Patent Application
20150010932
This application relates to methods for assaying protein-protein interactions and agents that modify such interactions.
Current methods of monitoring protein-protein interactions (PPIs), and for screening agents that modify such interactions, suffer from an inability to adequately interrogate low affinity PPIs, as well as to adequately assess bioavailability and functionality of test agents. One system, the bi-molecular luciferase complementation (BiLC) assay, has been developed for this purpose; however, current BiLC assays do not adequately address low affinity PPIs or test agent bioavailability.
The “ReBiL” assay disclosed herein involves a combination of BiLC assay and cre-recombinase mediated cassette exchange (RMCE). The disclosed ReBiL assays are novel, and provide an unexpectedly superior approach for interrogating PPIs (such as two members of a PPI pair) and agents that modify such interactions. Using the disclosed methods, it is possible to examine the specificity of PPIs, and the sensitivity of such interactions to agent modulation, with greater confidence than what was previously possible. The BiLC assay uses two complementary fragments of a split luciferase protein (termed nLuc and cLuc, respectively), each linked to a different member of a PPI pair (termed Protein1 and Protein2, respectively). Reconstitution of a functional luciferase protein from the two fragments is accomplished when Protein1 and Protein 2 (to which the nLuc and cLuc luciferase fragments are linked, respectively) form a PPI and bring the luciferase fragments into sufficient proximity to generate luciferase activity. In the disclosed ReBiL assay, the nLuc-Protein1 and cLuc-Protein2 fusion proteins are expressed from a nucleotide cassette introduced into the genome of a host cell by RMCE, which permits incorporation of the RMCE cassette at the same location in the genome of each host cell. The disclosed methods permit interrogation of transient and dynamic PPIs, identifying novel intracellular protein interaction partners, and screening and characterizing PPI antagonists and agonists.
In some embodiments, a method of determining if a test agent modifies a PPI between a first protein and a second protein is provided. The method includes inducing expression of a first fragment of a split-luciferase protein linked to the first protein and a second fragment of the split luciferase protein linked to the second protein, in a host cell. The first and second fragments can complement to form a functional split-luciferase protein. The host cell comprises a nucleic acid molecule introduced by RCME that encodes the first fragment linked to the first protein and the second fragment linked to the second protein, with each operably linked to an inducible promoter. The host cell is contacted with the test agent, and luciferase activity of the complemented split-luciferase protein is detected in the host cell. If an increase or decrease in the luciferase activity as compared to a control is detected, then the test agent is identified as an agent that modifies a PPI. If an increase or decrease in the luciferase activity as compared to the control is not detected, then the test agent is identified as an agent that does not modify a PPI. In some embodiments, if a decrease in luciferase activity as compared to a control is detected, then the test agent is identified as an agent that inhibits the PPI; and if an increase in luciferase activity as compared to a control is detected, then the test agent is identified as an agent that increases the PPI.
In additional embodiments, a method of determining if a test agent modifies a PPI between a first protein and a second protein is provided. The method includes inducing expression of a first fragment of a split-luciferase protein linked to the first protein and a second fragment of the split luciferase protein linked to the second protein, in a host cell. The first and second fragments can complement to form a functional split-luciferase protein. Additionally, the host cell comprises a nucleic acid sequence introduced by RCME that encodes the first fragment linked to the first protein and the second fragment linked to the second protein, each operably linked to an inducible promoter. The host cell can be lysed to form a cell lysate, and the lysate is contacted with the test agent. Any luciferase activity of the complemented split-luciferase protein is detected in the cell lysate. If an increase or decrease in the luciferase activity as compared to a control is detected, then the test agent is identified as an agent that modifies a PPI. If an increase or decrease in the luciferase activity as compared to the control is not detected, then the test agent is identified as an agent that does not modify a PPI. In some embodiments, if decrease in luciferase activity as compared to a control is detected, then the test agent is identified as an agent that inhibits the PPI; and if an increase in luciferase activity as compared to a control is detected, then the test agent is identified as an agent that increases the PPI.
In some embodiments, the disclosed methods are used to determining if a test agent is cell permeable, wherein determining that the test agent is cell permeable includes detected an increase or decrease in luciferase activity in the first cell population as compared to the control.
The foregoing and other features of this disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file named “Sequence.txt” (˜75 kb), which was created on Jun. 18, 2014, which is incorporated by reference herein. In the accompanying sequence listing:
SEQ ID NO: 1 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-p53 and cLuc-Mdm4 fusion proteins under control of a TREbi promoter.
SEQ ID NO: 2 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-p53 and cLuc-Mdm2 fusion proteins under control of a TREbi promoter.
SEQ ID NO: 3 is an exemplary nucleotide sequence encoding the nLuc-Linker1-HA-p53 fusion protein.
SEQ ID NO: 4 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-Mdm4 fusion protein.
SEQ ID NO: 5 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-Mdm2 fusion protein.
SEQ ID NO: 6 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Mdm4_RING domain and cLuc-Mdm2_RING domain fusion proteins under control of a TREbi promoter.
SEQ ID NO: 7 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Mdm4_RING domain and cLuc-Mdm2_RING_C464A domain fusion proteins under control of a TREbi promoter.
SEQ ID NO: 8 is an exemplary nucleotide sequence encoding the nLuc-Linker1-HA-Mdm4_RING domain fusion protein.
SEQ ID NO: 9 is an exemplary nucleotide sequence encoding the cLuc-Linker1-HA-Mdm2_RING domain fusion protein.
SEQ ID NO: 10 is an exemplary nucleotide sequence encoding the cLuc-Mdm2_RING_C464A domain fusion protein.
SEQ ID NO: 11 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2t and cLuc-FANCL fusion proteins under control of a TREbi promoter.
SEQ ID NO: 12 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2t and cLuc-FANCL_C307A fusion proteins under control of a TREbi promoter.
SEQ ID NO: 13 is an exemplary nucleotide sequence encoding the nLuc-Linker1-HA-Ube2t fusion protein.
SEQ ID NO: 14 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-FANCL fusion protein.
SEQ ID NO: 15 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-FANCL_C307A fusion protein.
SEQ ID NO: 16 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Mdm4 and cLuc-Mdm2 fusion proteins under control of a TREbi promoter.
SEQ ID NO: 17 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) for expressing nLuc-alone and the cLuc-Mdm2 fusion protein under control of a TREbi promoter.
SEQ ID NO: 18 is an exemplary nucleotide sequence encoding the nLuc-Linker1-HA-Mdm4 n-terminal fusion protein.
SEQ ID NO: 19 is an exemplary nucleotide sequence encoding the nLuc fragment of a split luciferase protein (including a linker1 and HA tag).
SEQ ID NO: 20 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-BRCA1 and BARD1-cLuc RING domain fusion proteins under control of a TREbi promoter.
SEQ ID NO: 21 is an exemplary nucleotide sequence encoding the nLuc-Linker1-HA-BRCA1 RING domain fusion protein.
SEQ ID NO: 22 is an exemplary nucleotide sequence encoding the BARD1_RING domain-cLuc fusion protein.
SEQ ID NO: 23 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2d3 and cLuc-BRCA1-BARD1 RING domain (BDfBC) fusion proteins under control of a TREbi promoter.
SEQ ID NO: 24 is an exemplary nucleotide sequence encoding the nLuc-Linker1-HA-Ube2d3 fusion protein.
SEQ ID NO: 25 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-BDfBC fusion protein.
SEQ ID NO: 26 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2d3 and cLuc-BRCA1_I26A-BARD1 RING domain fusion (BDfBC_I26A) fusion proteins under control of a TREbi promoter.
SEQ ID NO: 27 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-BDfBC_I26A fusion protein.
SEQ ID NO: 28 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2d3 and cLuc-BRCA1_C61G-BARD1 RING domain fusion (BDfBC_C61G) under control of a TREbi promoter.
SEQ ID NO: 29 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-BDfBC_C61G fusion protein.
SEQ ID NO: 30 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2d3 and cLuc-Mdm2 BiLC fusion proteins under control of a TREbi promoter.
SEQ ID NO: 31 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-Mdm2 fusion protein.
SEQ ID NO: 32 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2d3_C85A and cLuc-Mdm2 fusion proteins under control of a TREbi promoter.
SEQ ID NO: 33 is an exemplary nucleotide sequence encoding the nLuc-Linker1-HA-Ube2d3_C85A fusion protein.
SEQ ID NO: 34 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2d3 and cLuc-Mdm2_S395A fusion proteins under control of a TREbi promoter.
SEQ ID NO: 35 is an exemplary nucleotide sequence encoding the cLuc-Linker1-Flag-Mdm2_S395A fusion protein.
SEQ ID NO: 36 is an exemplary nucleotide sequence of a RMCE cassette (from Loxp3 to Loxp2) encoding the nLuc-Ube2d3_C85A and cLuc-Mdm2_S395A fusion proteins under control of a TREbi promoter.
SEQ ID NO: 37 is the amino acid sequence of an exemplary n-terminal fragment of a split luciferase protein that complements with SEQ ID NO: 39.
SEQ ID NO: 38 is an exemplary nucleotide sequence encoding SEQ ID NO: 37.
SEQ ID NO: 39 is the amino acid sequence of an exemplary c-terminal fragment of a split luciferase protein that complements with SEQ ID NO: 37.
SEQ ID NO: 40 is an exemplary nucleotide sequence encoding SEQ ID NO: 39.
SEQ ID NOs: 41 and 42 are the amino acid sequences of peptide linkers.
SEQ ID NOs: 43-45 are the amino acid sequences of protein tags.
SEQ ID NOs: 46-49 are the nucleic acid sequences of DNA primers.
SEQ ID NOs: 50-56 are the amino acid sequences of peptide linkers.
SEQ ID NOs: 57-91 are the nucleic acid sequences of plasmids for ReBiL assays.
The “ReBiL” assay disclosed herein involves a combination of the bi-molecular luciferase complementation (BiLC) assay and cre-recombinase mediated cassette exchange (RMCE). The BiLC assay uses two complementary fragments of a split luciferase protein (termed nLuc and cLuc, respectively), each linked to a different member of a PPI pair (termed Protein1 and Protein2, respectively). The complementary fragments of the split luciferase protein are not self-assembling. Reconstitution of a functional luciferase protein from the two fragments is accomplished when Protein1 and Protein 2 (to which the nLuc and cLuc luciferase fragments are linked, respectively) form a PPI and bring the luciferase fragments into sufficient proximity to generate luciferase activity. In the disclosed ReBiL assay, the nLuc-Protein1 and cLuc-Protein2 fusion proteins are expressed from a nucleotide cassette introduced into the genome of a host cell by RMCE, which permits incorporation of the RMCE cassette at the same location in the genome of each host cell. The disclosed methods permit interrogation of transient and dynamic PPIs, identifying novel intracellular protein interaction partners, and screening and characterizing PPI antagonists and agonists. The disclosed ReBiL assays are novel, and provide an unexpectedly superior approach for interrogating PPIs (such as two members of a PPI pair) and agents that modify such interactions.
In prior BiLC assays (that do not include the RMCE component described herein) the nucleic acid molecules encoding nLuc-Protein1 and cLuc-Protein2 were included on plasmids that needed to be simultaneously transfected into mammalian cells by either lipid-based methods or electroporation. Consequently, the sensitivity, accuracy and reproducibility of the BiLC assay (without RMCE) could be influenced by the percentage of cells in a population expressing each BiLC plasmid, as well as the copy numbers of plasmid DNA's taken uptake by the each transfected cell. These transfection-associated drawbacks have limited the usefulness of the BiLC assay.
The disclosed embodiments overcome transfection-associated limitations of BiLC assays by using RMCE-based inducible protein expression to (1) insert a nucleic acid cassette encoding both the nLuc-Protein1 and the cLuc-Protein2 (the RMCE cassette) into a single pre-selected genomic locus in the genome of any cell line of interest, and (2) express each of these proteins in an inducible manner (e.g., under control of a doxycycline-inducible promoter). The ReBiL strategy, and organization of the target sites in the genome, insures the insertion of a single copy of targeting cassette in a known orientation. This enables reproducible and consistent doxycycline-controllable expression of different split luciferase fusion proteins in independently constructed cell lines containing the same genomic target site.
The ReBiL assay is particularly effective for the detection and analysis of low affinity and transient PPIs. An example is the interaction between the E2 ubiquitin conjugating enzyme Ube2t and the E3 ubiquitin ligase FANCL. A standard transfection assay using split-luciferase complementation failed to reveal any significant interaction between Ube2t and FANCL. However, using the same split-luciferase fusion proteins in a ReBiL platform readily revealed their interaction above a negligible background observed with a mutant form of FANCL (C307A) that does not interact with Ube2t. This specific interaction revealed by ReBiL is noteworthy since analyses using purified proteins revealed a dissociation constant (Kd) of 454 nM, and this measurement required analysis at the non-physiological temperature of 8° C.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.
As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. As used herein, the term “comprises” means “includes.” Thus, “comprising a nucleic acid molecule” means “including a nucleic acid molecule” without excluding other elements. It is further to be understood that any and all base sizes given for nucleic acids are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All references, including patent applications and patents, are herein incorporated by reference.
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
3′ end: The end of a nucleic acid molecule that does not have a nucleotide bound to it 3′ of the terminal residue.
5′ end: The end of a nucleic acid sequence where the 5′ position of the terminal residue is not bound by a nucleotide.
Amino acid: Naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Antisense and Sense: Double-stranded DNA (dsDNA) has two strands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′ strand (the reverse compliment), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5′ to 3′ direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T).
Bi-molecular Luciferase Complementation (BiLC) assay: An assay for identifying a PPI, or modulation of a PPI, by an agent. The BiLC assay uses two complementary fragments of a split luciferase protein, each linked to a different member of a PPI pair. Reconstitution of a functional luciferase protein from the two fragments is accomplished when the members of the PPI pair form a PPI and bring the luciferase fragments into sufficient proximity to generate luciferase activity. Thus, formation of the PPI pair can be detected by detecting the corresponding luciferase activity. Split luciferase proteins, and methods of detecting the luciferase activity of such proteins, are disclosed herein and are known in the art, see, e.g., PCT Pub No. WO2007/027919.
Contacting: Placement in direct physical association, for example solid, liquid or gaseous forms. Contacting includes, for example, direct physical association of fully- and partially-solvated molecules.
Control: A sample or standard used for comparison with an experimental sample. The person of ordinary skill in the art will be able to select appropriate controls for the disclosed assays. In some embodiments, the control is a cell lysate or cell line that has not been treated with a test agent for comparison with a corresponding cell lysate or cell line that has been treated with the test agent. In additional embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample or plurality of such samples), or group of samples that represent baseline or normal values.
Detecting: To identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting luciferase activity in a cell or cell lysate. Detection can include a physical readout, such as fluorescence or a reaction output. Detection can be quantitative or qualitative.
Encoding: Unless evident from its context, includes nucleic acid sequences, such as RNA and DNA sequences, that encode a polypeptide, as well as RNA and DNA sequences that are transcribed into proteins, such as split-luciferase proteins an/or test proteins, and the like.
Expression: The process by which the coded information of a nucleic acid molecule is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein.
Host Cell or Recombinant Host Cell: A cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector. Typically, a host cell is a cell in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. For example, the host cell may be a bacteria cell, including an E. coli cell. “Host cell” also includes a colony of cells, for example, a colony of E. coli cells. Thus, “contacting a host cell” and “incubating a host cell” include contacting a colony of host cells or incubating a colony of host cells. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
Increase or Decrease: A statistically significant positive or negative change, respectively, in quantity from a control value. An increase is a positive change, such as a 50%, 100%, 200%, 300%, 400% or 500% increase as compared to the control value. A decrease is a negative change, such as a 50%, 100%, 200%, 300%, 400% or 500% decrease as compared to a control value.
Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer, which can include analogues of natural nucleotides that hybridize to nucleic acid molecules in a manner similar to naturally occurring nucleotides. In a particular example, a nucleic acid molecule is a single stranded (ss) DNA or RNA molecule, such as a probe or primer. In another particular example, a nucleic acid molecule is a double stranded (ds) nucleic acid, such as a target nucleic acid. Examples of modified nucleic acids are those with altered backbones, such as peptide nucleic acids (PNA).
DNA is a long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine (A), guanine (G), cytosine (C), and thymine (T) bound to a deoxyribose sugar to which a phosphate group is attached.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Promoter: A promoter is an array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements. A “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor). In one example, an inducible promoter is a bi-directional inducible promoter such as a TREbi promoter.
Protein: A polymer of amino acid residues, including amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Multiple polymers of amino acids binding to each other are a protein complex. Protein and polypeptide may be used interchangeably throughout this application and mean at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
Protein-Protein Interaction (PPI): A specific binding event between two proteins. For example, a PPI can occur between a receptor and a particular ligand. Binding can be specific and selective, so that one molecule is bound preferentially when compared to another molecule. In one example, a PPI is identified by a disassociation constant (Kd) of first protein for a second protein, compared to the Kd for one or more other cellular proteins.
Protein Tag: A polypeptide that, when fused to a heterologous protein or peptide, facilitates the detection or isolation of the heterologous protein. Nucleic acid encoding tags and nucleic acid constructs including nucleic acid sequences encoding tags are known to the skilled artisan and are available commercially. Exemplary tags include T7, FLAG, hemagglutinin (HA) VSV-G, V5, c-myc tags histidine (e.g., 6HIS; 5HIS), MBP, CBP and GST tags. Reagents (e.g., antibodies) to these and other tags are commercially available for a variety of sources.
Recombination mediated cassette exchange (RMCE): A method for genetic modification of mammalian cells. RMCE involves use of a DNA recombinase to target a nucleic acid molecule (the “RMCE cassette”) into a pre-determined genomic locus of a cell that was previously modified to contain the appropriate recombinase recognition sequences. For example, the widely used bacteriophage P1 Cre/LoxP recombination system utilizes LoxP sites to target recombination events. RMCE strategies can utilize two heterospecific LoxP sites (LoxP sites of different sequences) that will not recombine with each other to allow for directed recombination events. The LoxP sites are included on either end of the RMCE cassette, and the internal nucleotides encode proteins of interest, such as split-luciferase proteins for BiLC assays as described herein. Exemplary RMCE systems are known, and include, for example, in Wong et al., “Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange,” Nucleic Acids Res, 33, e147, 2005, and Toledo et al., “RMCE-ASAP: a gene targeting method of ES and somatic cells to accelerate phenotype analysis,” Nucl. Acids Res., 34, e922006; each of which is incorporated by reference herein in its entirety.
Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI web site. BLASTN is used to compare nucleic acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15÷20*100=75).
One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above.
Split-luciferase: A protein complex composed of two polypeptide chains that form a functional luciferase enzyme. Individually, the two polypeptide chains lack luciferase activity, but, when formed into a complex, the two proteins have functional luciferase enzymatic activity. The two polypeptide chains of a particular split-luciferase protein are known as complementing fragments or complementary fragments, or the first fragment and the second fragment of the split fluorescent protein. The split luciferase proteins for use with the disclosed methods are not self-assembling, that is, they do not include first and second fragments that spontaneously complement to form a functional luciferase. In some embodiments, the first fragment and the second fragment of a split luciferase protein for use in the disclosed methods are linked to different members of a PPI pair. Upon interaction of the members of the PPI pair, the first fragment and the second fragment of the split luciferase are brought within close enough proximity to form a functional luciferase, the activity of which can be detected, for example, using standard luciferase assays. Non self-assembling split luciferase proteins, and methods of detecting the luciferase activity of such proteins, are disclosed herein and are known in the art, see, e.g., PCT Pub No. WO2007/027919.
Test agent: Any agent that that is tested for its effects, for example its effects on a cell. In some embodiments, a test agent is a chemical compound, such as a chemotherapeutic agent or even an agent with unknown biological properties.
Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity. In one example the desired activity is hybridization of a probe to a target nucleic acid molecule.
Vector: A nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. An integrating vector is capable of integrating itself into a host nucleic acid. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes.
A. nLuc-Protein1, cLuc-Protein2, and Expression Thereof
The N- and C-terminal fragments of the split luciferase protein (“nLuc” and “cLuc,” respectively) are non-assembling fragments of a luciferase protein. Generally, a luciferase is an enzyme that catalyzes a light producing chemical reaction. For example, in several embodiments, the split fluorescent proteins for use with the disclosed assays include fragments of a firefly luciferase that uses D-luciferin as a substrate. An exemplary nLuc protein sequence for use with the disclosed embodiments is set forth as follows:
An exemplary DNA sequence encoding this nLuc protein is set forth as follows:
An exemplary cLuc protein sequence for use with the disclosed embodiments is set forth as follows:
An exemplary DNA sequence encoding this cLuc protein is set forth as follows:
One skilled in the art will appreciate that these sequences can be altered, while still retaining the desired function. Thus in some examples the sequences used have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, or 40.
It will be understood that the N-to-C terminal orientation of the first fragment of the split luciferase protein and the first member of the PPI pair in nLuc-Protein1 can be set forth as N-[first fragment of the split luciferase protein]-[first member of the PPI pair]-C, or N-[first member of the PPI pair]-[first fragment of the split luciferase protein]-C. Similarly, the N-to-C terminal orientation of the second fragment of the split luciferase protein and the second member of the PPI pair in cLuc-Protein2 can be set forth as N-[second fragment of the split luciferase protein]-[second member of the PPI pair]-C, or N-[second member of the PPI pair]-[second fragment of the split luciferase protein]-C.
In several embodiments, the nLuc-Protein1 and cLuc-Protein2 fusion proteins include a peptide linker separating the first or second fragment of the luciferase protein from the first or second member of the PPI pair, respectively. Exemplary linkers are disclosed in PCT Pub. No. WO2007/027919, herein incorporated by reference. In several embodiments, the peptide linker includes one or more a protein tag sequences. In some embodiments, the peptide linker includes two protein tag sequences. In some embodiments, the linker includes the amino acid sequence set forth as QISYASRGGSSGGG (SEQ ID NO: 41, designated Linker1) or GGGSSGGGQISYASRG (SEQ ID NO: 42, designated Linker2) and further includes one or more protein tags, such as a FLAG-tag (DYKDDDDK; SEQ ID NO: 43), Human influenza hemagglutinin (HA)-tag (YPYDVPDYA; SEQ ID NO: 44) and/or Myc-tag (EQKLISEEDL, SEQ ID NO: 45). This enables expression of the split-luciferase fusion proteins to be readily detected using commercial anti-FLAG, anti-HA, or anti-myc antibodies.
In some embodiments, the n-terminal split luciferase fragment (nLuc) and its fusion protein (Protein1) are constructed as Protein1-Linker1-HA-nLuc-FLAG. The c-terminal of split-luciferase (cLuc) and its fusion protein (Protein2) can be constructed as Protein2-Linker2-myc-cLuc-FLAG. In some embodiments, the Protein1-Linker1-HA-nLuc-FLAG and Protein2-Linker2-myc-cLuc-FLAG can be detected in western blot by anti-HA and anti-myc antibodies, respectively. In addition, their intracellular interaction locations (nucleus or cytosol) can be detected in situ, for example, by a proximity ligation in situ assay (P-LISA) (Soderberg, et al., Nat Methods 3, 995-1000, 2006) using anti-HA and anti-myc antibodies. Furthermore, since both split-luciferase fusion proteins carry a signal copy of the FLAG-tag, they can be quantified using the anti-FLAG antibody.
The disclosed methods can be used to interrogate the interaction of any two proteins, and/or to assay whether or not a particular test agent increases or decreases the interaction of any two proteins. Examples of possible PPI pairs include receptor-ligand, antibody-antigen, enzyme-substrate, dimerizing proteins, components of signal transduction cascades, component(s) of a composite structure, such as a ribosome or a virus, intercellular interacting molecules on different cells, such as an antigen presenting cell and an immune cell for response, such as a T cell, a B cell, an NK cell, a dendritic cell, a monocyte, a macrophage and so on, and other PPI pairs known to the art.
In some embodiments, the assays disclosed herein can be used for example, for detecting, monitoring, and/or modulating PPIs associated with human disease. Non-limiting examples of PPIs for use with the disclosed assays are listed in Table 1.
Nucleic acids encoding the nLuc-Protein1 and cLuc-Protein2 are provided. Nucleic acids encoding these molecules can readily be produced by one of skill in the art, using the amino acid and nucleotide sequences provided herein, sequences available in the art, and the genetic code. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same nLuc-Protein1 or cLuc-Protein2antibody sequence.
Nucleic acid sequences encoding the nLuc-Protein1 and cLuc-Protein2 can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.
Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.
The nLuc-Protein1 and cLuc-Protein2 polypeptides can be expressed from a nucleic acid cassette that is inserted into the genome of a host cell by RMCE.
The split luciferase fusion constructs in the RMCE cassette (see
Thus, the ReBiL technology uses several molecular manipulations of genetic constructs, and then introduction of the RMCE cassette encoding both the nLuc-Protein1 and cLuc-Protein2 into a single chromosomal site of a selected reporter cell line using RMCE. The nucleotide sequence of exemplary RMCE cassettes for ReBiL assays are provided herein as SEQ ID NOs: 1, 2, 6, 7, 11, 12, 16, 17, 20, 23, 26, 28, 30, 32, 34, and 36. One skilled in the art will appreciate that these sequences can be altered, while still retaining the desired function. Thus in some examples the sequences used have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 6, 7, 11, 12, 16, 17, 20, 23, 26, 28, 30, 32, 34, or 36.
Additionally, RMCE systems that can be used with the disclosed methods have been described, for example, in Wong et al., “Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange,” Nucleic Acids Res, 33, e147, 2005, and Toledo et al., “RMCE-ASAP: a gene targeting method of ES and somatic cells to accelerate phenotype analysis,” Nucl. Acids Res., 34, e922006; each of which is incorporated by reference herein in its entirety.
Reporter cell lines for use with the disclosed ReBiL assays can be constructed using known methods. In one non-limiting example, a reporter cell line for use with the disclosed methods is a U2OS human osteosarcoma cell line including an rtTA transactivator and TetR-KRAB transrepressor (this cell line is designated U2OS 134-8 HyTK-8). It will be appreciated that a host cell or a host cell line has been genetically altered by introduction of an exogenous polynucleotide. In several embodiments a host cell is a cell including a modified genome designed for RCME recombination. Reference to a host cell includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used. In some examples the host cell is a mammalian cell, such as a human cell. Additional cell lines that can be used with the disclosed methods are known in art, for example RMCE master cell lines that carry the HyTK exchange cassette, such as U2OS 134-8 HyTK-8 (human osteosarcoma cell line) (Wang, et al., Proc Natl Acad Sci USA 104, 12365-12370, 2007; Wade et al., Oncogene 31, 4789-4797, 2012), Saos-2 134-HyTK-20 cell line, and CHO 111-134 8-11 (Chinese hamster ovary cell line) (Wong et al., Nucleic Acids Res 33, e147, 2005; and Green et al., PLoS One 8, e58395, 2013). Additional examples of such cell lines, and methods of constructing such cell lines are described in Wong et al., Nucleic Acids Res, 33, e147, 2005, Toledo et al., Nucl. Acids Res., 34, e922006, 2006, and Green et al., PLoS One 8, e58395, 2013; each of which is incorporated by reference herein in its entirety.
The BiLC assay component of the ReBiL assay can be performed in cells, as well as in cell lysate. The duality of these two approaches allows for exquisite interrogation of PPIs in ways that were not possible before the instant disclosure. Using the disclosed embodiments, it is possible to examine the specificity of PPIs, and the sensitivity of such interactions to agent modulation, with greater confidence that what was previously possible.
For example, in some embodiments, the BiLC assay can be performed in a lysate prepared from cells induced to express the nLuc-Protein1 and cLuc-Protein2 fusion proteins in the presence or absence of serum to determine if a test agent (that modifies a PPI of interest) is or is not affected by the presence of serum. If, in the lysate in the presence of serum, the test agent increases or decreases the PPI (detected by increase or decrease in split luciferase activity) to the same degree as in the absence of serum, it indicates that the serum is not interfering with the test agent sufficiently or in such a way as to prevent the test agent from increasing or decreasing the PPI (e.g., by binding to the test agent or its target site or sites on one or both of the interacting proteins). If, in the lysate in the presence of serum, the test agent does not increase or decrease the PPI (detected by increase or decrease in split luciferase activity) to the same degree as in the absence of serum, it indicates that the serum is interfering with the test agent sufficiently or in such a way as to prevent the test agent from increasing or decreasing the PPI (e.g., by binding to the test agent or its target site or sites on one or both of the interacting proteins). In some embodiments, a finding of at least a 50% (such as at least a 60%, at least a 70%, at least a 80% or at least a 90%) change between the split luciferase activity measured in the presence of serum compared to the absence of serum indicates that the serum is interfering with the ability of the test agent to increase or decrease the PPI.
Further, the BiLC assay in live cells may be used to assess whether a test agent is penetrating the cell membrane and accessing the target PPI within the cell. For example, a finding that a test agent affects a PPI (detected by increase or decrease in split luciferase activity) in cell lysate, but not to the same degree in live cells, indicates that the test agent is unable to access (or is impeded in its access to) the protein-protein interacting pair in cells. For example, the test agent may not be able to penetrate the cell surface, or access the cellular compartment containing the protein-protein interacting pair. If serum is included in the tissue culture medium, the test agent may be binding to the serum, or the serum may bind to a receptor for the test agent on the cell surface, or may interact with the cell in another way to impede entry of the test agent into the cell or into the region within the cell where the interacting protein targets localize. In some embodiments, a finding that the test agent produces no more than 50% of the increase or decrease in the PPI (as measured by detecting an increase or decrease in split luciferase activity) in live cells compared to that in cell lysate, indicates that the test agent is unable to access (or is impeded in its access to) the protein-protein interacting pair in cells.
Thus, the disclosed embodiments include assays in which the combination of a cell-based and a lysate-format BiLC assay enables the BiLC approach to be used for rapid optimization of the Structure Activity Relationship (SAR) of a lead agent, for example to detect modifications that do not interfere with the ability of an agent to bind its target but do enable the agent to enter the cell.
In several embodiments, the disclosed methods include a method of assaying a test agent for modification of an interaction between a first protein and a second protein. In some embodiments, the first and second proteins are proteins known to interact, and the test agent is an agent being screened for an increase or decrease in the interaction between the first and second proteins. In additional embodiments, the first and second proteins are proteins known not to interact, and the test agent is an agent being screened for an increase in the interaction between the first and second proteins. The BiLC assay can be performed in the live cells expressing the proteins, or a cell lysate can be generated, and the BiLC assay performed on the cell lysate. The cells or the lysate can be contacted with the test agent and luciferase activity is measured.
Detecting a decrease in luciferase activity (e.g., at least a 20% decrease, such as at least a 30, 40, 50, 60, 70, 80, 90, or 95% decrease in luciferase activity) compared to a control (e.g., a sample not treated with the test agent) indicates that the test agent inhibits the interaction between the first and second proteins, detecting an increase in luciferase activity (e.g., at least a 20% increase, such as at least a 30, 40, 50, 100, 150, 200, 300, 400, or 500% increase in luciferase activity) compared to a control (e.g., a sample not treated with the test agent) indicates that the agent increases the interaction between the first and second proteins, and detecting no significant change in luciferase activity (e.g., no more than a 20% increase (such as no more than a 5, 10, or 15% increase) and/or no more than a 20% decrease (such as no more than a 5, 10, or 15% decrease) in luciferase activity) compared to a control (e.g., a sample not treated with the test agent) indicates that the agent does not modify the interaction between the first and second proteins. It will be appreciated that the assay can be performed in cells, and in lysate, to determine if the test agent cell permeable, and to test the effects of various other compounds on the ability of the test agent to affect the PPI.
Methods of detecting and quantitating the luciferase activity of a split luciferase protein are known to the person of ordinary skill in the art. Examples of such methods are described herein as well as, for example, in Luker et al., “Kinetics of regulated PPIs revealed with firefly luciferase complementation imaging in cells and living animals,” Proc Natl Acad Sci USA, 101, 12288-12293, 2004, and PCT Pub. WO2007/027919, each of which is incorporated by reference herein in its entirety.
In some embodiments, an algorithm is used to quantify the BiLC signal from ReBiL assay, for example, for comparing results between different ReBiL assays. In some embodiments, the algorithm for quantifying signals from an ReBiL assay includes Equation 1:
[BiLC/CellTiter]/[(Split luc fusionLess)/Actin] (Equation 1)
wherein
“BiLC” is the bi-luciferase luminescent signal represents PPI in each sample; “CellTiter” is the CellTiter-Glo (or other cell detecting reagent) luminescent signal that provides an estimation of cell number in each sample. The CellTiter-Glo (Promega Cat # G7572) is a luminescence based assay that measures the numbers of viable cells by quantify the ATP produced by viable cells. “Split luc fusionLess” is the protein abundance of the less abundant split luciferase fusion protein of a pair of N-ter and C-ter split luciferase fusion proteins. For example, if each split-luciferase protein includes a single copy HA-epitope, the amounts of split luc fusion proteins can be determined by anti-HA antibody in a quantitative western blot with Licor Odyssey system, and the split luciferase protein of least abundance identified. “Actin” is the loading control for both nLuc and cLuc in the western blot. An embodiment of quantification of ReBiL assay results using BiLC signal and western blotting using the Licor Odyssey image system is depicted in
Several embodiments utilize temperature modulation for ReBiL assay. For example, the RMCE-base protein expression, or the BiLC assay (or both) can be performed at various different temperatures depending on the particular PPI of interest. In some examples, the RMCE-based inducible protein expression, the BiLC assay, or both is performed at 37° C. In other examples, the RMCE-based inducible protein expression, the BiLC assay, or both is performed at less than 37° C. (such as 36, 35, 34, 33, 32, 31, or 30° C., for example 30-36 or 32-34° C.). In still more examples, the RMCE-based inducible protein expression, the BiLC assay, or both is performed at more than 37° C. (such as at least 37, 38, 39, 40 or 41° C., for example 37 to 60° C., 37 to 50° C. or 37 to 40° C.).
In several embodiments, the BiLC assay is performed in cell lysate. Methods and reagents for making cell lysate are known to the person of ordinary skill in the art. In some embodiments, the buffer used for making the cell lysate includes:
(a) CA-630 lysis buffer (CLB): 50 mM Tris-HCl pH8.0, 5 mM EDTA, 150 mM NaCl, 0.5% CA-630, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and a protease inhibitor cocktail (such as Complete Mini Protease Inhibitor Cocktail (Roche Cat #11836153001) or;
(b) PPI lysis buffer (PLB): 100 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 150 mM NaCl; 0.1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and a protease inhibitor cocktail (such as Complete Mini Protease Inhibitor Cocktail (Roche)); or
(c) Promega Glo Lysis Buffer (GLB, Promega Cat # E2661).
Further, in some embodiments, the ReBiL assay may be used to report the status of a cell such as ES cell if one has an assay for a cellular property, such as “stemness,” based on protein interactions. As an example, if two proteins are required to interact in order to endow stem cells with the capacity to self-renew, a cardinal property of stem cells, then the disclosed ReBiL assay can be used to detect such interaction by the methods described herein.
Performing the BiLC assay in live cells versus in cellular lysate to assess a test agent's permeability also can be extended to other types of biochemical or biological assays. For example, the stability of a protein usually increases when binding to its ligands (or some compounds). If the ligand (or compound) is not cell permeable, it would not stabilize its intracellular target proteins. Thus, increased target protein stability could be a second indicator that the compound is hitting its target in the cell. This might be valuable if the molecule enters the cell at concentrations high enough to reduce BiLC fractionally, but not at high enough levels to elicit a biological response. Thus, the combination of reduced luciferase and increased target stability would provide an internal validation control to give greater confidence in a positive result for a low efficacy lead molecule and would support optimization efforts.
In several embodiments, the ReBiL assay is used in high throughput screens of test agents for modulators of particular PPIs. In this context, the sensitivity of PPI interactions to agent modulation can be examined with greater confidence that what was previously possible. For example, in several such embodiments, the high-throughput assays can be completed with a Z-prime score of greater than 0.5 (see Example 1 for additional description of Z-prime scores).
Additional methods and reagents for BiLC assays (without RMCE) have been described, for example, in Luker et al., “Kinetics of regulated PPIs revealed with firefly luciferase complementation imaging in cells and living animals,” Proc Natl Acad Sci USA, 101, 12288-12293, 2004, and PCT Pub. WO2007/027919, each of which is incorporated by reference herein in its entirety.
The methods disclosed herein are of use for identifying test agents that are modulators of PPIs. The test agents identified using the methods disclosed herein can be of use for increasing or decreasing a PPI. Any test agent that has potential (whether or not ultimately realized) to affect the PPI can be tested using the methods of this disclosure.
Exemplary test agents include, but are not limited to, peptides such as, soluble peptides, including but not limited to members of random peptide libraries (see for example, Lam et al., Nature, 354:82-84, 1991; Houghten et al., Nature, 354:84-86, 1991), and combinatorial chemistry-derived molecular library made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; for example, Songyang et al., Cell, 72:767-778, 1993), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, and epitope-binding fragments thereof), small organic or inorganic molecules (such as, so-called natural products or members of chemical combinatorial libraries), hormone(s), molecular complexes (such as protein complexes), or nucleic acids. The person of skill in the art is familiar with agents, for example small molecule libraries of agents, which can be used in the disclosed methods. Several embodiments include screening a library of agents for an effect on a PPI of interest.
Appropriate tests agents can be contained in libraries, for example, synthetic or natural compounds in a combinatorial library. Numerous libraries are commercially available or can be readily produced; means for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, such as antisense oligonucleotides and oligopeptides, also are known. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or can be readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Such libraries are useful for the screening of a large number of different compounds.
Libraries (such as combinatorial chemical libraries) useful in the disclosed methods include, but are not limited to, peptide libraries (for example see U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res., 37:487-493, 1991; Houghton et al., Nature, 354:84-88, 1991; and PCT Publication No. WO 91/19735), encoded peptides (see for example PCT Publication WO 93/20242), random bio-oligomers (see for example PCT Publication No. WO 92/00091), benzodiazepines (see for example U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (see for example Hobbs et al., Proc. Natl. Acad. Sci. USA, 90:6909-6913, 1993), vinylogous polypeptides (see for example Hagihara et al., J. Am. Chem. Soc., 114:6568, 1992), nonpeptidal peptidomimetics with glucose scaffolding (see for example Hirschmann et al., J. Am. Chem. Soc., 114:9217-9218, 1992), analogous organic syntheses of small compound libraries (see for example Chen et al., J. Am. Chem. Soc., 116:2661, 1994), oligocarbamates (see for example Cho et al., Science, 261:1303, 1003), and/or peptidyl phosphonates (see for example Campbell et al., J. Org. Chem., 59:658, 1994), nucleic acid libraries (see Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989), peptide nucleic acid libraries (see for example U.S. Pat. No. 5,539,083), antibody libraries (see for example Vaughn et al., Nat. Biotechnol., 14:309-314, 1996; PCT App. No. PCT/US96/10287), carbohydrate libraries (see for example Liang et al., Science, 274:1520-1522, 1996; U.S. Pat. No. 5,593,853), small organic molecule libraries (see for example benzodiazepines, Baum, C&EN, January 18, page 33, 1993; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones and methathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514) and the like.
Libraries useful for the disclosed screening methods can be produced in a variety of manners including, but not limited to, spatially arrayed multipin peptide synthesis (see for example Geysen, et al., Proc. Natl. Acad. Sci., 81(13):3998 4002, 1984), “tea bag” peptide synthesis (see for example Houghten, Proc. Natl. Acad. Sci., 82(15):5131 5135, 1985), phage display (see for example Scott and Smith, Science, 249:386-390, 1990), spot or disc synthesis (see for example Dittrich et al., Bioorg. Med. Chem. Lett., 8(17):2351 2356, 1998), or split and mix solid phase synthesis on beads (see for example Furka et al., Int. J. Pept. Protein Res., 37(6):487 493, 1991; Lam et al., Chem. Rev., 97(2):411-448, 1997). Libraries may include a varying number of compositions (members), such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members, such as up to about 500,000 members, or even more than 500,000 members.
In one convenient embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such combinatorial libraries are then screened in one or more assays as described herein to identify those library members (particularly chemical species or subclasses) that display a desired characteristic activity (such as in an increase or decrease in luciferase activity resulting from nLuc-Protein1 and cLuc-Protein2 interaction). In one example a test agent of use is identified that increases a PPI of interest. In another example a test agent of use is identified that decreases a PPI of interest.
The compounds identified using the methods disclosed herein can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics. In some instances, pools of candidate agents may be identify and further screened to determine which individual or subpools of agents in the collective have a desired activity.
An agent that decreases a PPI is one that reduces the quality, amount, or strength of the interaction between two proteins, for example the binding of a first protein to a second protein. In some examples, the agent can reduce the interaction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control, such as the interaction of the first and second protein in the absence of the agent. Such decreases can be measured using the methods known in the art and further disclosed herein. An agent that increases a PPI is one that enhances the quality, amount, or strength of the interaction between two proteins, for example the binding of a first protein to a second protein. In some examples, the agent can increase the interaction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control, such as the interaction of the first and second protein in the absence of the agent. Such increases can be measured using the methods known in the art and further disclosed herein.
The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims should not be limited to those features exemplified.
This example illustrates development of the ReBiL assay for interrogating PPIs in live cells, and in cell lysate, and for examining the effect of test agents on PPIs.
Most cellular functions involve PPIs. Consequently, strategies to detect PPIs and their interruption by antagonists in living cells can illuminate biological mechanisms and generate drugs to treat diverse diseases. This example discloses recombinase enhanced BiLC, “ReBiL,” which combines Cre-recombinase mediated cassette exchange (RMCE) and bi-molecular luciferase complementation (BiLC), and was developed to enable PPI detection and facilitate elucidation of antagonists and agonists in vivo and in vitro. The ReBiL assay was used to evaluate small molecule and peptide antagonists of complexes comprising the p53 tumor suppressor and its repressors Mdm2 and Mdm4. Small molecule antagonist Nutlin-3a exhibited the expected activity in living cells and in cell lysates, while SAH peptides only exhibited potency in vitro. Such peptides were shown to induce p53-independent cytosolic enzyme leakage, a process antagonized by serum and exacerbated by the TAT transduction domain. The ReBiL assay's ability to enable rapid assessment of target specificity, cell permeability, and off-target effects facilitates development of next generation therapeutics, cell permeable PPI modifiers, and elaboration of diverse biological mechanisms.
Most cellular functions involve macromolecular machines whose functions require interactions between multiple proteins. These interactions may be static or dynamic and are typically tightly regulated. It is therefore not surprising that aberrant PPIs (PPIs) lead to myriad human diseases. For example, cancer initiation and progression can result from aberrant interaction between proteins that function as oncogenic drivers, and their antagonists that serve as suppressors of tumorigenesis. Defects in the p53 tumor suppressor pathway have been estimated to occur in up to 22 million cancer patients, with about 50% being due to inactivating mutations in p53 itself. Many of the remaining tumors contain lesions that engender over-expression of either of two oncogenes, Mdm2 or Mdm4, that bind to p53 and inactivate it by serving as an E3 ubiquitin ligase (Mdm2) and/or a transcriptional antagonist (Mdm2 and Mdm4). Therefore, for patients with cancers expressing wild type p53, restoring its function using pharmacological interruption of p53-Mdm2 and p53-Mdm4 is an attractive strategy.
Unlike classic enzyme-ligand binding pockets that can be effectively targeted by small molecules, surfaces at which protein subunits interact are typically large, flat and featureless. This has led to the general impression that PPIs cannot be targeted using small drug-like compounds. Nevertheless, several PPI antagonists that disrupt p53-Mdm2, p53-Mdm4 or both have been reported (Brown et al., Nat Rev Cancer 9, 862-873, 2009; Wade et al., Nat Rev Cancer 13, 83-96, 2013). Most recently, SAH peptides have been designed and suggested as the model for superior PPI antagonists because of their larger interaction interfaces, better structural stability, protease resistance, and cell permeability (Verdine et al., Methods Enzymol 503, 3-33, 2012). PPI antagonist effectiveness has conventionally been evaluated using in vitro biochemical and biophysical assays that quantify the ability of the PPI to displace one of the interacting protein fragments. However, such assays do not reveal whether molecules that work effectively in in vitro systems can cross the cell membrane to effect target disruption in a native intracellular environment. While fluorescence-activated cell sorting (FACS) analyses have been used to indicate whether fluorophore-tagged PPI antagonists can enter cells, they do not reveal the subcellular localization (endosome versus cytoplasm) of the antagonists, nor whether they reach their targets at concentrations sufficient to disrupt the PPI to elicit biological effects. Furthermore, assays of biologic activity such as cell death can be misleading, and do not provide direct evidence of the intracellular efficacy of a PPI antagonist. For example, since p53 can be activated by diverse cellular insults and by many different mechanisms, the ability of a putative PPI antagonist to activate p53 target genes or p53-dependent biological processes does not prove that these effects were mediated by disruption of p53-Mdm2 and/or p53-Mdm4 complexes.
Clearly, a critical unmet need is availability of real time assays that directly measure disruption of intracellular protein complexes. Disclosed herein is a plug-and-play platform system to detect PPIs in cells and in lysates to address this need. This system integrates RMCE and BiLC. The p53 tumor suppressor pathway was used as a model system to validate this Recombinase-enhanced BiLC (ReBiL) platform system. The ReBiL system enabled us to rapidly evaluate the efficacy of target disruption and cell permeability of small molecule and peptide-based p53-Mdm2 and p53-Mdm4 PPI antagonists and a novel Mdm2-Mdm4 interaction agonist.
Development of ReBiL System.
Numerous strategies have been devised to study intracellular PPI networks including protein-fragment complementation assays (PCAs) and two hybrid-based approaches. The approach disclosed herein employs split luciferase complementation for at least two unique advantages. First, the absence of background luminescence in mammalian cells affords high signal-to-noise ratios. Second, while split fluorescent proteins associate irreversibly (Magliery et al., J Am Chem Soc 127, 146-157, 2005), split luciferase fragments exhibit little if any interaction by themselves (Luker et al., Proc Natl Acad Sci USA 101, 12288-12293, 2004) and their complementation is readily reversed (Macdonald-Obermann et al., Proc Natl Acad Sci USA 109, 137-142, 2012). These factors make luciferase complementation ideal for analyzing PPI stability and for ascertaining the effectiveness of antagonists.
BiLC relies on the reconstitution of luciferase enzymatic activity from two split luciferase fragments and the interaction of the proteins to which they are genetically fused (Luker et al., Proc Natl Acad Sci USA 101, 12288-12293, 2004) (
This “ReBiL” platform (see Table 2) confers at least two significant experimental and analytical advantages. First, it facilitates structure, function, and interaction analyses because it enables BiLC fusions encoding wild type and mutant proteins to be integrated into, and expressed from, the same chromosomal locus. Second, single copy integration and doxycycline-tunable regulation of transgenes generates rheostatic and uniform expression (Rossi et al., Mol Cell 6, 723-728, 2000) (
Nucleic Acids Res 23 , 3605-3606, 1995).
One 8, e58395, 2013).
Nucleic Acids Res 33, e147, 2005), U2OS,
Evaluation of PPI Antagonists in Living Cells.
Numerous small molecule and peptide-based compounds have been reported to interfere with p53-Mdm2 and p53-Mdm4 interactions in cell free assays and to activate p53 in living cells (see reviews Brown et al., Nat Rev Cancer 9, 862-873, 2009; Wade et al., Nat Rev Cancer 13, 83-96, 2013). However, recent data raise questions about the ability of some SAH peptides to interfere with p53-Mdm2 and p53-Mdm4 interactions in cells (Brown et al., ACS Chem Biol 8, 506-512, 2013). The ReBiL platform was used to evaluate reported small molecule, SAH peptide, and cyclotide based p53-Mdm2 and p53-Mdm4 PPI antagonists.
p53-Mdm2 and p53-Mdm4 ReBiL reporters were generated (
Nutlin-3a disrupts p53-Mdm2 but not p53-Mdm4 interactions (Vassilev et al., Science 303, 844-848, 2004; Patton et al., Cancer Res 66, 3169-3176, 2006; Wade et al., Cell Cycle 7, 1973-1982, 2008). Nutlin-3a reduced the BiLC signal generated from p53-Mdm2 complementation, but had no noticeable effect on the p53-Mdm4 BiLC signal (
Whether small molecules like Nutlin-3a disrupt pre-formed p53-Mdm2 complexes in living cells has remained an open question. The ReBiL system was used to investigate this by first inducing the expression of the p53-Mdm2 pair to generate a functional BiLC complex. Doxycycline was removed to prevent further p53-Mdm2 transcription. Subsequent incubation of the “pre-loaded” cells with Nutlin-3a enabled measurement of the decay of the p53-Mdm2 BiLC complex in living cells. The p53-Mdm2 complexes decayed over time and this was accelerated significantly and dose dependently by Nutlin-3a (
Disruption of p53-Mdm4 complexes has been an important goal for reactivation of the wild type p53 in cancer therapy (Wade et al., Nat Rev Cancer 13, 83-96, 2013). The ReBiL system was used to determine whether a previously reported small molecule p53-Mdm4 antagonist, SJ-172550 (Reed et al., J Biol Chem 285, 10786-10796, 2010) disrupts this interaction in cells. No decrease in p53-Mdm4 luciferase signal was detected (
Analysis of SAH Peptides and the Antagonistic Effects of Serum.
Next, it was determined whether SAH peptide-based antagonists that disrupt both p53-Mdm2 and p53-Mdm4 interactions in vitro including SAHp53-8 (Bernal et al., J Am Chem Soc 129, 2456-2457, 2007; Bernal et al., Cancer Cell 18, 411-422, 2010), sMTide-02 (Brown et al., ACS Chem Biol 8, 506-512, 2013), and ATSP-7041 (Chang et al., Proc Natl Acad Sci USA 110, E3445-54, 2013) do so in cells. Their larger binding surfaces confer far higher binding affinities than Nutlin-3a, exemplified by ATSP-7041 with a Ki=0.9 nM for Mdm2 compared with Ki=52 nM for Nutlin-3a (determined by Chang et al., Proc Natl Acad Sci USA 110, E3445-54, 2013). Surprisingly, despite this much higher binding affinity, SAH peptides are typically used at higher concentrations (20 μM to 100 μM) to elicit cellular activities (Brown et al., ACS Chem Biol 8, 506-512, 2013; Bernal et al., Cancer Cell 18, 411-422, 2010; Chang et al., Proc Natl Acad Sci USA 110, E3445-54, 2013; Gembarska et al., Nat Med 18, 1239-1247, 2012). Indeed, in spite of its 57-fold higher binding affinity, ATSP-7041 (10 μM) reached full p53-Mdm2 inhibition much slower (4 hours) than Nutlin-3a (1 hour, compare
These results indicate that higher binding affinity in vitro does not necessarily correlate with increased intracellular PPI disruption activity, indicating that there might be a barrier to effective entry of the SAH peptides into the cells. The increased activity of ATSP-7041 in 0% serum (Chang et al., Proc Natl Acad Sci USA 110, E3445-54, 2013) (
As ReBiL enables real time analyses of the kinetics of target disruption in cells, it was used to determine when target disruption occurs and then correlate this with other parameters such as cell viability. Wild-type SAH peptides dose dependently reduced viability of the p53-null Saos-2 ReBiL cells (
Development of a Cell-Free BiLC Assay to Facilitate Analyses of PPI Efficacy and Elucidate Serum-Based Inhibitory Mechanisms.
The following two reasonable explanations of serum's ability to both prevent SAH peptide-induced cytotoxicity (
Insight into these possibilities was gained by developing a cell-free BiLC assay. It was reasoned that if SAH peptides efficiently disrupt PPIs in cell lysates, but not in intact cells, then membrane penetration and access to their targets within the cell might be limiting. Expression of the BiLC complexes was induced by doxycycline, and then prepared cell lysates using an optimized buffer (PPI lysis buffer, PLB) (see
(1) CA-630 lysis buffer (CLB): 50 mM Tris-HCl pH8.0, 5 mM EDTA, 150 mM NaCl, 0.5% CA-630, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and Complete Mini Protease Inhibitor Cocktail (Roche).
(2) PPI lysis buffer (PLB): 100 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 150 mM NaCl; 0.1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and Complete Mini Protease Inhibitor Cocktail (Roche).
(3) Promega Glo Lysis Buffer (GLB, Promega Cat # E2661): The optimized lysis buffer for the BiLC lysate assay was identified by measuring the signal-to-noise ratio of a set of validated positive and negative BiLC PPI pairs. They are the interaction between the Mdm4_RING and Mdm2_RING (Tanimura et al., FEBS Lett 447, 5-9, 1999) as the positive signal and any detectable interactions between Mdm4_RING domain and Mdm2_RING_C464A as the negative control or background noise because the cysteine (Cys464) to alanine mutation collapses the RING domain structure and prevents interaction with the Mdm4_RING domain (Kostic et al., J Mol Biol 363, 433-450, 2006).
The U2OS reporter cells encoding nLuc-Mdm4_RING/cLuc-Mdm2_RING were induced by 500 ng/ml doxycycline for 24 hours and lysed with these three lysis buffers respectively. For negative control, the U2OS reporter cell that harbors the nLuc-Mdm4_RING/cLuc-Mdm2_RING_C464A was used. The C464A mutation in Mdm2 abolished its RING domain structure and rendered it unable to interact with Mdm4 RING domain.
The cells were lysed on culture plates and lysates were transferred to microcentrifuge tubes and cleared by centrifugation (16,000 rcf for 3˜5 minutes at 4° C.). The cleared lysates (20 μl) and luciferin reagent (20 μl) (Promega Bright-Glo E2620 or Steady-Glo E2520) were pipetted into each well of a 384-well plate (Corning 3570). The plate was incubated at 26° C. for 15 min and luminescence was measured in a Tecan M200 luminometer with 0.5 second integration time on each well at 26° C.
The results showed that both PLB lysates and GLB lysates generated around 10 times higher luminescent signals than those of CLB lysates (
Consistent with the cell-based BiLC assay, Nutlin-3a efficiently disrupted p53-Mdm2, but not p53-Mdm4, complexes in the lysate BiLC assay (
The use of the ReBiL assay to show that SJ-172550 does not inhibit the p53-Mdm4 PPI provides an exemplary demonstration that the RMCE-BiLC assay is unexpectedly superior to the current fluorescence polarization (FP) based PPI antagonist discovery technology. For example, SJ-172550 was previously identified as an antagonist of p53/Mdm4 interaction using high-throughput FP-based screen (Reed, D. et al. Identification and characterization of the first small molecule inhibitor of MDMX. J Biol Chem 285, 10786-10796, 2010, incorporated by reference herein in its entirety). The prior screen of p53/Mdmx (aka Mdm4) interaction in fluorescence polarization (FP) assay had a low Z-prime score <0.4 in 384-well plate format (see p.10788 of Reed et al. J Biol Chem 285, 10786-10796, 2010). The Z-prime score is commonly used to measure the quality of a high-throughput screen assay, and use of a Z-prime score to quantify the effectiveness of a high throughput screen is known in the art (see, e.g., Zhang et al, “A simple Statistical Parameter for use in evaluation and validation of high throughput screening assays,” J Bimolecular Screening, 4, 67-73, 1999, which is incorporated by reference herein in its entirety). An assay with Z-prime score between 1 and 0.5 is considered excellent for high-throughput screen (Z-prime score cannot exceed 1). Typically, a screening facility will not perform any screening assay unless the Z-prime score is higher than 0.5. In contrast to the FP-based p53/Mdm4 interaction assay, the same p53/Mdm4 interaction in ReBiL format has a Z-prime score of 0.74 in 1536-well plate format. Thus, in a system using 4-fold higher throughput (384 vs. 1536), the ReBiL assay demonstrated a much higher Z-prime score than that of FP-based assay. This contrasts with the typical trend that the higher throughput of an assay usually has a lower Z-primer score (i.e., that it is more noisy).
Using the ReBiL assay, it was demonstrated that SJ-172550 is a poor p53/Mdm4 antagonist using the lysate ReBiL assay (
For the results shown in
The inhibitory effect of serum on SAH peptides could result from direct binding of serum albumin to the peptide (Bird et al., ACS Chem Biol 9, 831-837, 2014). 10% fetal bovine serum (FBS) was added into the lysate BiLC assays to determine directly whether serum reduces the ability of SAH peptides to disrupt p53-Mdm2 and p53-Mdm4 interactions. Serum did not reduce the potency of ATSP-7041, SAHp53-8, or sMTide-02 in cell lysates (compare
The data presented above indicates that serum might compromise the ability of SAH peptides to enter the cell. Additionally, there might be a mechanistic linkage between the reduced cytotoxicity and reduced PPI disruption by SAH peptides in cells exposed to serum containing medium. If SAH peptides compromise membrane integrity, they would be able to gain access to the cytoplasm and their targets; by extension, the serum effect could be explained if it antagonized such membrane effects. This possibility was tested by examining membrane integrity after exposure to SAH peptides in the presence or absence of 10% serum. The lactate dehydrogenase (LDH) leakage assay (Decker et al., J Immunol Methods 115, 61-69, 1988) was used to quantitatively measure release of this stable cytosolic enzyme into culture media. The data show that the SAH peptides ATSP-7041, sMTide-02, and SAHp53-8 all cause LDH leakage in the absence of serum (
Thus, the combination of ReBiL assays in cellular lysates and in live cells provides a simple and efficient strategy to quickly assess the specificity and potency of putative PPI disrupters, and indicates when poor activity may derive from inefficient intracellular target access. If a test agent reduces BiLC signals in the lysate in the presence of serum, but not in the cell, it could mean one of several things. For example, the serum might prevent the entry of the drug, for example, by binding to cellular receptors that bind the drug. In this way, serum protein(s) act as inhibitors of drug uptake, but do not directly bind to and inactivate the drug.
Accordingly, the studies provide an assay in which the combination of cell-based and lysate-format BiLC assays enables the BiLC approach to be used as a very rapid SAR type platform able to detect modifications that do not interfere with the ability of a compound to bind its target but do enable the compound to enter the cell. Thus, this enables the assay to be applied as a cell permeability assay. This concept of performing the BiLC assay in live cells versus in cellular lysate to assess a compound's permeability should be able to be extended to other types of biochemical or biological assays. For example, the stability of a protein usually increases when binding to its ligands (or some compounds). If the ligand (or compound) is not cell permeable, it would not stabilize its intracellular target proteins. Thus, increased target protein stability could be a second indicator that the compound is hitting its target in the cell. This might be valuable if the molecule enters the cell at concentrations high enough to reduce BiLC fractionally, but not at high enough levels to elicit a biological response. Thus, the combination of reduced luciferase and increased target stability would provide an internal validation control to give greater confidence in a positive result for a low efficacy lead molecule and would support optimization efforts.
The human protein “interactome” may involve ˜130,000 to ˜650,000 PPIs. Even if a small fraction of these elicit diseases through aberrant interactions, the availability of rapid PPI antagonist screens will open up vast new opportunities for developing therapeutic agents. Although image-based assays such as the proximity ligation in situ assay (PLISA) and fluorescence based two/three hybrid (F2H and F3H) screens have been used to evaluate PPI antagonists in cells, they do not enable simultaneous evaluation of on-target validation in cell lysates and facile high throughput analysis of target disruption in living cells. As shown here, ReBiL provides an integrated real time in vivo and in vitro method for studying PPIs and their antagonists.
Using the ReBiL strategy, the ability of two small molecules, Nutlin-3a and MI-219 (Ding et al., J Med Chem 49, 3432-3435, 2006), to interfere selectively with p53-Mdm2 interaction was confirmed. However, as MI-219 also exhibited p53-independent activity, it was not characterized further. Surprisingly, other published antagonists (SJ-172550 and RO-5963) showed little if any PPI disruption activity in cells. The results presented herein suggest that the reported p53 activating effects of these compounds may result from induction of other cellular stresses (Beckerman et al., Cold Spring Harb Perspect Biol 2, a000935, 2010), and not from p53-Mdm2 or p53-Mdm4 disruption. Therefore, the results reinforce the caution that there can be a lack of concordance between in vitro competition binding assays and in vivo biological readouts, and they emphasize the critical importance of directly analyzing target disruption in cells for deducing mechanism of action.
The ReBiL approach also led to new insights concerning conflicting results of studies using SAH peptides (Brown et al., ACS Chem Biol 8, 506-512, 2013; Okamoto et al., ACS Chem Biol 8, 297-302, 2013; Okamoto et al., ACS Chem Biol 9, 838-839, 2014). Considerable effort and resources have been expended to develop SAH peptides as PPI antagonists as they present larger interaction interfaces with which to more effectively disrupt PPIs. They have also been reported to confer protease resistance and enhanced cell permeability. Given the difficulty of developing small molecule p53-Mdm4 disrupters, SAH peptides were developed as dual p53-Mdm2 and p53-Mdm4 antagonists since Mdm2 and Mdm4 contain similar N-terminal hydrophobic clefts that interact with p53. Consistent with expectations, SAH peptides that target this region have high binding affinities and an ability to disrupt both p53-Mdm2 and p53-Mdm4 complexes in vitro (
It has been observed that serum decreases the biological activity of SAH peptides (Brown et al., ACS Chem Biol 8, 506-512, 2013; Chang et al., Proc Natl Acad Sci USA 110, E3445-54, 2013; Edwards et al., Chem Biol 20, 888-902, 2013; see also
A non-limiting explanation is that the membrane disruption may commonly result from positively charged cell-penetrating peptides (CPPs) appended to peptides with exposed hydrophobic residues. For example, the stapled PMI-PenArg, a lysine to arginine derivative of the CPP penetratin (Amand et al., Biochem Biophys Res Commun 371, 621-625, 2008), elicited cell death within three hours in cells growing in serum free media. Similarly, the cationic cell penetrating D-peptide DPMI-γ-DR9 rapidly induced p53-independent cytotoxicity (Liu et al., Proc Natl Acad Sci USA 107, 14321-14326, 2010). The positively charged CPPs from N-terminal prion proteins also elicited membrane leakage in a defined large unilamellar phospholipid vesicles (Magzoub et al., Biochem Biophys Acta 1716, 126-136, 2005). It is also worth noting that the unstapled DPMI-γ-DR9, TAT-PMI, and PMI-s-s-TAT induce severe membrane damage and cytotoxicity, indicating that chemical stapling per se is not required for cytotoxicity (Okamoto et al., ACS Chem Biol 9, 838-839, 2014).
The ReBiL strategy creates a facile platform system for PPI analyses. The system was shown to detect interactions between p53, Mdm2, Mdm4, BRCA1-BARD1 (Brzovic et al., Nat Struct Biol 8, 833-837, 2001), and Ube2t-FANCL (Machida et al., Mol Cell 23, 589-596, 2006), among other proteins. Furthermore, the very high signal to noise ratio in the lysate format enabled ReBiL to be used for high throughput drug screens in a 1,536 well format with Z′ values exceeding 0.7. It is expected that ReBiL will have broad applications for problems ranging from identification of noncoding RNAs that facilitate cytosolic PPIs to factors that impact plasma membrane-associated K-RAS dimerization, neither of which are feasible using other strategies such as the two/three-hybrid systems with transcriptional readouts. Together, these attributes should enable ReBiL to broaden understanding of the impact of disease relevant mutations on protein interactions, to elucidate more precisely mechanisms of drug action, to improve efficacy of PPI antagonists, and to advance the understanding of the makeup of the human protein interactome.
Generation Master Cell Line for RMCE Mediated Insertion of BiLC Fusion Partner Cassette.
Human osteosarcoma U2OS (p53 WT) and Saos-2 (p53 null) cells were transfected with linearized pWHE134 carrying genes encoding rtTA2s-M2, TetR(B/E)-KRAB, and G418 resistance (Wong et al., Nucleic Acids Res 33, e147, 2005). Selection for G418 resistance (400˜800 μg/ml) produced several stable clones that were further screened for their ability to exhibit low basal and high doxycycline-induced levels of TRE-luciferase reporter construct. This generated the U2OS 134-8 and Saos-2 134-14 lines. To introduce a single copy of the HyTK cassette genomic integration, the U2OS 134-8 and Saos-2 134-14 cell lines were infected with the Lenti-viral vector HIVL3-TRE-luciferase-CMV-HyTK-2L with a very low MOI (multiplicity of infection). Selection for Hygromycin B resistance (200˜400 μg/ml), and again screening for clones that produced high signal to noise ratios for TRE-luciferase generated the doxycycline inducible RMCE master acceptor cell lines U2OS 134-8 HyTK8 and Saos-2 134-14 HyTK20. Genomic real time qPCR with primers against Hygromycin resistant gene (qPCR-Hygro-1: 5′-GGATTTCGGCTCCAACAATG-3′ (SEQ ID NO: 46) and qPCR-Hygro-2: 5′-TGCTCCATACAAGCCAACCA-3′ (SEQ ID NO: 47)) and endogenous GAPDH (qPCR-hGAPDH-1: 5′-ACTCCCACTACCCCCTTCCA-3′ (SEQ ID NO: 48) and qPCRhGAPDH-2: 5′-GTGAGGGCGCAGTGAGATCT-3′ (SEQ ID NO: 49)) genomic loci exhibited the expected Hygro/GAPDH ratio of ˜0.5 (one copy of the RMCE acceptor/two copies of GAPDH, data not shown), confirming that the HyTK cassette is present at one copy.
Cell Culture.
Normal human fibroblasts (WS1) were cultured in MEM, 15% FBS, 2× non-essential amino acids, 2− Vitamins, 10 μM β-mercaptoethanol and 10 μg/ml Ciprofloxacin. Saos-2 cells (ACTT HTB-85) were cultured in DMEM/F12 (50:50), 10% FBS and 10 μg/ml Ciprofloxacin. U2OS cells (134-8 HyTK8) were maintained in DMEM, 10% FBS, 400 μg/ml G418, 10 μg/ml Ciprofloxacin, and 200 μg/ml Hygromycin B. After targeting, U2OS RMCE derivatives were maintained in DMEM, 10% FBS, 400 μg/ml G418 and 10 μg/ml Ciprofloxacin. The Saos-2 134-14 HyTK20 was maintained in DMEM/F12 (50:50), 10% FBS, 400 μg/ml G418, 10 μg/ml Ciprofloxacin, and 200 μg/ml Hygromycin B. The Saos-2 post-RMCE derivatives were maintained in DMEM/F12 (50:50), 10% FBS, 400 μg/ml G418 and 10 μg/ml Ciprofloxacin. WS 1, U2OS and its RMCE derivatives were grown at 37° C. with 7% CO2. It was empirically discovered that Saos-2 and its RMCE derivatives grew better in a 37° C. low-oxygen incubator with 3%˜5% 02 and 7%˜5% CO2. If the introduced transgene does not impede cell growth, 5 ng/ml Doxycycline and 2˜3 μg/ml Blasticidin were added in media to prevent RMCE cassette loss due to genomic instability or epigenetic silencing in U2OS and Saos-2 post-RMCE cell lines.
Construction of RMCE Targeting Plasmids.
Standard molecular biology methods including PCR, restriction enzymes and T4 DNA ligase as well as the Gibson Assembly strategy (Gibson et al., Nat Methods 6, 343-345, 2009) (NEB E2611S) were used to construct all ReBiL targeting plasmids, detailed features of which are described in Table 3.
RMCE.
The U2OS 134-8 HyTK8 or Saos-2 134-14 HyTK20 (˜70% confluent) in a E-well or 12-well plate were transfected with FuGENE HD or X-tremeGENE 9 per manufacture's protocols with RMCE targeting plasmid and pOG231 (Crerecombinase, available through Addgene) at a 2:1 ratio. The transfected cells were trypsinized and seeded at clonogenic density; for example, transfected cells from one 6-well plate were usually seeded into 3˜4 15-cm plates. The Ganciclovir selection procedure for RMCE clones has been described in detail previously (Wong et al., Nucleic Acids Res 33, e147, 2005; Green et al., PLoS One 8, e58395, 2013). RMCE colonies from a single targeting vector can be individually picked or pooled together as all should be identical.
Real-Time BiLC Assay in Living Cells.
Phenol-red free DMEM/F12 (Life Technology No. 11039-021 or Sigma D2906-1L) containing 2× concentrated reagents including doxycycline and D-luciferin (potassium salt; Biosynth L-8220) were pipetted into 384-well plate (Corning 3570); 20 μl per well. The Saos-2 ReBiL reporter cells were washed, trypsinized and cell numbers were determined. The numbers of required cells were collected into 1.5 ml Protein LoBind tubes (Eppendorf No. 022431081) and spun at 200 rcf for 5 minutes at room temperature. Supernatants were discarded, ReBiL reporter cells were resuspended with DMEM/F12 (phenol-red free) and 20 μl cells were pipetted into each well. The final concentration of each component is as follows: FBS 10%, Ciprofloxacin 10 μg/ml, Doxycycline 0˜500 ng/ml, D-luciferin 100 μM, ReBiL reporter cells 5,000˜20,000 cells per well. The plate was sealed with a MicroAmp Optical Adhesive Film (Life Technology No. 4311971), and luminescence was measured in a Tecan M200: integration time 2 seconds, 15˜30 minutes per cycle for a total of 24˜48 hours at 37° C.
Doxycycline Withdrawal Strategy to Enable Real-Time BiLC Analysis Protein Complex Dissociation
(a) The following protocol was used to evaluate small molecule PPI antagonists. Saos-2 ReBiL reporter cells were cultured in 10-cm (or 15-cm) dishes with regular media containing doxycycline (500 ng/ml) and d-luciferin (100 μM) for 24 hours. The next day, the cells were washed, trypsinized, and cell numbers were determined. Cells were handled as described above except there doxycycline was eliminated from the BiLC assay media. (b) To evaluate SAH peptides, the cells were seeded into 96-well plate (Corning 3917) with 20,000 cells per well, and incubated in the presence of doxycycline (500 ng/ml) and D-luciferin (100 μM) at 37° C. CO2 incubator for 24 hours. The next day, the media were aspirated; cells were washed once with DMEM, and 50 μl of DMEM/F12 media containing D-luciferin (100 μM) and the indicated SAH peptides at concentrations chosen based on prior reports describing the use of each peptide. The plate was sealed with a MicroAmp Optical Adhesive Film, and luminescence was measured in a Tecan M200: integration time 2 seconds, 5˜10 minutes per cycle for total 6 hours at 37° C.
Cell Viability Assay.
Luminescence-based end-point cell viability assay was performed using CellTiter Glo (measures the amount of ATP produced by viable cells, Promega G7572) according to the manufacturer's protocol. Luminescence was detected in a Tecan M200 with integration time 0.5 second.
BiLC Assay Using Cell Lysates.
The Saos-2 or U2OS ReBiL reporter cells were cultured in regular media with doxycycline (500 ng/ml, 48˜72 hours). The 4× concentrated drugs diluted in DMEM/F12 media were pipetted into 384 well plates, 10 μl per well. Cells were washed with PBS-twice and lysed with PLB buffer (100 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 150 mM NaCl; 0.1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and Roche Complete Mini Protease Inhibitor Cocktail). The cell lysates were transferred into 1.5 ml microcentrifuge tubes and cleared by centrifugation (13,000 rcf for 5 minutes at 4° C.). The clear lysates were collected, diluted with DMEM (˜300 μl DMEM added into 100 μl lysates); 10 μl of diluted lysates were pipetted into each of the 384-wells, and the plates were incubated at room temperature for 10 minutes. 20 μl of luciferin reagents were added (Promega Bright-Glo E2620 or Steady-Glo E2520) into each well and luminescence was measured in a Tecan M200: integration time 0.5 seconds, 3˜5 minutes per cycle for total 30 minutes at 26° C.
Lactate Dehydrogenase (LDH) Leakage Assay.
Saos-2 and WS 1 cells were seeded into 96-well plates (20,000 cells per well) and incubated at 37° C. CO2 incubator overnight. The next day, growth media were aspirated and cells were washed with DMEM. The different PPI antagonists (25 μM and 10 μM) with or without 10% FBS in DMEM/F12 (50:50) phenol-red free media were added to each well and plates were incubated at 37° C. in a CO2 incubator for 6 hours. LDH leakage into media was detected by the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega G1780) per manufacture's protocol. Assay background was evaluated by using the LDH reading from wells containing only media and PPI antagonists with or without 10% FBS and was subtracted from the values obtained in the presence of the PPI antagonists. Cells lysed by 0.8% Triton X-100 (provided by the Kit), a concentration sufficient to ensure lyse all cells but low enough not to interfere with the LDH assay, represents the maximum LDH leakage in this experiment and its reading is set to 100%. DMSO treatment served as the vehicle control. It also measures some trace amount of LDH in the serum and some low amount of spontaneous LDH leakage from cells and its reading is set to 0%. The % LDH Leakage=100×[(PPI−PPIBackground)−(DMSO−DMSOBackground)]/[(Lysed−LysedBackground)−(DMSO−DMSOBackground)]%.
Western Blotting and Antibodies.
The total protein from cell lysates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore No. IPFL00010) using methods described previously (Wade et al., J Biol Chem 281, 33036-33044, 2006). Primary antibodies were mouse anti-FLAG M2 (SIGMA F3165), mouse anti-FLAG FG4R (LifeTein LT0420), mouse anti-HA (Covance HA.11) and rabbit monoclonal anti-HA (Cell Signaling C29F4). Secondary antibodies were conjugated to Alexa Flour 680 (Life Technology) or IRDye800 (LiCOR) for scanning in the LiCOR Odyssey system.
Synthesis of SAH Peptides ATSP 7041 and ATSP 7342.
Fmoc amino acids were obtained from Chemimpex and Novabiochem. Olefin building blocks were from AAPPTEC. Coupling reagents were from Novabiochem (HBTU). All other chemicals were purchased from Sigma (Fluka) and were used without further purification. LC-MS analysis was performed on an AGILENT 1100 system equipped with a C18 column (Phenomenex, Gemini) in combination with an electrospray mass spectrometer from Agilent (LC/MSD trap XCT). LC conditions: flow 0.5 mL/min, r.t., eluent systems: eluent A=water (0.1% FA), eluent B=acetonitrile (0.1% FA), linear gradient of 5 to 95% B in 25 min. UV detection was performed at 220 and 288 nm. Crude peptides were purified by preparative RP-HPLC on the AGILENT 1100 HPLC system operated at 4 mL/min using a C18 preparative column from Waters (XBridge Prep) with a linear gradient of 10 to 60% B in 45 min at room temperature. UV detection was performed at 220 and 288 nm.
The SAH peptide ATSP 7041 and the corresponding F19A mutant ATSP 7342 were synthesized on a 0.3 mmol scale by standard protocols using Fmoc chemistry. As solid support a Rink amide resin was used (Novabiochem Rink Amide MBHA resin, 100-200 mesh, loading=0.6 mmole/g). Fmoc amino acids were coupled in five-fold excess with HBTU/DIEA (1:2) in DMF for 30 min. The Fmoc-protected olefin building blocks were coupled in 2.5-fold excess using HBTU/DIEA (1:1) in DMF for 45 min. Fmoc deprotection was realized by treating the peptide-bound resin with 20% (v/v) piperidine/DMF for 15 min. After assembly of the linear peptide chain the N-terminus was acetylated using a solution of acetic anhydride and DIEA in DMF. Ring-closing metathesis (RCM) was performed as described previously (Kim et al., Nat Protoc 6, 761-771, 2011). In brief, peptide-loaded resin was treated with Grubbs first-generation catalyst bis(tricyclohexylphosphine)benzylidine ruthenium(IV) dichloride in dry 1,2-dichloroethane while gently bubbling N2 through the solution at room temperature until complete consumption of the starting material was detected by liquid chromatography-mass spectrometry (LC-MS). Final deprotection and cleavage of the peptides from the resin was realized using TFA/H20/TIS (85/5/5, v/v) for 2 h at room temperature. The resin was removed and the crude peptides were precipitated by the addition of cold diethyl ether to yield the desired peptides. Crude peptides were purified on a reversed-phase C18 column (Waters; XBridge Prep C18) to yield the pure peptides. Composition and purity of the SAH peptides was confirmed by LC-MS mass spectrometry using a C18 column (Phenomenex, Gemini).
ESI-MS (ATSP 7041)=(ES+) [M+2H]2+=calcd: 873.3 (monoisotopic); obsd: 873.0
ESI-MS (ATSP 7342)=(ES+) [M+2H]2+=calcd: 835.2 (monoisotopic); obsd: 835.0
Synthesis of TAT-PMI (TUC.HNP6.186.1) and TAT-PMI_F3A (TUC.HNP6.186.2) Peptides
[TUC.HNP6.186.1: H-RKKRRQRRR-Ahx-TSFAEYWNLLSP-NH2] and F3A mutant [TUC.HNP6.186.2: H-RKKRRQRRR-Ahx-TSAAEYWNLLSP-NH2] were synthesized on Rink amide resin (0.68 mmol/g) using standard Fmoc (9-fluorenylmethoxycarbonyl) chemistry. Cleavage was achieved by treatment with TFA/TIS/H20 (95:2.5:2.5) for 40 min, and precipitation with Et20. The peptides were purified on a Beckman HPLC using a C18 semi-preparative reverse phase column (YMC) with a gradient from 5% to 60% acetonitrile in water (containing 0.1% (v/v) trifluoroacetid acid).
Synthesis of MCoTi-2-PMI-TAT Graft (TUC.PS10.084) (Compound 5): (See
Peptide sequence Boc-Cys(Trt)(43)-Ile-Cys(Trt)-Arg(Pbf)-Gly-Asn(Trt)-Gly-Tyr(tBu)-Cys(Trt)-Gly-Ser(tBu)-Ahx-Thr(tBu)-Ser(tBu)-Phe-Ala-Glu(OtBu)-Tyr(tBu)-Trp(Boc)-Asn(Trt)-Leu-Leu-Ser(tBu)-Pro-Gly-Val-Cys(Trt)-Pro-Lys(Boc)-Ile-Leu-Lys(Alloc)(12)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Cys(Trt)-Pro-Gly-Gly(l)-O-2-Chlorotrityl (Compound 1) was synthesized using Tribute peptide synthesizer (Protein Technologies, Tucson) on 2-Chlorotrityl chloride resin (S=0.5 mmol/g) by standard Fmoc-AminoAcid(Boc/tBu/OtBu/Trt/Pbf) protocol with following exception: Fmoc-Lys(Alloc) for position (12) and Boc-Cys(Trt)-for position (43) were used. Amino acids were coupled by HCTU/DIEA activation in DMF with recoupling. For Fmoc(Boc)-Cys(Trt)-OH coupling, 2,4,6-collidine was used as base instead of DIEA.
Subsequently, the Alloc-protecting group of Lys(12) was deprotected by solution of DMBA (7 eq) and Pd(0)(PPh3)4 (0.2 eq) in DCM. The protected TAT sequence Boc-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Gly-Ahx-bAla- was synthesized on the side chain of Lys(12) according to standard protocol using Tribute synthesizer, with N-terminal Boc-Gly used to terminate the sequence.
The fully protected peptide acid Boc-Cys(Trt)(43)-Ile-Cys(Trt)-Arg(Pbf)-Gly-Asn(Trt)-Gly-Tyr(tBu)-Cys(Trt)-Gly-Ser(tBu)-Ahx-Thr(tBu)-Ser(tBu)-Phe-Ala-Glu(OtBu)-Tyr(tBu)-Trp(Boc)-Asn(Trt)-Leu-Leu-Ser(tBu)-Pro-Gly-Val-Cys(Trt)-Pro-Lys(Boc)-Ile-Leu-Lys[Boc-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Gly-Ahx-bAla-]-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Cys(Trt)-Pro-Gly-Gly(l)-OH (Compound 2) was cleaved by TFE/HOAc/DCM(20/20/60, 1.5 hour, precipitated by diethyl ether and dried.
To a solution of 1.2 g (˜0.15 mmol) of (Compound 2), 10 eq of EDCI.HCl, 15 eq of HOBt in 10 ml DMF and 30 eq of 3-mercapto ethyl propionate were added and reaction stirred overnight. The fully protected peptide thioester Boc-Cys(Trt)(43)-Ile-Cys(Trt)-Arg(Pbf)-Gly-Asn(Trt)-Gly-Tyr(tBu)-Cys(Trt)-Gly-Ser(tBu)-Ahx-Thr(tBu)-Ser(tBu)-Phe-Ala-Glu(OtBu)-Tyr(tBu)-Trp(Boc)-Asn(Trt)-Leu-Leu-Ser(tBu)-Pro-Gly-Val-Cys(Trt)-Pro-Lys(Boc)-Ile-Leu-Lys[Boc-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Gly-Ahx-bAla-]-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Cys(Trt)-Pro-Gly-Gly(l)-S-CH2-CH2-COOEt (Compound 3) was precipitated with water, washed with water, 1% NaHCO3, water and 2% aq HOAc, and dried. The protected peptide (Compound 3) was deprotected by a mixture TFA/thioanisole/EDT/TIPS (85:5:5:5) for 2.5 hours, precipitated, washed with diethyl ether, dissolved in acetonitrile/water, and lyophilized. The deprotected thioester H-Cys(43)-Ile-Cys-Arg-Gly-Asn-Gly-Tyr-Cys-Gly-Ser-Ahx-Thr-Ser-Phe-Ala-Glu-Tyr-Trp-Asn-Leu-Leu-Ser-Pro-Gly-Val-Cys-Pro-Lys-Ile-Leu-Lys[H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Ahx-bAla-]-Lys-Cys-Arg-Arg-Asp-Ser-Asp-Cys-Pro-Gly-Gly(1)-S-CH2-CH2-COOEt (Compound 4) was purified by RP-HPLC.
Cyclization/oxidation/folding of (compound 4): 120 mg of thioester (compound 4) was dissolved in 120 ml of solution 0.1M NH4HCO3/acetonitrile(3:7) containing glutathione (reduced, 2 mM, oxidized 0.4 mM). The solution was stirred for 24 hour, treated slowly with 0.1M solution of K3Fe(CN)6 until yellow color persist and lyophilized. The final miniprotein (Compound 5) was purified by RP-HPLC chromatography, yielding 60.1 mg of final product.
C266H434N92072S6, MW=6265.27 (average) LC/MS analysis: (M+4H)4+=1567.56 (calc. 1567.32, average), (M+5H)5+=1245.14 (calc. 1254.06, average).
Synthesis of MCoTi-1-PMI Graft TUC.PS9.004.F7 (Compound 10), (See
Peptide sequence Boc-Cys(Trt)(41)-Ile-Cys(Trt)-Arg(Pbf)-Gly-Asn(Trt)-Gly-Tyr(tBu)-Cys(Trt)-Gly-Ahx-Thr(tBu)-Ser(tBu)-Phe-Ala-Glu(OtBu)-Tyr(tBu)-Trp(Boc)-Asn(Trt)-Leu-Leu-Ser(tBu)-Gly-Val-Cys(Trt)-Pro-Lys(Boc)-Ile-Leu-Gln(Trt)-Arg(Pbf)-Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Cys(Trt)-Pro-Gly-Ala(1)-O-2-Chlorotrityl (Compound 6) was assembled according to general procedure outlined for MCoTi-2-PMI-TAT (Compound 1).
Cleavage from the resin afforded the fully protected peptide acid Boc-Cys(Trt)(41)-Ile-Cys(Trt)-Arg(Pbf)-Gly-Asn(Trt)-Gly-Tyr(tBu)-Cys(Trt)-Gly-Ahx-Thr(tBu)-Ser(tBu)-Phe-Ala-Glu(OtBu)-Tyr(tBu)-Trp(Boc)-Asn(Trt)-Leu-Leu-Ser(tBu)-Gly-Val-Cys(Trt)-Pro-Lys(Boc)-Ile-Leu-Gln(Trt)-Arg(Pbf)-Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Cys(Trt)-Pro-Gly-Ala(1)-OH (7), which was converted to thioester Boc-Cys(Trt)(41)-Ile-Cys(Trt)-Arg(Pbf)-Gly-Asn(Trt)-Gly-Tyr(tBu)-Cys(Trt)-Gly-Ahx-Thr(tBu)-Ser(tBu)-Phe-Ala-Glu(OtBu)-Tyr(tBu)-Trp(Boc)-Asn(Trt)-Leu-Leu-Ser(tBu)-Gly-Val-Cys(Trt)-Pro-Lys(Boc)-Ile-Leu-Gln(Trt)-Arg(Pbf)-Cys(Trt)-Arg(Pbf)-Arg(Pbf)-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Cys(Trt)-Pro-Gly-Ala(1)-S-CH2-CH2-COOEt (Compound 8) according to procedure outlined for (Compound 3).
Deprotection of (8) and subsequent purification according to procedure outlined for peptide (4) gave thioester H-Cys(41)-Ile-Cys-Arg-Gly-Asn-Gly-Tyr-Cys-Gly-Ahx-Thr-Ser-Phe-Ala-Glu-Tyr-Trp-Asn-Leu-Leu-Ser-Gly-Val-Cys-Pro-Lys-Ile-Leu-Gln-Arg-Cys-Arg-Arg-Asp-Ser-Asp-Cys-Pro-Gly-Ala(1)-S-CH2-CH2-COOEt (Compound 9).
Cyclization/oxidation/folding and purification of (Compound 9) according to procedure outlined for (Compound 4) yielded PMI-grafted cyclotide (Compound 10).
C192H294N58O56S6. MW=4503.14 (average), LC/MS analysis: (M+3H)3+=1502.05 (calc. 1502.05, average), (M+4H)4+=1126.57 (calc. 1126.79, average).
Synthesis of Pyrrolopyrimidine 3b.
Pyrrolopyrimidine analog TUC.HNP6.185.2 was prepared via solid-phase synthesis starting with Rink amide PS resin (0.68 mmol/g) using reported procedure (Lee et al., J Am Chem Soc 133, 676-679, 2011).
GS
SG
A
SG (SEQ ID NO: 53), where the bold,
SG (SEQ ID NO: 54), where the bold,
SG
S
Abbreviations: D.R., Drug Resistance; nLuc, N-terminal split luciferase fragment (amino acid 1-416, the DNA sequence was based on luc2); cLuc, C-terminal split luciferase fragment (amino acid 398-550, the DNA sequence was based on luc2); Amp; Ampicillin resistant gene; BSD, Blasticidin resistance gene from Aspergillus terreus; Kan/Neo, Kanamycin and Neomycin resistant gene; MCS, multiple cloning site. All BiLC fusion constructs listed here were verified by DNA sequencing.
The TAT-PMI and TAT-PMI_F3A peptides are linear conjugates of TAT7 and PMI. Cyclotides are protein scaffolds comprising intrinsic secondary structures that stabilize bioactive peptide domains (Craik et al., Curr Opin Drug Discov Devel 9, 251-260, 2006). They are thermally, chemically, and proteolytically stable. Cyclotides are synthetically accessible allowing insertion/grafting of functional peptide sequences derived from parent proteins (Thongyoo et al., J Med Chem 52, 6197-6200, 2009). Cyclotide can be further modified to modulate physico-chemical, PK, and PD properties. Cyclotide McoTi-1-PMI and McoTi-2-PMI-TAT can be referred to as proteomimetic scaffolds with PMI bioactive loop. The PMI peptide has been previously described (see Pazgier et al., Proc Natl Acad Sci USA 106, 4665-4670, 2009).
In several embodiments, the intracellular reconstitution of BiLC fusion proteins is temperature sensitive. For example, the nLuc-Mdm4_RING domain and cLuc-Mdm2_RING domain BiLC pair showed decreasing luminescence when the temperature was increased (
As described above, the ReBiL assay has exquisite specificity in both the lysate and in-cell formats. The specificity of this assay was additionally assessed as follows:
The ubiquitin E3 ligase FANCL and E2 conjugating enzyme Ube2t from Fanconi anemia complex have been shown to form a PPI, whereas the FANCL_C307A RING domain mutant does not interact with Ube2t measured by biochemical assays (Machida et al., Mol Cell 23, 589-596, 2006; Alpi et al., Mol Cell 32, 767-777, 2008. The FANCL-C307A is a RING domain mutation that prevents interaction with Ube2t and served as a negative control.
BiLC signals generated by randomly integrated inducible reporters encoding either nLuc-Ube2t and cLuc-FANCL or nLuc-Ube2t and the cLuc-FANCL_C307A mutation were compared (see
Since the expression levels of Ube2t and FANCL_C307A were somewhat higher (
Additionally, Mdm2 RING domain interacts specifically with Mdm4 RING domain and Mdm2 and Mdm4 RING domain BiLC pair gives robust signals (
The tumor suppressor p53 plays a critical role in preventing cells exposed to various stresses from proliferating. Under normal physiological conditions, p53 is very unstable. Genetic and biochemical studies provide convincing evidence that Mdm2, a RING domain E3 ubiquitin ligase, is the predominant E3 controlling p53 proteasomal degradation. However, the mechanism by which Mdm2 recruits an E2 ubiquitin conjugating enzyme, is regulated inside cells, and which E2(s) are recruited remain important unsolved questions.
Here, the p53 mimetic compound Nutlin-3a, which structurally resembles the p53 Phe19, Trp23 and Leu26 side chains that bind deeply into the Mdm2 N-terminal hydrophobic p53-binding pocket, was used with the bi-molecular split luciferase complementation (BiLC) assay to reveal conditions that regulate intracellular interactions between the Mdm2 E3 ligase and E2 Ube2d3. The results show that at least three conditions must be met to detect Mdm2-E2 interactions. First, the substrate (p53) or a substrate mimetic (Nutlin-3a, or SAH peptides derived from the Mdm2 binding residues in p53) must be present. Second, Mdm2 phosphorylation at ATM kinase sites reduces or eliminates E2 association. Finally, while it is not possible to measure E2 association using catalytically active E2, using the catalytically inactive mutant Ube2d3-C85A significantly increases detection of E2-Mdm2 interactions. These results indicate that substrate binding facilitates Mdm2 E3 ligase recruitment of the E2. Without wishing to be bound to a particular theory, it is proposed that in this system, E2 recruitment and consequent control of p53 stability and abundance are influenced by environmental cues that affect DNA damage-induced phosphorylation and dephosphorylation signaling pathways. The data are consistent with recent publications suggesting that phosphorylation at specific DNA damage kinase sites regulates RING-RING association, and it is proposed that this impacts the ability of Mdm2 homodimers to recruit biologically relevant E2's. This system has the potential to interrogate which E2s interact with Mdm2 under different biological conditions, and to identify the signaling pathways involved.
Bimolecular-luciferase split complementation (BiLC) assay relies on the reconstitution of firefly luciferase enzymatic activity from two split luciferase fragments. As the split luciferase fragments do not interact by themselves, luciferase enzymatic reconstitution requires interaction of the proteins to which they are fused.
A pair of split luciferase-fusions controlled by the doxycycline-inducible bi-directional promoter (TREbi) was delivered into a pre-selected chromosomal locus in the human osteosarcoma cell line U2OS through RMCE. As shown in
Two documented E3/E2 pairs (BRCA1-BARD1 RING domain fusion (BDfBC)/Ube2d3 and FANCL/Ube2t) were used to validate the BiLC system. As shown in
Which E2(s) physiologically interact with Mdm2 E3 ligase is unclear. Ube2d3 (UbcH5c) was used as a model of E2 to study its interaction with Mdm2 since the Ube2d family members have been widely used in in vitro Mdm2/p53 ubiquitination assays. Unexpectedly, no interaction between Mdm2 and Ube2d3 was detected (
Structural studies of Mdm2 N-terminus have revealed that Mdm2 can undergo conformational changes when binding to p53 peptides (Uhrinova et al., J Mol Biol 350, 587-598 (2005)). The small molecule Mdm2 inhibitor Nutlin-3a structurally resembles p53 Phe19, Trp23 and Leu26 side chains that bind deeply into Mdm2 N-terminal p53-binding pocket. Thus, the binding of Nutlin-3a to Mdm2 mimics p53/Mdm2 N-termini interaction. As shown in
The intracellular interaction of E2 Ube2d3 and Mdm2 E3 in the presence of p53 mimetic compounds was shown. In addition, the results indicated that ubiquitin-charged E2 is not necessary for its interactions with E3 ligase.
This example illustrates a quantitative ReBiL assay for detecting KRAS dimerization.
To illustrate the quantitative ReBiL assay, KRAS proteins were expressed as nLuc and cLuc fusions with an HA tag (
The results show that the ReBiL assay can be used to quantitatively interrogate PPIs.
It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments. We claim all such modifications and variations that fall within the scope and spirit of the claims below.
This application claims priority to U.S. Provisional Application No. 61/842,079, filed Jul. 2, 2013, which is incorporated by reference in its entirety.
This invention was made with government support under Grant Nos. R01-CA61449 and R03-MH089489-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61842079 | Jul 2013 | US |
