypeptide encompassed by the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a modified CD-NTase polypeptide, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
The modified CD-NTase polypeptide, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin. sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Transdermal patches have the added advantage of providing controlled delivery of a respiration uncoupling agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more respiration uncoupling agents in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of a modified CD-NTase polypeptide, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
When the respiration uncoupling agents encompassed by the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be determined by the methods encompassed by the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci USA 91:3054 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference.
a. Bacterial Strains and Growth Conditions
E. coli was cultivated at 37° C., shaking, in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl w/v), and stored in LB plus 30% glycerol at −80° C. unless otherwise indicated. When appropriate, carbenicillin (100 μg/ml), ampicillin (100 μg/ml), and chloramphenicol (34 μg/ml) were used BL21 E. coli (strain CodonPlus™ (DE3)-RIL transformed with pRARE2. Agilent) was used for all protein expression and DFI10β E. coli (strain Top10, Invitrogen) was used for cloning and plasmid propagation. For repression of protein expression from pET vectors, BL21 E. coli was cultivated in MDG medium (0.5% glucose, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 0.25% aspartic acid, and trace metals) with ampicillin and chloramphenicol. For optimum protein expression from pET vectors. BL21 E. coli was cultivated in M9ZB medium (0.5% glycerol, 1% Cas-Amino Acids, 47.8 mM Na2HPO4, 22 mM KH2PO4, 18.7 mM NH4Cl, 85.6 mM NaCl, 2 mM MgSO4, and trace metals) with ampicillin and chloramphenicol (Studier et al. (2005) Protein Expr. and Purif. 41:207-234).
b. Cloning and Plasmid Construction
Cloning and plasmid construction were performed as previously described (Studier et al. (2005) Protein Expr and Purif 41:207-234). Briefly, for vectors constructed in this study, genes were either amplified from genomic DNA or synthesized as gBLOCKs (Integrated DNA Technologies) with ≥18 base pairs of homology flanking the insert sequence and ligated into BamHI/NotI linearized vector by Gibson assembly®. Reactions were transformed into electrocompetent DH10β and selected with carbenicillin plates. Sanger sequencing confirmed each vector was free of mutations within the multiple cloning site. N-terminal 6×His-MBP tag and 6×His-SUMO2 tag fusions were constructed using custom pET16MBP27 or pETSUMO228, respectively. CD-NTases and their effector coding sequences were codon optimized for bacterial expression (Integrated DNA Technologies) with the exception of ECOR31 and Vibrio cholerae derived sequences. Synthases were overexpressed in mammalian cells from pcDNA4 plasmids, previously described (Kranzusch et al. (2015) Mol Cell 59.891-903). For expression of MBP N-terminally tagged dncV and cdnE in mammalian cells, MBP and the fused CD-NTase were codon optimized for expression in human cells. For coexpression with their putative effector genes, N-terminal MBP-tagged CD-NTases were cloned into pBAD33 (Guzman et al. (1995) J. Bacteriol. 177:4121-4130) modified with a ribosomal binding site and oriT for conjugation. For cloned CD-NTase details see Table 4A and for cloned CD-NTase effector details see Table 5.
c. Recombinant Protein Purification
Proteins were purified as previously described (Zhou et al. (2018) Cell 174:P300-311). Briefly, chemically competent BL21 E. coli was transformed with a protein expression plasmid, recovered on MDG plates overnight, cultivated as a 30 mL starter culture in MDG liquid medium overnight, and was used to seed an M9ZB culture at ˜1:1000. 25 mL-4 L M9ZB cultures were cultivated for ˜4 h until OD was 2-3.5 at which time IPTG was added at 0.5 mM and cultures were shifted to 16° C. overnight. Harvested E. coli was washed in 1×PBS, stored as a flash-frozen pellet at −80° C., or immediately disrupted by sonication in lysis buffer (20 mM HEPES-KOH pH 7.5, 400 mM NaCl. 30 mM imidazole, 10% glycerol, 1 mM DTT). Lysates were clarified by centrifugation, filtered through glass wool, and proteins were purified by affinity chromatography using Ni-NTA (Qiagen) resin and a gravity column. Resin was washed (lysis buffer made with 1 M NaCl), eluted (lysis buffer+300 mM imidizole), and the eluate was dialyzed overnight at 4° C. (20 mM HEPES-KOH pH 7.5, 300 mM NaCl, 1 mM DTT). For SUMO fusion proteins, dialysis was supplemented with ˜250 μg of human SENP2 protease (D364-L589, M497A) Small-scale preparations of proteins were flash-frozen at this stage and stored at −80° C. in storage buffer (10% glycerol, 20 mM HEPES-KOH pH 7.5, 250 mM KCl, 1 mM TCEP). Where appropriate, proteins were filter-concentrated using centrifugation through 10 kDa or 30 kDa cut-off column (Millipore Sigma).
For large-scale protein preps (cGAS, DncV, DisA. CdnE. Rm-CdnE, Em-CdnE. STING, RECON, Lp-CdnE02, Ec-CdnD02, CyaA), size exclusion chromatography followed by concentration was performed in storage buffer without glycerol. Initial CD-NTase proteins were purified and screened as N-terminal MBP fusions as they stabilized proteins and increased protein expression levels in E. coli. Although a TEV site and linker separated the fused protein of interest, these proteins were used undigested. Proteins were either freshly thawed from −80° C. stocks and immediately used, or maintained at −20° C. in a storage buffer with 50% total glycerol. It was found that glycerol stocks of second messenger synthases at −20° C. retain >90% activity for at least 6 months and were appropriate for biochemical assays. Additional protein details are found in Table 2.1.
Homo sapiens
Vibrio cholerae
Bacillus thuringiensis
E aka WspE* (HIS)
Pseudomonas aeruginosa
E. coli
(His-MBP)
E. coli
HIS)
(codon optimized)
Rhodothermus marinus
(codon optimized)
Elizabethkingia meningoseptica
(codon optimized)
Elizabethkingia meningoseptica
(codon optimized)
Elizabethkingia meningoseptica
Mus musculus
Mus musc
(codon optimized)
Legionella pneumophila
(codon optimized)
Enterobacter cloacae
E. coli
indicates data missing or illegible when filed
d. Biochemistry and Second Messenger Synthesis Assays
Recombinant, candidate nucleotidylransferase reactions combined 4 μL of 5× reaction buffer (250 mM CAPSO pH 9.4, 175 mM KCl, 25 mM Mg(OAc)2, 5 mM DTT), 2 μL of 10×NTPs, 1 μL [α-32P] NTPs (˜1 μCi), 1 μL of candidate enzyme in storage buffer (˜20 μM), and a remaining volume of nuclease free water. The final reactions (50 mM CAPSO pH 9.4, 50 mM KCl, 5 mM Mg(OAc)2, 1 mM DTT, ≤5% glycerol, 25-250 μM individual NTPs, trace amounts of [(α-32P] NTP, 1 μM enzyme) were started with addition of enzyme. Where indicated, pH was altered by replacing CAPSO buffer with appropriate buffer from “StockOptions pH Buffer Kit” (Hampton Research). When appropriate, Mg2+ was replaced with an equimolar concentration of Mn2+ (MnCl2), cGAS reactions were always carried out with Tris at pH 7.5 and supplemented with 1 μM ISD45 dsDNA (Stetson & Medzhitov. (2006) Immunity 24:93-103). Reactions were carried out with 25 μM of each indicated NTP for
Reactions were incubated for 2 h at 37° C. prior to analysis unless otherwise stated. Reactions were stopped by addition of 5 U of alkaline phosphatase (New England Biolabs) which removed triphosphates on remaining NTPs and converted the remaining nucleotide [α-32P] to 32Pi and allowed visualization of cyclic nucleotides. After a ≥20 min incubation, 0.5 μL (PEI-cellulose) or 1 μL (silica) of the reaction was spotted 1.5 cm from the bottom of the TLC plate, spaced 0.8 cm apart. 20 cm×20 cm F-coated PEI-cellulose TLCs (Millipore) were developed in 1.5 M KH2PO4 (pH 3.8) until the buffer front reacted ˜1 cm from the top; 20 cm×10 cm F-coated silica HP-TLC plates (Millipore) were developed in 11:7:2 1-propanol:NH4OH:H2O in a chemical fume hood for 1 hour. Plates were dried and exposed to phosphorscreen prior to detection by Typhoon™ Trio Variable Mode Imager system (GE Healthcare). All TLC data are representative of ≥3 independent experiments, with the exception of the biochemical screen which represents ≥22 independent experiments.
e. Nucleotide Synthesis and Purification for Mass Spectrometry
Cyclic nucleotides were produced in large scale using previously described methods (Sureka el al. (2014) Cell 158:1389-1401) with the following changes. Small-scale second messenger synthesis assays were scaled up to 10-40 mL reactions with final conditions of 50 mM CAPSO pH 9.4, 12.5-50 mM KCl, 5-20 mM Mg(OAc)2, 1 mM DTT, ≥5% glycerol, 250 μM individual NTPs, and 1 μM enzyme. A 20 μL aliquot of the larger reaction was removed and [α-32P] NTP were added to follow reaction progress. Reactions were incubated for 24 hours at which time 5 U of CIP/mL of reaction was added and the reaction was further incubated for 2-24 hours. Reactions were heat inactivated at 65° C., for 30 min, diluted to a final salt concentration of 12.5 mM, and purified by anion exchange chromatography and FPLC (either 1 mL Q-sepharose® column, or Mono Q 4.6/100 PE, GE Healthcare). The column was washed with water and 1 mL fractions were collected during a gradient elution with 2M ammonium acetate. Fractions harboring the appropriate product were identified by A260 and silica TLC, visualizing the nucleotide products by UV-shadowing, imaging using a handheld camera, and comparing migration to paired, radiolabeled reactions. Selected fractions were concentrated by evaporation and re-suspended in 30 μL of nuclease free water for MS. For NMR nucleotides were further purified using size chromatography (Superdex™ 30 Increase 10/300 GL, GE Healthcare). The column was washed with water and 1 mL fractions were collected, identified by A260, pooled, and evaporated. Concentrations of purified nucleotides were estimated from A260 using the estimated extinction coefficients based on RNA oligonucleotides: cUMP-AMP ε=22,800 L mole−1 cm−1, cAAG ε=37,000 L mole−1 cm−1.
The ESI-LC/MS analysis was performed using an Agilent 6530 QTOF mass spectrometer coupled to a 1290 infinity binary LC system operating the electrospray source in positive ionization mode. All samples were chromatographed on an Agilent ZORBAX Bonus-RP C18 column (4.6×150 mm; 3.5 μm particle size) at 50° C. column temperature. The solvent system consisted of 10 mM ammonium acetate (A) and methanol (B). The HPLC gradient with a flow rate of 1 ml/min starts at 5% B, holds for 2 min and then increases over 12 min to 100% B. Identification of CDNs and cAAG was performed by targeted mass analysis for exact masses and formulae for all possible CDNs and cAAG using Profinder software (Agilent).
f. Crystallization and Structure Determination
CdnE homologs were crystallized in apo form or in complex with nucleotide substrates at 18° C. using hanging drop vapor diffusion. Purified Rm-CdnE and Em-CdnE were diluted on ice to 7-10 mg/ml and used immediately to set trays. Alternatively, co-complex crystals were grown by first incubating Rm-CdnE and Em-CdnE in the presence of ˜10 mM total combined nucleotide concentration and 10.5 mM MgCl2 on ice for 30 min. Following incubation, 2 μl hanging drops were set at a ratio of 1:1 or 1.2:0.8 (protein:reservoir) over 350 μl of reservoir in Easy-Xtal 15-Well trays (Qiagen). Optimized crystallization conditions were as follows: Apo Rm-CdnE 10-20% ethanol, 100 mM Tris-HCl pH 7.5; Rm-CdnE-Apcpp-Upnpp 24% PEG-3350, 0.24 M sodium malonate; Apo Em-CdnE 16% PEG-5000 MME, 21 mM sodium citrate pH 7.0, 100 mM HEPES-KOH pH 7.5; Em-CdnE-GTP-Apcpp 100 mM tri-sodium citrate pH 6.4, 10% PEG-3350; Em-CdnE-pppApA 100 mM tri-sodium citrate pH 7.0, 8% PEG-3350. Crystals grew in 3-30 days, and all crystals were harvested using reservoir solution supplemented with 10-25% ethylene glycol using a nylon loop except Apo Rm-CdnE crystals were harvested using NVH oil. X-ray diffraction data were collected at the Advanced Light Source (beamlines 8.2.1 and 5.0.1) and the Advanced Photon Source (beamlines 24-ID-C and 24-ID-E).
Data were processed with XDS and AIMLESS32 using the SSRL autoxds script (A. Gonzalez, Stanford SSRL). Experimental phase information for Rm-CdnE was determined using data collected from selenomethionine-substituted crystals Four sites were identified with HySS in PHENIX (Adams et al. (2010) Acta Crystallogr. D Biol. Crystallogr. 66:213-221), and an initial map was calculated using SOLVE/RESOLVE (Terwilliger (1999) Acta Crystallogr. D Biol. Crystallogr. 55:1863-1871). Model building was completed in Coot (Emsley & Cowtan (2004) Acta Crystallogr. D Biol. Crystallogr. 60:2126-2132) prior to refinement in PHENIX. Following model completion, the Apo Rm-CdnE structure was used for molecular replacement to determine the nucleotide bound structures. Rm-CdnE models were not sufficient to phase Em-CdnE data, but a minimal core Rm-CdnE active-site model was able to successfully determine the substructure and assist experimental phasing with data collected from a native crystal using sulfur single-wavelength anomalous dispersion at a minimal accessible wavelength (˜7,235 eV). 16 heavy sites were identified in HySS that correspond to 12 sulfur, and 4 phosphate sites in the Em-CdnE-pppApA structure, and Em-CdnE model building was completed as for Rm-CdnE. X-ray data for refinement were extended according to I/o resolution cut-off of ˜1.5 and CC* correlation and Rpim parameters. Final structures were refined to stereochemistry statistics for Ramachandran plot (favored/allowed), rotamer outliers, and MolProbity score as follows: Rm-CdnE Apo, 98.6%/1.4%, 0.4% and 0.98; Rm-CdnE-Apcpp-UpNpp, 98.9%/1.1%, 0.4% and 1.25; Em-CdnE Apo 97.8%/2.2%, 0.8% and 1.08, Em-CdnE-GTP-Apcpp, 97.8%/2.2%, 0.8%, and 105. Em-CdnE-pppApA, 98.1%/1.9%, 0.8%, and 1.29.
In the text and in figures side-chains are numbered according to Rm-CdnE sequence. The analogous residues to N166 from Rm-CdnE in E. coli CdnE is N174, in Em-CdnE is S169, in DncV is S259, and in human cGAS is S378.
g. Gel Shift Assays
In vitro binding assays were performed as previously described (Stetson & Medzhitov. (2006) Immunity 24:93-103). Briefly, recombinant Mus musculus STING or RECON, at 4, 20, or 100 μM was incubated with radiolabeled nucleotide (≤1 μM final concentration) in gel shift buffer for final conditions of 50 mM Tris pH 7.5, 60 mM KCl, 5 mM Mg(OAc)2, and 1 mM DTT Experiments were prepared by combining 1 μL of [α-32P] labeled nucleotide, 2 μL 5× gel shift buffer, 5 μL of nuclease free water, and started by addition of 2 μL of recombinant protein in storage buffer. [α-32P] labeled nucleotides were produced with 25 μM of each NTP and ˜1 μCi of each [α-32P] NTP in the following conditions: cGAS (ATP, GTP/[α-32P] GTP), DncV (ATP, GTP/[α-32P] ATP), DisA (ATP/[α-32P] ATP). WspR (GTP/[α-32P] GTP), CdnE (ATP, UTP/[α-32P] UTP), and Ec-CdnD02 (ATP, GTP/[α-32P] GTP).
After 30 min of equilibration, bound and free nucleotide were separated by 6% native PAGE in 0.5×TBE buffer. Gels were dried, and exposed to phosphorscreen prior to detection by Typhoon Trio Variable Mode Imager system (GE Healthcare). Gel shifts were quantified with ImageQuant® 5.2 and the % bound nucleotide was calculated as a proportion of total bound and free nucleotide for each lane, after subtraction of background signal. Gel shift data is representative of ≥2 independent experiments.
h. Cellular Assays for Interferon-β Induction
In-cell assays were performed as previously described29. Briefly, HEK293T cells were transfected using Lipofectamine™ 2000 in 96-well format with: a control plasmid constitutively expressing Renilla luciferase (2 ng pRL-TK), a reporter plasmid expressing interferon β inducible firefly luciferase (20 ng), a plasmid expressing Mus musculus STING (5 ng), and a 5-fold dilution series of pcDNA4-based plasmids expressing a nucleotidyltransferase (1, 2, 6, 30, 150 ng). 2′3′ cGAMP was produced from mouse cGAS, 3′3′ cGAMP was produced from V. cholerae DncV, cyclic di-AMP (cAA) was produced from Bacillus subtilis DisA, cyclic di-GMP (cGG) was produced from P. aeruginosa WspR*. Luciferase production was quantified after 24 hours and firefly luciferase was normalized to Renilla, which was then normalized to empty nucleotidyltransferase vector used at 150 ng. Data are mean±standard error of the mean from 3 replicates and are representative of 3 independent experiments.
i. RECON Enzyme Assay
Activity assays were performed as previously described (McFarland et al. (2017) Immunity 46:433-445) Briefly, a 2-fold dilution series of nucleotide from 50-0.05 μM was incubated in 1×PBS with 200 μM NADPH (cosubstrate) and 25 μM 9,10-Phenanthrenequinone (substrate). The reactions were started with the addition of RECON to a final concentration of 0.5 μM and absorbance at 340 nm was monitored at 20 s intervals for 20 min. Slope of each reaction in the linear range (20-250 s) was used to calculate activity (Linear regression/straight Line analysis, Prism 7.0c). Values were normalized to reactions with zero nucleotide added, which defined 100% activity.
j. Bioinformatics and Tree Construction
To bioinformatically map CD-NTase-like enzymes in bacteria, a previous analysis by Burroughs et al. that combined iterative BLAST analysis, secondary structure predictions, and hidden Markov models to collect DncV-like proteins and their genomic context was extended (Burroughs el al. (2015) Nucleic Acids Res 43:10633-10654). Homologs of each of these 1300 identified proteins by BLAST analysis of the NCBI non-redundant protein database, then combined these datasets to identify >5600 CD-NTase-like genes. The dataset was then manually curated (Geneious Software) Bacterial genomes and sequences were aligned using MAFFT FFT-NS-2 algorithm v7.388 (Katoh & Standley (2013) Mol Biol Evol 30:772-780), a BLOSUM62 scoring matrix, an open gap penalty of 2, and an offset value of 0.123. Proteins with large truncations or lacking the essential DNA polymerase β-like nucleotidyltransferase residues [ie GS . . . (D/E)X(D/E) . . . (D/E)] were removed. The tree was created from the MAFFT alignment using a Jukes-Cantor genetic distance model, Neighbor-Joining method, no outgroup, and resampled by Bootstrap for 100 replicates sorted by topologies. The unrooted tree is used to represent global CD-NTase diversity and does accurately reflect the specific evolutionary relationship between the major CD-NTase A-H clades. The aligned sequences along with pairwise identity comparisons were extracted and used to define clades and clusters. A cluster was defined as >˜10 CD-NTases that share >24.5% identity to the sequence preceding each in the alignment. For clarity, 14 poorly aligned CD-NTases were excluded from the tree and are indicated in Tables 4A-4C. The full dataset organized by order from the alignment and containing pairwise comparison of protein identity to each preceding gene is available in Tables 4A-4C.
Each sequence was identified from the nonredundant database of protein sequences and, at times, represents identical proteins translated from genes found in multiple bacteria. For this reason, additional metadata was extracted for each sequence from the NCBI Identical Protein Groups (IPG) database. Number of “Protein Accessions” in IPG was used as a quantification of the number of isolated bacterial genomes that harbor each NTase and demonstrated >16,000 genomes harbor CD-NTase isolates. At the time of access (02/03/2018) 5,686 nonredundant CD-NTases sequences were identified representing a total of 16,717 genomes. At that time, 130.135 bacterial genomes had been deposited in the NCBI Genome database, leading to the crude approximation of 12.8% of genomes harboring CD-NTase genes. As some of these genomes may harbor more than one CD-NTase and the IPG can overestimate number of genomes encoding a given protein, it has been estimated that >10% of bacterial genomes sequenced encode CD-NTases. Taxanomic analysis was performed using metadata associated with each CD-NTase in NCBI When multiple bacteria were represented by one identical sequence, the highest common taxonomical group was used. IPG and Taxanomic data are also found in Tables 4A-4C.
Type CD-NTase enzymes were manually selected from clusters based on the relevance of the organism from which they were isolated (i.e., human or plant pathogen/commensal organism), their predicted aptness for in vitro expression (thermophilic organisms or isolates from E. coli), the similarity of their operon to the DncV/CdnE operons, and the number of identical protein sequences represented by each unique sequence.
k. CD-NTase Screen
Each type CD-NTase was codon optimized for E. coli, synthesized (IDT), cloned as an N-terminal 6×His-MBP-tag, and purified from a 25 mL culture. E. coli growth, protein induction, and bacterial disruption were performed as described above. Lysates were clarified by centrifugation and Ni-NTA affinity purification was performed as described above with gravity columns replaced by spin columns at 100×g. Buffer exchange of eluted proteins was performed by concentrating the eluate using 0.5 mL 10 kDa cut-off spin column (Ambion) followed by dilution with storage buffer and re-concentration 3× (final imidazole concentration ˜0.3 mM). Proteins were analyzed for second messenger synthesis fresh and flash-frozen for storage at −80° C. For biochemical screen. ATP/CTP/GTP/UTP were used at 25 μM each and incubated overnight with the reaction conditions indicated using methods described above. 1 μL of screened protein (˜1 μg) was added to the reaction and the same volume was assessed by SDS-PAGE followed by coomassie staining, shown in
l. NMR
All NMR experiments were conducted on a Varian 400-MR spectrometer (9.4 T, 400 MHz) Samples were prepared by re-suspending evaporated nucleotide samples into 500 μL D2O supplemented with 5 mM TMSP (3-(trimethylsilyl)propionic-2,2,3,3-d4) at 27° C. Data were processed and figures were generated using VnmrJ software. 1H and 31P chemical shifts are reported in parts per million (ppm). J coupling constants are reported in units of frequency (Hertz) with multiplicities listed as s (singlet), d (doublet), and m (multiplet). These data appear in the figure legends of each NMR spectra.
m. Phospholipase Assay
Patatin-like lipases were assayed as previously described (Gaspar & Machner (2014) Proc. Natl. Acad. Sci. U.S.A. 111:4560-4565). Briefly, CapV and CapE were produced recombinantly and catalytic activity was measured using the EnzChek® Phospholipase A1 Assay Kit (Invitrogen) according to the manufacturer's instructions. Phospholipases (250 nM) were incubated with 2.5, 0.25, or 0.025 μM CDN. c-di-AMP (Invivogen), 3′3′ cGAMP (Invivogen), and c-di-GMP (Biolog) were purchased as chemical standards, cUMP-AMP was purified as described above. Assays were monitored fluorometrically Ex=460 nm/Em=515 nm, for 60 min at ˜90 s intervals at room temperature using a Biotek Synergy plate reader. Slope of each reaction in the linear range was used to calculate activity (Linear regression/straight line analysis, Prism 7.0c). A PLA 1 standard curve from 20-0.02 U was used to interpolate phospholipase activity. Emission was monitored at a gain of 100 and/or 50 in order to extend the linear range of the assay. Data are mean±standard error of the mean (SEM) from 3 replicates and are representative of 3 independent experiments.
n. Western Blot Analyzsis
CD-NTase in-cell expression levels were verified by Western blot of lysed cells. Confluent HEK293T cells were seeded 24 h prior to transfection at a dilution of 1:4 in a 6-well dish. Cells were transfected with 2 μg of plasmid using Lipofectamine® 2000. At 24 h post transfection cells were harvested by washing cells from the dish using Hanks Buffered Saline Solution, pelleted at low speed, and flash frozen. Pelleted cells were lysed by re-suspending the pellet in 400 μL 1×LDS buffer (ThermoFisher Scientific)+5% β-ME, boiling for 5 min, and vigorously vertexing. Samples were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with primary antibodies 1:5k Rabbit anti-MBP (Millipore Cat #AB3596, RRID:AB_91531) and 1:10k Mouse anti-Tubulin (Millipore Cat #MABT205, RRID:AB_11204167), followed by secondary antibodies at 1:10k IRDye 680RD Goat anti-Rabbit IgG (LI-COR Biosciences Cat #925-68071, RRID:AB_2721181) and 543 IRDye 800CW Goat anti-Mouse IgG (LI-COR Biosciences Cat #P/N 925-32210, RRID:AB_2687825). Stained membrane was imaged using a LI-COR Odyssey® CLx imager. Blots are representative of two independent experiments.
o. Coexpression of CD-NTases and Effectors in E. coli
CD-NTases chosen for in-depth analysis were cloned into an arabinose-inducible, chloramphenicol resistant pBAD33 plasmid. Putative CD-NTase effector genes were selected based on proximity, if they were classified as involved in biological conflict (Burroughs et al. (2015) Nucleic Acids Res. 43:10633-10654), and based on analogous operon architecture to known effector phospholipases Effector genes were codon optimized for E. coli and cloned into pETSUMO, a carbenicillin resistant vector that is IPTG inducible in BL21-DE3 E. coli (ThermoFisher Scientific). Three pairs of vectors were assessed for each CD-NTase-effector pair: (1) cogant CD-NTase+effector, (2) CD-NTase+GFP, (3) mCherry+effector. Fluorescence proteins were used as negative controls. Vectors were cotransformed into electrocompetent BL21-DE3 E. coli, selected with both relevant antibiotics, and maintained wider non-inducing conditions (0.2% glucose). Overnight bacterial cultures were serially diluted into LB and 5 μL was spot plated on selective medium containing 5 μM IPTG and 0.2% arabinose. Colony formation was enumerated and images were taken at ˜24 h. Data are the mean±SEM of 3 independent experiments.
Cyclic dinucleotides (CDNs) play central roles in bacterial homeostasis and virulence as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern recognition receptors in animal cells. 3′3′ cGAMP is synthesized in V. cholerae by the enzyme DncV and controls a signaling network on the Vibrio Seventh Pandemic Island-1 (VSP-D), a mobile genetic element present in current V. cholerae pandemic isolates (Davies et al. (2012) Cell 149, 358-370; Dziejman et al (2002) Proc. Natl. Acad. Sci. U.S.A. 99:1556-1561). During the investigation of the homologs of dncV outside the Vibrionales, an unexpected partial operon was identified in E. coli where dncV is replaced with a gene of unknown function (WP_001593458, here renamed cdnE) (Schubert et al. (2004) Mol Microbiol 51:837-848). The operon architecture implies that cdnE may be an alternative 3′3′ cGAMP synthase (
DncV is a structural homolog of human cGAS, and each enzyme uses a single active site to sequentially form two separate phosphodiester bonds and release a CDN product (Kranzusch et al. (2014) Cell 158:1011-1021) (
In Vibrio, DncV controls the activity of cGAMP activated phospholipase in Vibrio (CapV), a patatin-like lipase that is a direct 3′3′ cGAMP receptor encoded in the dncV operon (Severin el al. (2018) Proc. Natl. Acad. Sci. U.S.A. 115:E6048-E6055). cdnE is also preceded by a gene encoding a patatin-like phospholipase (here renamed cUMP-AMP activated phospholipase in E. coli, capE,
To determine the mechanism of hybrid purine-pyrimidine CDN formation, and understand the relationship between CdnE, cGAS and DncV, a series of X-ray crystal structures of a CdnE homolog from the thermophilic bacterium Rhodothermus marinus were determined (Rm-CdnE,
aValues in parentheses are for highest-resolution shell.
aValues in parentheses are for highest-resolution shell.
The structure of Rm-CdnE in complex with nonhydrolyzable ATP and UTP reveals an asparagine (N166) side chain that forms hydrogen bonds with the uracil base and positions the UTP α-P for attack by the 3′ hydroxyl of ATP (
CdnE homologs were surveyed and it was determined that N166 is nearly universally conserved (
High-resolution structures of cGAS, OAS1, DncV, and two CdnE homologs allowed for the rational definition of shared structural and functional homology. All of these enzymes share three features: (1) a common DNA polymerase β-like nucleotidyltransferase superfamily protein-fold in spite of dramatic sequence divergence, (2) template-independent synthesis of a diffusible molecule through caging of the active site, using a protein scaffold not conserved with more distantly related templated polymerases, and (3) an active site architecture that allows diversification of products and phosphodiester linkage through amino acid substitutions within the active-site lid. This family of enzymes have been designated as CD-NTases (cGAS/DncV-like Nucleotidyltransferases), a structurally and evolutionarily distinct subset of the DNA polymerase β-like nucleotidyltransferase superfamily (
Many bacteria that encode CD-NTases thrive in close proximity to eukaryotic hosts, including fungi, plants, and animals such as humans (
DncV and CdnE evolved from a common ancestor but exhibit dramatic divergence in primary amino acid sequence. It was believed that these enzymes comprise only a small fraction of existing bacterial CD-NTase diversity, and that kingdom-wide analysis of the protein family would allow systematic identification of bacterial second messenger nucleotides as well as agonists/antagonists of the innate immune system. Accordingly, bioinformatic analysis was coupled with a large-scale, forward biochemical screen to directly uncover additional nucleotide second messengers. Previously. Burroughs et al. used a hidden Markov model derived from cGAS and DncV to identify ˜1,300 potentially related bacterial proteins (Burroughs et al. (2015) Nucleic Acids Res 43:10633-10654). Based on this previous analysis and the CdnE findings, >5,600 unique bacterial enzymes predicted to share common CD-NTase structural features were identified (
66 CD-NTase proteins were purified and each tested for nucleotide second messenger synthesis (
The 16 most active CD-NTases were selected for in-depth analysis (
Paradoxically, it was unexpected that CDNs would be identified by mass spectrometry in some reactions despite visualizing prominent product formation by PEI-cellulose TLC. Using orthogonal TLC conditions, these unknown products exhibited distinct migration patterns that indicated existence of unique non-CDN second messengers (
Similar to cUMP-AMP, the bacterial cyclic trinucleotide cAAG escaped STING recognition but was detected by RECON, confirming the new definition of STING and RECON ligand specificity (
Data presented here showed that CD-NTases are widely distributed and CD-NTases synthesize nucleotide second messengers with extraordinary biochemical diversity. Along with the GGDEF and DAC/DisA domains responsible for c-di-GMP and c-di-AMP synthesis in diverse bacterial phyla (Jenal & Lori (2017) Nat. Rev. Micro. 325:279; Corrigan & Grundling (2013) Nat. Rev. Micro. 11:513-524), CD-NTTases now represent a third major enzymatic family responsible for nucleotide second messenger synthesis. Recent evidence indicates divergent GGDEF family enzymes produce 3′3′ cGAMP in addition to c-di-GMP (Hallberg et al (2016) Proc. Natl. Acad Sci. U.S.A. 113:1790-1795), indicating that the selective pressures that drive CD-NTase product diversity are also in effect for GGDEF and DAC/DisA-like synthases. In bacteria, CD-NTases are found in similar operons and their shared location in mobile genetic elements indicates a unifying function CD-NTase products have cognate receptors in mammals, and CD-NTase genes can provide a selective advantage for some bacterium-eukaryote interactions. The data showed that bacteria can take advantage of the limits of host immune receptor specificity, and that a single mutation in a CD-NTase enables incorporation of pyrimidines, and thus evasion or enhancement of STING signaling by modulating enzyme specificity.
Nucleotidyltransferases (such as cGAS, DncV, and CdnE) are a highly diverse superfamily of proteins that share a common fold to catalyze many different chemical reactions that are not limited to cGAMP synthesis, including DNA polymerization, tRNA modification, and nucleotide modification. Based on the results described herein, it was unexpected that DncV homologs, like CdnE, would (1) synthesize a nucleotide second messenger and (2) that messenger would be a molecule other than cGAMP, such as cyclic UMP-AMP (cUMP-AMP). Moreover, despite recognition of CdnE, a major obstacle was systematic identification of other nucleotidyltransferases capable of catalyzing similar reactions. The crystal structure of CdnE was determined to understand the core protein motifs necessary for cUMP-AMP synthesis and compared these data against information for cGAMP synthesis. These observations allowed for the identification of a cGAS/DncV-like nucleotidyltransferase (CD-NTase) family of proteins and unified nucleotidyltransferases capable of second messenger synthesis, such as the surprising finding that a subset of CD-NTases synthesize cyclic trinucleotides, a new class of nucleotide second messenger.
A unifying characteristic of almost all CD-NTase-encoding genes is their location within similar operons in predicted mobile genetic elements (
The ordinarily skilled artisan can identify CD-NTases within an organism of interest based on the state of the art and the present disclosure. For example, several methods to locate CD-NTases in a given strain or identify specific strains/organisms that encode a CD-NTase of interest are shown as follows. If a given bacterial strain encodes an already annotated CD-NTase, the CD-NTase can be identified by downloading the Tables 4A-4C. Strain names, organism species, or other database identifiers can be searched in these tables. These tables can also be searched for a specific CD-NTase to identify bacterial strains encoding that gene. Upon locating the bacterial strain/gene, the associated NCBI accession number can be used to further locate sequence, ordered locus, and genomic information. Table 4A provides information on specific enzymes screened in
CD-NTases within any given organism can also be identified by conserved domain. For example, one can browse for the conserved domains that describe CD-NTase family proteins using Pfam domains: Mab-21 protein domain (PF03281). PAP_central domain (PF04928), N-terminal Pol-β-like nucleotidyltransferase core domain (PF14792 and PF01909), C-terminal OAS1_C domain (PF10421), and C-terminal tRNA-NucTransf2 domain (PF9249). EuKaryotic Orthologous Groups (KOG) database: KOG3963, KOG2245, KOG3792, and KOG37933; Clusters of Orthologous Groups (COG): COG5186, COG1746, COG1665, and COG1669; and NCBI conserved domain database: CD05402, CD05400, and CD5397.
An additional method of identifying CD-NTase genes encoded by an organism of interest is to BLAST the complete list of type CD-NTases using their NCBI identifiers, found in Table 4A using the following steps. A complete list of type CD-NTase identifiers (WP_001901330.1, WP_001593454.1, WP_023121145.1, WP_016849025.1, WP_020363757.1, WP_031517737.1, KDD27955.1, WP_032579276.1, WP_016268104.1, WP_012995826.1, WP_000058223.1, WP_017897513.1, WP_023633898.1, WP_002302472.1, WP_044727581.1, WP_000995828.1, WP_005836899.1, WP_026109030.1, EFJ98156.1, WP_001534692.1, WP_023223657.1, WP_005110610.1, YP_635404.1, WP_003090158.1, WP_001279388.1, WP_000246637.1, WP_031517014.1, WP_000246636.1, EIQ80517.1, WP_044779457.1, WP_008409465.1, WP_002106335.1, WP_000019626.1, WP_000763718.1, WP_003305997.1, WP_009895113.1, WP_043964485.1, EEH69894.1, WP_032676400.1, NP_766712.1, WP_023727438.1, WP_004556387.1, WP_032942206.1, WP_001056752.1, WP_031656629.1, WP_044359458.1, WP_013858317.1, WP_015376200.1, EKS31071.1, WP_006018769.1, WP_008253492.1, WP_012997810.1, WP_023568228.1, WP_000072410.1, WP_017790128.1, WP_006482377.1, WP_001593458.1, WP_042646516.1, WP_041847730.1, WP_000899483.1, WP_062726309.1, WP_054878246.1, WP_009654824.1, EGF179124.1, WP_009929206.1, WP_000102010.1, WP_002347527.1, WP_014072508.1, WP_016200549.1) is copied and pasted in to the box “Enter accession number” using a BLAST-P search (available on the World Wide Web at blast.ncbi.nlm.nih.gov/Blast.cgi). The organism and strain to search in the query box is defined as “Organism” and the “BLAST” button is clicked.
Additionally, ordinarily skilled artisan can classify the clade association of a newly described CD-NTase. For example, the CD-NTase amino acid sequence of interest can be aligned to the sequences in Table 4B. A newly described CD-NTase(s) is compared to neighboring proteins based on alignment. A CD-NTase is considered a member of a clade/cluster if it shares >24.5% amino acid identity with other members of that clade/cluster.
The horizontal acquisition of CD-NTases indicates that they provide a selective advantage but do not function to alter species-specific nucleotide signaling networks and instead alter bacterial physiology via receptors adjacently encoded, similar to capV-dncV and capE-cdnE. Burroughs et al. noted that genes adjacent to CD-NTases are generally involved in biological conflict, including phospholipases, nucleases, and pore-forming agents (Burroughs el al (2015) Nucleic Acids Res 43:10633-10654). Coexpression of dncV and capV is toxic to E. coli (Severin el al. (2018) Proc. Natl. Acad. Sci. U.S.A. 115:E6048-E6055) and it was tested if coexpression of each CD-NTase with its adjacently encoded, putative receptor was also toxic to E. coli. Expression of dncV-capV was unique in inhibiting colony formation, and other CD-NTase-predicted receptor pairs, including the active cdnE-capE pair, did not impair bacterial growth (
All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims the benefit of U.S. Provisional Application No. 62/727,647, filed on 6 Sep. 2018, and U.S. Provisional Application No. 62/769,163, filed on 19 Nov. 2018; the entire contents of each of said applications are incorporated herein in their entirety by this reference.
This invention was made with government support under grant number R01AI018045, R01AI026289. P41 GM103403. S10 RR029205 and 5T32CA207021-02 awarded by the National Institutes of Health and under grant number DE-AC02-06CH11357 awarded by the Department of Energy. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/049478 | 9/4/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62727647 | Sep 2018 | US | |
| 62769163 | Nov 2018 | US |
