Patent Application
20070015768
Potential drug target validation involves determining whether a DNA, RNA or protein molecule is implicated in a disease process and is therefore a suitable target for development of new therapeutic drugs. Drug discovery, the process by which bioactive compounds are identified and characterized, is a critical step in the development of new treatments for human diseases. The landscape of drug discovery has changed dramatically due to the genomics revolution. DNA and protein sequences are yielding a host of new drug targets and an enormous amount of associated information.
The identification of genes and proteins involved in various disease states or key biological processes, such as inflammation and immune response, is a vital part of the drug design process. Many diseases and disorders could be treated or prevented by decreasing the expression of one or more genes involved in the molecular etiology of the condition if the appropriate molecular target could be identified and appropriate antagonists developed. For example, cancer, in which one or more cellular oncogenes become activated and result in the unchecked progression of cell cycle processes, could be treated by antagonizing appropriate cell cycle control genes. Furthermore many human genetic diseases, such as Huntington's disease, and certain prion conditions, which are influenced by both genetic and epigenetic factors, result from the inappropriate activity of a polypeptide as opposed to the complete loss of its function. Accordingly, antagonizing the aberrant function of such mutant genes would provide a means of treatment. Additionally, infectious diseases such as HIV have been successfully treated with molecular antagonists targeted to specific essential retroviral proteins such as HIV protease or reverse transcriptase. Drug therapy strategies for treating such diseases and disorders have frequently employed molecular antagonists which target the polypeptide product of the disease gene(s). However the discovery of relevant gene or protein targets is often difficult and time consuming.
One area of particular interest is the identification of host genes and proteins that are co-opted by viruses during the viral life cycle. The serious and incurable nature of many viral diseases, coupled with the high rate of mutations found in many viruses, makes the identification of antiviral agents a high priority for the improvement of world health. Genes and proteins involved in a viral life cycle are also appealing as a subject for investigation because such genes and proteins will typically have additional activities in the host cell and may play a role in other non-viral disease states.
Viral maturation involves the proteolytic processing of the Gag proteins and the activity of various host proteins. It is believed that cellular machineries for exo/endocytosis and for ubiquitin conjugation may be involved in the maturation. In particular, the assembly, budding and subsequent release of retroid viruses, RNA viruses and envelope viruses, such as various retroviruses, rhabdoviruses, lentiviruses, and filoviruses may involve the Gag polyprotein. After its synthesis, Gag is targeted to the plasma membrane where it induces budding of nascent virus particles.
The role of ubiquitin in virus assembly was suggested by Dunigan et al. (1988, Virology 165, 310, Meyers et al. 1991, Virology 180, 602), who observed that mature virus particles were enriched in unconjugated ubiquitin. More recently, it was shown that proteasome inhibitors suppress the release of HIV-1, HIV-2 and virus-like particles derived from SIV and RSV Gag. Also, inhibitors affect Gag processing and maturation into infectious particles (Schubert et al 2000, PNAS 97, 13057, Harty et al. 2000, PNAS 97, 13871, Strack et al. 2000, PNAS 97, 13063, Patnaik et al. 2000, PNAS 97, 13069).
It is well known in the art that ubiquitin-mediated proteolysis is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days, and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle.
Targeted proteins undergoing selective degradation, presumably through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and generally also requires auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains.
The conjugation of ubiquitin to protein substrates is a multi-step process. In an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of an E1 enzyme. Activated ubiquitin may then be transferred to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein substrates, typically with the assistance of an E3 protein, also known as a ubiquitin enzyme. In certain instances, substrates are recognized directly by the ubiquitin-conjugated E2 enzyme.
It is also known that the ubiquitin system plays a role in a wide range of cellular processes including cell cycle progression, apoptosis, and turnover of many membrane receptors. In viral infections, the ubiquitin system is involved not only with assembly, budding and release, but also with repression of host proteins such as p53, which may lead to a viral-induced neoplasm. The HIV Vpu protein interacts with an E3 protein that regulates IκB degradation, and is thought to promote apoptosis of infected cells by indirectly inhibiting NF-κB activity (Bour et al. (2001) J Exp Med 194:1299-311; U.S. Pat. No. 5,932,425). The ubiquitin system regulates protein function by both mono-ubiquitination and poly-ubiquitination, and poly-ubiquitination is primarily associated with protein degradation.
It would be beneficial to identify compounds that target one or more of the proteins involved in these processes for use in, among other things, anti-viral therapy.
In certain aspects, the application provides compounds that inhibit the ubiquitin ligase activity of one or more proteins, particularly proteins having a RING finger domain (“RING domain”). Compounds disclosed herein may be used, for example, to inhibit the ubiquitin ligase activity of a suitable target protein in vitro or in an ex vivo cell. Compounds disclosed herein may also be used, for example, to inhibit the ubiquitin ligase activity of a suitable target protein in vivo, particularly in a patient suffering from a disease involving an activity of the target protein.
In certain embodiments, compounds disclosed herein may be administered to a subject for the purpose of treating a viral infection, particularly an infection by an envelope virus. Compounds disclosed herein may also be administered to a subject at risk of contracting a viral infection, particularly an infection of an envelope virus, so as to diminish the likelihood that the subject will contract an infection. In a preferred embodiment, compounds disclosed herein are administered to a subject having, or at risk for contracting, a human immunodeficiency virus (HIV) infection.
In certain embodiments, compounds disclosed herein may be formulated as a pharmaceutical composition for administration to a human or animal subject. Optionally, such formulation may include additional active ingredients, such as additional antiviral agents. Additionally, compounds disclosed herein may be administered as part of a combined antiviral therapeutic regimen involving administration of multiple compounds (at different times and doses, as needed).
In certain embodiments, compounds disclosed herein may be used in the formulation of a medicament. The medicament may be formulated for administration to a subject having or at risk for a disease in which a ubiquitin ligase contributes to the causation or pathology. The medicament may be formulated for administration to a subject having or at risk for a viral infection, such as an infection with an envelope virus and preferably a retrovirus. In a preferred embodiment, compounds disclosed herein may be used in the formulation of a medicament for administration to a subject having or at risk for an HIV infection.
In certain embodiments, the application provides agents and methods for inhibiting the ubiquitin ligase activity of a POSH polypeptide in vivo, in an ex vivo cell or in vitro. Agents that inhibit the ubiquitin ligase activity of a POSH polypeptide may be used to treat a disease in which the ubiquitin ligase activity of POSH contributes to the causation and/or pathology. In further embodiments, the application provides numerous methods by which additional agents that inhibit the ubiquitin ligase activity of POSH may be identified.
In certain embodiments, the application relates to a small molecule inhibitor of a POSH polypeptide. In certain embodiments, the POSH polypeptide small molecule inhibitor modulates the activity of a POSH polypeptide. In certain further embodiemtns, the activity of the POSH polypeptide is ubiquitin ligase activity. In other embodiments, the activity of the POSH polypeptide is binding of the POSH polypeptide to an E2. In certain embodiments, the POSH polypeptide small molecule inhibitor is a compound selected from among the compounds presented in Table 1. In other embodiments, the POSH polypeptide small molecule inhibitor is a compound selected from among the compounds presented in Table 7.
In certain embodiments, the application provides a method of inhibiting the ubiquitin ligase activity of a polypeptide, comprising contacting a cell with a small molecule. In preferred embodiments, the small molecule is a compound selected from among the compounds presented in Table 1. In other embodiments, the small molecule is a compound selected from among the compounds presented in Table 7. In certain further embodiments, the small molecule inhibits the ubiquitin ligase activity of a polypeptide comprising a RING domain. In certain embodiments, the polypeptide is a POSH polypeptide. In further embodiments of the application, a POSH polypeptide is selected from the group consisting of a polypeptide represented by SEQ ID NOs: 2, 5, 7, 9, and 11. In yet further embodiments, the polypeptide is a human POSH polypeptide. In certain embodiments, the polypeptide is a Cbl-b polypeptide. In further embodiments, the polypeptide is a human Cbl-b polypeptide. In yet further embodiments, the polypeptide is selected from the group consisting of a polypeptide represented by SEQ ID NOs: 4346, 48, 50, 52, 54, 56, and 58. In certain embodiments, the polypeptide is a PEM-3-like polypeptide. In further embodiments, the PEM-3-like polypeptide is a human PEM-3-like polypeptide.
In additional embodiments, the application relates to a method of inhibiting the activity of a POSH polypeptide, comprising contacting a cell with a small molecule. In certain embodiments, the activity of the POSH polypeptide is ubiquitin ligase activity. In other embodiments, the activity of the POSH polypeptide is binding of the POSH polypeptide to a POSH-AP. In certain embodiments, the POSH-AP is an E2. In other embodiments, the POSH-AP is selected from the group consisting of: ARIH2, ASF1, ATP5A1, BANF1, BAT3, BCAR1, BCL9, BIA2, BRD4, C11orf17, C6orf11, C6orf60, CBX4, CDT1, CGI-27, CIC, CL25084, CLK2, COLIA1, DAP, DDX31, DKFZp434B1231, DKFZp761A052, DLG5, DNM2, DRP2, EEF1A1, EGLN2, EIF4EBP1, EVPL, EWSR1, FAT, FL53657, FLJ10120, FLJ13231, FLJ13479, FLJ37147, FSTL1, GC20, GLUL, HEBP2, Hs.31535, Hs.380933, HSPA1B, HSPC016, HSPC142, ITGB, J03930, KHDRBS1, KIAA0191, KLAA1111, KIAA1598, LAMA5, LOC118987, LOC90987, MADH6, MAP1A, MBD2, MRPL36, MT2A, NAP4, NQO2, NUMA1, OPTN, PA1-RBP1, PAWR, PCBP1, PCNT2, PGD, PIASY, POLQ, POLR2J2, PRDX5, PROL4, RAP80, RBAF600, RNH, RPL, RPS20, RPS27A, SETDB1, SF3A2, SH2D2A, SIAH2, SLC2A1, SRPK2, SSR4, STC2, THOC2, TLE1, TPX2, UBB, UBC, VCL, XM—208944, XTP3TPB, ZFM1, ZNF147, and ZNF151. In yet other embodiments, the POSH-AP is selected from the group consisting of: PKA, SNX1, SNX3, ATP6V0C, PTPN12, PPP1CA, GOSR2, CENTB1, DDEF1, ARF1, ARF5, PACS-1, EPS8L2, HERPUD1, UNC84B, MSTP028, GOCAP, EIF3S3, SRA1, CBL-B, RALA, SIAH1, SMN1, SMN2, SYNE1, TTC3, VCY2IP1, UBE2N (UBC13), ARHV (Chp), WASF1, HIP55, SPG20, HLA-A, and HLA-B. In additional embodiments, the small molecule is a compound selected from among the compounds presented in Table 1. In further embodiments, the small molecule is a compound selected from among the compounds presented in Table 7.
In additional embodiments, the application relates to a method of inhibiting maturation of a virus in a cell, comprising contacting the cell with a small molecule that inhibits the ubiquitin ligase activity of a polypeptide. In certain embodiments, the virus is an envelope virus. In further embodiments, the virus is a retrovirus. In yet further embodiments, the virus is a human immunodeficiency virus. In certain embodiments, the polypeptide comprises a RING domain. In further embodiments of the application, the polypeptide is selected from the group consisting of a polypeptide represented by SEQ ID NOs: 2, 5, 7, 9, and 11. In yet further embodiments, the polypeptide is a human POSH polypeptide. In certain embodiments, the polypeptide is a Cbl-b polypeptide. In further embodiments, the polypeptide is a human Cbl-b polypeptide. In yet further embodiments, the polypeptide is selected from the group consisting of a polypeptide represented by SEQ ID NOs: 43-46, 48, 50, 52, 54, 56, and 58. In certain embodiments, the polypeptide is a PEM-3-like polypeptide. In further embodiments, the PEM-3-like polypeptide is a human PEM-3-like polypeptide.
The application further relates to a method of treating a viral infection in a subject in need thereof, comprising administering to the subject a small molecule that inhibits the ubiquitin ligase activity of a polypeptide. In certain embodiments, the viral infection is an infection by an envelope virus. In further embodiments, the viral infection is an infection by a retrovirus. In yet further embodiments, the viral infection is an infection by a human immunodeficiency virus. In certain embodiments, the polypeptide comprises a RING domain. In other embodiments, the polypeptide is selected from among a POSH polypeptide, a Cbl-b polypeptide, a PEM-3-like polypeptide, a SIAH1 polypeptide, and a TTC3 polypeptide. In yet other embodiments of the application, the polypeptide is selected from the group consisting of a polypeptide represented by SEQ ID NOs: 2, 5, 7, 9, and 11. In yet further embodiments, the polypeptide is a human POSH polypeptide. In certain embodiments, the polypeptide is a Cbl-b polypeptide. In further embodiments, the polypeptide is a human Cbl-b polypeptide. In yet further embodiments, the polypeptide is selected from the group consisting of a polypeptide represented by SEQ ID NOs: 43-46, 48, 50, 52, 54, 56, and 58. In certain embodiments, the polypeptide is a PEM-3-like polypeptide. In further embodiments, the PEM-3-like polypeptide is a human PEM-3-like polypeptide.
In certain embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound as described herein and a pharmaceutically acceptable excipient to a small molecule inhibitor of a POSH polypeptide. In further embodiments, the mammal is a rodent.
In additional embodiments, the application provides a method of inhibiting the transport of APP in a cell, comprising contacting the cell with a small molecule that inhibits the ubiquitin ligase activity of a polypeptide. In certain embodiments, the polypeptide comprises a RING domain. In further embodiments, the polypeptide is a human POSH polypeptide.
The application additionally relates to a method of inhibiting the ubiquitin ligase activity of a POSH polypeptide in a subject in need thereof, comprising administrating to the subject an amount of a POSH polypeptide inhibitor effective to inhibit the ubiquitin ligase activity of said POSH polypeptide.
In certain embodiments, the application provides a method of treating a neurological disorder in a subject in need thereof, comprising administrating a small molecule that inhibits the ubiquitin ligase activity of a polypeptide. In certain embodiments, the polypeptide comprises a RING domain. In further embodiments, the polypeptide is a human POSH polypeptide. Examples of neurological disorders include Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, depression, and schizophrenia.
In additional embodiments, the application provides a method of inhibiting amyloid polypeptide production in a cell, comprising administrating a small molecule that inhibits the ubiquitin ligase activity of a polypeptide. In certain embodiments, the polypeptide comprises a RING domain. In further embodiments, the polypeptide is a human POSH polypeptide.
In other embodiments, the application provides a method of inhibiting cell proliferation, comprising administrating a small molecule that inhibits the ubiquitin ligase activity of a polypeptide. In certain embodiments, the polypeptide comprises a RING domain. In further embodiments, the polypeptide is a human POSH polypeptide.
In preferred embodiments of the application, a small molecule employed in the methods of the application is a compound selected from among the compounds presented in Table 1 or Table 7.
In certain embodiments of the application, a POSH polypeptide is selected from the group consisting of a polypeptide represented by SEQ ID NOs: 2, 5, 7, 9, and 11.
In certain embodiments, the application relates to a method of inhibiting viral budding in a cell, comprising contacting the cell with an agent selected from the group consisting of a compound listed in Table 7.
In certain embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound presented in table 7 and a pharmaceutically acceptable excipient.
The additional embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 2 of table 1 and a pharmaceutically acceptable excipient.
In other embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 3 of table 1 and a pharmaceutically acceptable excipient.
In yet other embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 4 of table 1 and a pharmaceutically acceptable excipient.
In additional embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 5 of table 1 and a pharmaceutically acceptable excipient.
In certain embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 6 of table 1 and a pharmaceutically acceptable excipient.
In other embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 7 of table 1 and a pharmaceutically acceptable excipient.
In yet other embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 8 of table 1 and a pharmaceutically acceptable excipient.
In certain embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 9 of table 1 and a pharmaceutically acceptable excipient.
In other embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 10 of table 1 and a pharmaceutically acceptable excipient.
In yet other embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 11 of table 1 and a pharmaceutically acceptable excipient.
In additional embodiments, the application provides a composition formulated for administration to a mammal, the composition comprising a compound selected from the group defined by scaffold no. 12 of table 1 and a pharmaceutically acceptable excipient.
Other features and advantages of the application will be apparent from the following detailed description, and from the claims.
1. Definitions
The term “amyloid polypeptide” is used to refer to any of the various polypeptides that are significant components of amyloid plaque as well as precursors thereof. The amyloid beta A4 precursor protein (“APP”) gives rise to smaller proteins, such as the roughly 40 amino acid beta amyloid proteins that form a major component of the amyloid plaque associated with Alzheimer's disease, Down's syndrome (in older patients) and certain hereditary cerebral hemorrhage amyloidoses. APP has several isoforms generated by alternative splicing of a 19-exon gene: exons 1-13, 13a, and 14-18 (Yoshikai et al., 1990). The predominant transcripts are APP695 (exons 1-6, 9-18, not 13a), APP751 (exons 1-7, 9-18, not 13a), and APP770 (exons 1-18, not 13a). All of these encode multidomain proteins with a single membrane-spanning region. They differ in that APP751 and APP770 contain exon 7, which encodes a serine protease inhibitor domain. APP695 is a predominant form in neuronal tissue, whereas APP751 is the predominant variant elsewhere. Beta-amyloid is derived from that part of the protein encoded by parts of exons 16 and 17. All of the isoforms of APP and any of the smaller proteins derived therefrom are included in the term “amyloid polypeptide”, as well as any of the various naturally occuring variations thereof and any artificially produced variants that retain one or more functional properties of the naturally occuring protein or that are useful as a proxy for monitoring the production of APP or a protein derived therefrom. The subset of amyloid polypeptides that are APP or derived therefrom may be referred to specifically as “APP amyloid polypeptides”. Yoshikai et al. Gene 87: 257-263, 1990.
The term “envelope virus” as used herein refers to any virus that uses cellular membrane and/or any organelle membrane in the viral release process.
“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is “unrelated” or “non-homologous” shares less than 40% identity, though preferably less than 25% identity with a sequence of the present application. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity.
The term “homology” describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid and protein sequences of the present application may be used as a “query sequence” to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the application. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the application. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used. See http://www.ncbi.nlm.nih.gov.
As used herein, “identity” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.
“Lentiviruses” include primate lentiviruses, e.g., human immunodeficiency virus types 1 and 2 (HIV-1/HIV-2); simian immunodeficiency virus (SIV) from Chimpanzee (SIVcpz), Sooty mangabey (SIVsmm), African Green Monkey (SIVagm), Syke's monkey (SIVsyk), Mandrill (SIVmnd) and Macaque (SIVmac). Lentiviruses also include feline lentiviruses, e.g., Feline immunodeficiency virus (FIV); Bovine lentiviruses, e.g., Bovine immunodeficiency virus (BIV); Ovine lentiviruses, e.g., Maedi/Visna virus (MVV) and Caprine arthritis encephalitis virus (CAEV); and Equine lentiviruses, e.g., Equine infectious anemia virus (EIAV). All lentiviruses express at least two additional regulatory proteins (Tat, Rev) in addition to Gag, Pol, and Env proteins. Primate lentiviruses produce other accessory proteins including Nef, Vpr, Vpu, Vpx, and Vif. Generally, lentiviruses are the causative agents of a variety of disease, including, in addition to immunodeficiency, neurological degeneration, and arthritis. Nucleotide sequences of the various lentiviruses can be found in Genbank under the following Accession Nos. (from J. M. Coffin, S. H. Hughes, and H. E. Varmus, “Retroviruses” Cold Spring Harbor Laboratory Press, 199, 7 p 804): 1) HIV-1: K03455, M19921, K02013, M3843 1, M38429, K02007 and M17449; 2) HIV-2: M30502, J04542, M30895, J04498, M15390, M31113 and L07625; 3) SIV:M29975, M30931, M58410, M66437, L06042, M33262, M19499, M32741, M31345 and L03295; 4) FIV: M25381, M36968 and U1 1820; 5)BIV. M32690; 6)EIAV: M16575, M87581 and U01866; 6)Visna: M10608, M51543, L06906, M60609 and M60610; 7) CAEV: M33677; and 8) Ovine lentivirus M31646 and M34193. Lentiviral DNA can also be obtained from the American Type Culture Collection (ATCC). For example, feline immunodeficiency virus is available under ATCC Designation No. VR-2333 and VR-3112. Equine infectious anemia virus A is available under ATCC Designation No. VR-778. Caprine arthritis-encephalitis virus is available under ATCC Designation No. VR-905. Visna virus is available under ATCC Designation No. VR-779. As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
The term “viral maturation” as used herein refers to the production, post-translational processing, assembly and/or release of proteins and other components that form a viral particle. Accrodingly, this includes the processing of viral proteins leading to the pinching off of nascent virion from the cell membrane.
A “POSH nucleic acid” is a nucleic acid comprising a sequence as represented in any of SEQ ID Nos:1, 3, 4, 6, 8, and 10 as well as any of the variants described herein.
A “POSH polypeptide” or “POSH protein” is a polypeptide comprising a sequence as represented in any of SEQ ID Nos: 2, 5, 7, 9 and 11 as well as any of the variations described herein.
A “POSH-associated protein” or “POSH-AP” refers to a protein capable of interacting with and/or binding to a POSH polypeptide. Generally, the POSH-AP may interact directly or indirectly with the POSH polypeptide. Exemplary POSH-APs are provided throughout.
A “RING domain” or “Ring Finger” is a zinc-binding domain with a defined octet of cysteine and histidine residues. Certain RING domains comprise the consensus sequences as set forth below (amino acid nomenclature is as set forth in Table A): Cys Xaa Xaa Cys Xaa10-20 CyS Xaa His Xaa2-5 Cys Xaa Xaa Cys Xaa13-50 Cys Xaa Xaa Cys (SEQ ID NO: 53) or Cys Xaa Xaa Cys Xaa10-20 Cys Xaa His Xaa2-5 His Xaa Xaa Cys Xaa13-50 Cys Xaa Xaa Cys (SEQ ID NO: 54) or Cys Xaa Xaa Cys Xaa9-39 Cys Xaa1-3 His Xaa2-3 Cys Xaa Xaa Cys Xaa4-48 CyS Xaa Xaa Cys (SEQ ID NO: 55). Certain RING domains are represented as amino acid sequences that are at least 80% identical to amino acids 12-52 of SEQ ID NO: 2 and is set forth in SEQ ID No: 26. Preferred RING domains are 85%, 90%, 95%, 98% and, most preferably, 100% identical to the amino acid sequence of SEQ ID NO: 26. Preferred RING domains of the application bind to various protein partners to form a complex that has ubiquitin ligase activity. RING domains preferably interact with at least one of the following protein types: F box proteins, E2 ubiquitin conjugating enzymes and cullins.
The term “RNA interference” or “RNAi” refers to any method by which expression of a gene or gene product is decreased by introducing into a target cell one or more double-stranded RNAs which are homologous to the gene of interest (particularly to the messenger RNA of the gene of interest).
“Small molecule” as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 2.5 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures comprising arrays of small molecules, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the application.
An “SH3” or “Src Homology 3” domain is a protein domain of generally about 60 amino acid residues first identified as a conserved sequence in the non-catalytic part of several cytoplasmic protein tyrosine kinases (e.g., Src, Abl, Lck). SH3 domains mediate assembly of specific protein complexes via binding to proline-rich peptides. Exemplary SH3 domains are represented by amino acids 137-192, 199-258, 448-505 and 832-888 of SEQ ID NO:2 and are set forth in SEQ ID Nos: 27-30. In certain embodiments, an SH3 domain interacts with a consensus sequence of RXaaXaaPXaaX6P (where X6, as defined in table A below, is a hydrophobic amino acid). In certain embodiments, an SH3 domain interacts with one or more of the following sequences: P(T/S)AP, PFRDY, RPEPTAP, RQGPKEP, RQGPKEPFR, RPEPTAPEE and RPLPVAP.
As used herein, a “transgenic animal” is any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic animals described herein, the transgene causes cells to express a recombinant human POSH protein. The “non-human animals” of the application include vertebrates such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation. The term “chimeric animal” is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal. The term “tissue specific chimeric animal” indicates that the recombinant human POSH genes is present and/or expressed in some tissues but not others.
As used herein, the term “transgene” means a nucleic acid sequence (encoding, e.g., human POSH polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
*Abbreviations as adopted from http://smart.embl-heidelberg.de/SMART_DATA/alignments/consensus/grouping.html.
2. Overview
In part, the present application relates to the discovery of compounds that inhibit the ubiquitin ligase activity of one or more ubiquitin ligase polypeptides, such as polypeptides containing one or more RING domains.
POSH is a ubiquitin ligase comprising a RING domain. As described in PCT patent application PCT/US/02/24589; PCT patent application PCT US/02/36366; U.S. patent application Ser. No. 10/293,965; and European patent application EP02257796, all of which are incorporated by reference herein, the inhibition of POSH inhibits maturation of viruses in infected cells. Many compounds disclosed herein inhibit the ubiquitin ligase activity of POSH and have demonstrated antiviral activity.
Given the functional and structural similarity between proteins having RING domains, it is expected that compounds disclosed herein may be effective for inhibiting the ubiquitin ligase activity of additional ubiquitin ligases.
Ubiquitin ligases contribute to the causation of or the pathology of a wide range of diseases, and compounds disclosed herein may be evaluated and used for treating such diseases, as appropriate.
3. Compounds
In certain embodiments, the present application provides compounds that inhibit the ubiquitin ligase activity of a polypeptide. In certain embodiments, the compounds of the application inhibit the ubiquitin ligase activity of a polypeptide comprising a RING domain. In a preferred embodiment, the application relates to compounds that inhibit the ubiquitin ligase activity of a POSH polypeptide. Many compounds disclosed herein inhibit viral maturation in infected cells.
In certain embodiments, the present application provides compounds described by any of scaffold numbers 1-12 below. Specific examples of compounds are provided in Example 1, below.
The term “acylamino” is art-recognized and refers to a moiety that can be represented by the general formula:
wherein R9 is as defined below, and R′11 represents a hydrogen, an alkyl, an alkenyl or —(CH2)m—R8, where m and R8 are as defined below.
Herein, the term “aliphatic group” refers to a straight-chain, branched-chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, and an alkynyl group.
The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH2)m—R8, where m and R8 are described below.
The term “alkyl” refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
Moreover, the term “alkyl” (or “lower alkyl”) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), —CF3, —CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, —CF3, —CN, and the like.
Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
The term “alkylthio” refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the “alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, —S-alkynyl, and —S—(CH2)m—R8, wherein m and R8 are defined below. Representative alkylthio groups include methylthio, ethylthio, and the like.
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
wherein R9, R10 and R′10 each independently represent a hydrogen, an alkyl, an alkenyl, —(CH2)m—R8, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In preferred embodiments, only one of R9 or R10 can be a carbonyl, e.g., R9, R10 and the nitrogen together do not form an imide. In still more preferred embodiments, the term ‘amine’ does not encompass amides, e.g., wherein one of R9 and R10 represents a carbonyl. In even more preferred embodiments, R9 and R10 (and optionally R′10) each independently represent a hydrogen, an alkyl, an alkenyl, or —(CH2)m—R8. Thus, the term “alkylamine” as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R9 and R10 is an alkyl group.
The term “amido” is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
wherein R9, R10 are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.
The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
The term “aryl” as used herein includes 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics.” The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, —CF3, —CN, or the like. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
The term “carbocycle”, as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
The term “carbonyl” is art-recognized and includes such moieties as can be represented by the general formula:
wherein X is a bond or represents an oxygen or a sulfur, and R11 represents a hydrogen, an alkyl, an alkenyl, —(CH2)n—R8 or a pharmaceutically acceptable salt, R′11 represents a hydrogen, an alkyl, an alkenyl or —(CH2)m—R8, where m and R8 are as defined above. Where X is an oxygen and R11 or R11 is not hydrogen, the formula represents an “ester”. Where X is an oxygen, and R11 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R11 is a hydrogen, the formula represents a “carboxylic acid”. Where X is an oxygen, and R11 is hydrogen, the formula represents a “formate”. In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a “thiocarbonyl” group. Where X is a sulfur and R11 or R′11 is not hydrogen, the formula represents a “thioester.” Where X is a sulfur and R11 is hydrogen, the formula represents a “thiocarboxylic acid.” Where X is a sulfur and R11′ is hydrogen, the formula represents a “thioformate.” On the other hand, where X is a bond, and R11 is not hydrogen, the above formula represents a “ketone” group. Where X is a bond, and R11 is hydrogen, the above formula represents an “aldehyde” group.
The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
The terms “heterocyclyl” or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or the like.
As used herein, the term “nitro” means —NO2; the term “halogen” designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term “hydroxyl” means —OH; and the term “sulfonyl” means —SO2—.
A “phosphonamidite” can be represented in general formula:
wherein 9 and R10 are as defined above, Q2 represents O, S or N, and R48 represents a lower alkyl or an aryl, Q2 represents O, S or N.
A “phosphoramidite” can be represented in general formula:
wherein R9 and R10 are as defined above, and Q2 represents O, S or N.
A “phosphoryl” can in general be represented by the formula:
wherein Q1 represented S or O, and R46 represents hydrogen, a lower alkyl or an aryl. When used to substitute, for example, an alkyl, the phosphoryl group of the phosphorylalkyl can be represented by the general formula:
wherein Q1 represented S or O, and each R46 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents O, S or N. When Q1 is an S, the phosphoryl moiety is a “phosphorothioate”.
The terms “polycyclyl” or “polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings”. Rings that are joined through non-adjacent atoms are termed “bridged” rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or the like.
The phrase “protecting group” as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).
A “selenoalkyl” refers to an alkyl group having a substituted seleno group attached thereto. Exemplary “selenoethers” which may be substituted on the alkyl are selected from one of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH2)m—R8, m and R8 being defined above.
As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
The term “sulfamoyl” is art-recognized and includes a moiety that can be represented by the general formula:
in which R9 and R10 are as defined above.
The term “sulfate” is art recognized and includes a moiety that can be represented by the general formula:
in which R41 is as defined above.
The term “sulfonamido” is art recognized and includes a moiety that can be represented by the general formula:
in which R9 and R′11 are as defined above.
The term “sulfonate” is art-recognized and includes a moiety that can be represented by the general formula:
in which R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moiety that can be represented by the general formula:
in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.
Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof, wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound. In general, the compounds of the present invention may be prepared by the methods illustrated in general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Also for purposes of this invention, the term “hydrocarbon” is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds which can be substituted or unsubstituted.
4. Therapeutic Targets
Compounds of the present application may inhibit the ubiquitin ligase activity of a variety of polypeptides. In particular, proteins having RING domains may be inhibited by compounds disclosed herein. A RING domain is a structural component of a subset of ubiquitin ligases, and it is associated with the ubiquitin ligase activity of many E3 polypeptides. Examples of E3 polypeptides containing RING domains include POSH, Cbl-b, and PEM-3-like.
“Selectivity” of a compound of the invention refers to the ability of a compound, such as a small molecule inhibitor of a ubiquitin ligase as described herein, to preferentially interact with one protein over another. For example, in certain instances, a compound will bind to one protein and not bind to another. Also, for example, in other instances, a compound will bind to more than one protein, but will bind one protein more strongly than it binds another. In certain embodiments, a compound of the invention may inhibit one E3 and not inhibit another, different E3. For example, a small molecule inhibitor of the invention may inhibit the ubiquitin ligase activity of one ubiquitin ligase but not inhibit the ubiquitin ligase activity of another, different ubiquitin ligase. In certain embodiments, the invention relates to compounds that inhibit the ubiquitin ligase activity of more than one E3 ligase but inhibit to a greater degree the ubiquitin ligase activity of one E3 over another, different E3. For example, an agent of the invention may inhibit entirely the ubiquitin ligase activity of one E3 but inhibit only partially or not at all the ubiquitin ligase activity of a different E3.
A selective inhibitor of the invention has selectivity for at least one polypeptide over another polypeptide. For example, in certain embodiments, a selective inhibitor of the invention may inhibit the ubiquitin ligase activity of two different E3 polypeptides, but selectively targets only one of them, resulting in selective inhibition of the ubiquitin ligase activity of the targeted E3 polypeptide.
In a preferred embodiment, compounds disclosed herein may be used to inhibit the ubiquitin ligase activity of a POSH polypeptide, and particularly naturally occurring POSH polypeptides, such as the canonical human POSH polypeptide and allelic variants thereof. The term “POSH polypeptide” is intended to include both naturally occurring and modified forms of POSH, including fragments retaining ubiquitin ligase activity. The term POSH polypeptide is specifically intended to include a polypeptide comprising a RING domain, an SH3 domain and having at least 50% sequence identity to the RING domain of a naturally occurring POSH polypeptide. Examples of POSH nucleic acids and polypeptides are prented in the tables below. POSH comprises a RING domain and is a ubiquitin ligase.
In certain instances, inhibiting the activity of a ubiquitin ligase such as an E3 polypeptide results in inhibition of viral budding. For example, inhibition of viral budding by inhibiting POSH has been presented in U.S. Provisional Application No. 60/469,462, filed May 9, 2003, and entitled “Inhibition of Viral Maturation, Methods and Compositions Related Thereto”. Furthermore, inhibition of viral budding by inhibiting Cbl-b has been shown and is presented in the Examples of the present application. Additionally, it has been shown and is presented in the Examples of the present application that viral infectivity can be inhibited by inhibiting the ubiquitin ligase, PEM-3-like.
Mus musculus
Mus musculus
Mus musculus
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
In addition the following Tables provide the nucleic acid sequence and related SEQ ID NOs for domains of human POSH protein and a summary of sequence identification numbers used in this application.
Drosophila melanogaster POSH
Drosophila melanogaster POSH
The following table provides the sequences of the RING domain and the various SH3 domains.
In certain embodiments, compounds disclosed herein may be evaluated for effectiveness in inhibiting the ubiquitin ligase activity of a Cbl-b polypeptide, and, as appropriate, used to inhibit such activity.
Cbl-b polypeptides contain an amino-terminal variant SH2 domain, a RING finger, and a carboxyl-terminal proline-rich domain with potential tyrosine phosphorylation sites. Cbl-b is highly homologous to the mammalian Cbl and the nematode Sli-1 proteins. Examples of Cbl-b are a human Cbl-b (UniGene No.: Hs.3144), an alternative human Cbl-b (UniGene No.: Hs.381921) that may be a splice variant of Cbl-b, a human Cbl-b polypeptide that is a splice variant represented by the amino acid sequence depicted in SEQ ID NO: 50, which is encoded by the nucleic acid sequence depicted in SEQ ID NO: 49, a human Cbl-b polypeptide that is a splice variant represented by the amino acid sequence depicted in SEQ ID NO: 52, which is encoded by the nucleic acid sequence depicted in SEQ ID NO: 51.
Certain Cbl-b polypeptides have been shown to function as adaptor proteins by interacting with other signaling molecules, e.g., interaction with cell surface receptor tyrosine kinases, e.g., EGFR (Ettenberg, S A et al (2001) J Biol Chem 276:77-84) or with proteins such as Syk (Elly, C et al (1999) Oncogene 18:1147-56), Crk-L (Elly, C et al (1999) Oncogene 18:1147-56), PI3K (Fang, D et al. (2001) J Biol Chem 16:4872-8), Grb2 (Ettenberg, S A et al (1999) Oncogene 18:1855-66), or Vav (Bustelo, X R et al. (1997) Oncogene 15:2511-20). Certain Cbl-b polypeptides have been demonstrated to interact directly with the nucleotide exchange factor, Vav (Bustelo, X R et al. (1997) Oncogene 15:2511-20). Certain Cbl-b polypeptides have been shown to function as an E3 ubiquitin ligase that recognizes tyrosine phosphorylated substrates through its SH2 domain and through its RING domain, recruits a ubiquitin-conjugating enzyme, E2 (Joazeiro, C et al. (1999) Science 286:309-312) Additionally, certain Cbl-b polypeptides have been shown to associate directly with the p85 subunit of P13K and to function as an E3 ligase in the ubiquitination of PI3K (Fang, D et al. (2001) J Biol Chem 16:4872-8).
The term Cbl-b is used herein to refer to full-length, human Cbl-b (UniGene No.: Hs.3144) as well as an alternative Cbl-b (UniGene No.: Hs.381921) composed of two separate Cbl-b sequences (e.g., nucleic acid sequences) that may be a splice variant. The term Cbl-b is used herein to refer as well to the human Cbl-b splice variant represented by the amino acid sequence of SEQ ID NO: 50, which is encoded by the nucleic acid sequence of SEQ ID NO: 49 and to the human Cbl-b splice variant represented by the amino acid sequence of SEQ ID NO: 52, which is encoded by the nucleic acid sequence of SEQ ID NO: 51. The term Cbl-b is used herein to refer as well to various naturally occurring Cbl-b homologs, as well as functionally similar variants and fragments that retain at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring Cbl-b. The term specifically includes human Cbl-b nucleic acid and amino acid sequences (e.g., SEQ ID NOS: 51 and 52) and the sequences presented in the Examples.
In certain embodiments, compounds disclosed herein may be evaluated for effectiveness in inhibiting the ubiquitin ligase activity of a PEM-3-like polypeptide, and, as appropriate, used to inhibit such activity.
PEM-3-like protein bears a unique composition of KH domains and RING domains and is predicted to localize to the nucleoplasm and to the cytoplasm. The protein, SAM68, and homologous proteins containing a KH domain, play an important role in the post-transcriptional regulation of HIV-1 replication. These proteins are involved in the CRM1 pathway and have been found to interact with viral RNA. CRM1 is a receptor protein normally involved in the nuclear export of certain RNAs and proteins. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein, is involved in directing unspliced viral RNA from the nucleus to the plasma membrane. Although MA does not contain the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved CRMlp pathway (Dupont, S et al. (1999) Nature 402:681-685). Nuclear export of another retroviral Gag polyprotein, the Rous sarcoma virus Gag polyprotein, is mediated by a CRM1-dependent nuclear export pathway (Scheifele, L Z et al. (2002) Proc Natl Acad Sci USA 99:3944-3949). While not wishing to be bound to mechanism, PEM-3-like polypeptides may be involved in the CRM1 pathway and may play a role in the post-transcriptional regulation of HIV-1 and in the replication of other viruses.
The term PEM-3-like is used herein to refer to full-length, human PEM-3-like. The term PEM-3-like is used herein to refer as well to various naturally occurring PEM-3-like homologs, as well as functionally similar variants and fragments that retain at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring PEM-3-like. The term specifically includes human PEM-3-like nucleic acid and amino acid sequences and the sequences presented in the Examples.
5. Transgenic Animals and Uses Thereof
In certain embodiments, the application further provides methods for using animal models for determining the safety and/or efficacy of virus therapeutics, i.e. compounds which are useful for treating and/or preventing the development of diseases or conditions, which are caused by, or contributed to by viral infection (e.g., AIDS). In addition the assays are useful for further improving known anti-viral compounds, e.g, by modifying their structure to increase their stability and/or activity and/or toxicity. Accordingly, the application features methods of administering a compound disclosed herein to an animal model for viral infection. Animals may be a mammal such as a mouse, rat, rabbit, goat, sheep, dog, cat, cow, or non-human primate. Such an animal may be susceptible to infection with envelope viruses, retroid viruses and RNA viruses such as various rhabdoviruses, lentiviruses, and filoviruses. Retroid viruses include lentiviruses such as HIV. Other RNA viruses include filoviruses such as Ebola virus.
A transgenic animal may carry a construct for expressing a ubiquitin ligase of interest, such as a gene encoding a POSH polypeptide. A transgenic animal may carry a loss of function mutation or knockout of the endogenous copy of a ubiquitin ligase of interest. A transgenic animal may carry one or more human genes involved in HIV infection, such as Cyclin T1, CD34, CCR5, and fusin (CRCX4). In a further embodiment, the additional human transgene is a gene involved in a disease or condition that is associated with AIDS (e.g., hypertension, Kaposi's sarcoma, cachexia, etc.) Such an animal may be an useful animal model for studying HIV infection, AIDS and related disease development.
Another aspect of the present application concerns transgenic animals which are comprised of cells (of that animal) which contain a transgene of the present application and which preferably (though optionally) express an exogenous POSH and/or POSH-AP protein in one or more cells in the animal. A POSH or POSH-AP transgene can encode the wild-type form of the protein, or can encode homologs thereof, as well as antisense constructs. Moreover, it may be desirable to express the heterologous transgene conditionally such that either the timing or the level of gene expression can be regulated. Such conditional expression can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Pat. No. 4,833,080.
Moreover, transgenic animals exhibiting tissue specific expression can be generated, for example, by inserting a tissue specific regulatory element, such as an enhancer, into the transgene. For example, the endogenous POSH or POSH-AP gene promoter or a portion thereof can be replaced with another promoter and/or enhancer, e.g., a CMV or a Moloney murine leukemia virus (MLV) promoter and/or enhancer.
Alternatively, non-human transgenic animals that only express HIV transgenes in the brain can be generated using brain specific promoters (e.g., myelin basic protein (MBP) promoter, the neurofilament protein (NF-L) promoter, the gonadotropin-releasing hormone promoter, the vasopressin promoter and the neuron-specific enolase promoter, see So Forss-Petter et al., Neuron, 5, 187, (1990). Such animals can provide a useful in vivo model to evaluate the ability of a potential anti-HIV drug to cross the blood-brain barrier. Other target cells for which specific promoters can be used are, for example, macrophages, T cells and B cells. Other tissue specific promoters are well-known in the art, see e.g., R. Jaenisch, Science, 240, 1468 (1988).
Non-human transgenic animals containing an inducible transgene can be generated using inducible regulatory elements (e.g., metallothionein promoter), which are well-known in the art. Transgene expression can then be initiated in these animals by administering to the animal a compound which induces gene expression (e.g., heavy metals). Another preferred inducible system comprises a tetracycline-inducible transcriptional activator (U.S. Pat. No. 5,654,168 issued Aug. 5, 1997 to Bujard and Gossen and U.S. Pat. No. 5,650,298 issued Jul. 22, 1997 to Bujard et al.).
In general, transgenic animal lines can be obtained by generating transgenic animals having incorporated into their genome at least one transgene, selecting at least one founder from these animals and breeding the founder or founders to establish at least one line of transgenic animals having the selected transgene incorporated into their genome.
The present application provides for transgenic animals that carry the transgene in all their cells, as well as animals that carry the transgene in some, but not all cells, i.e., mosaic animals. The transgene can be integrated as a single transgene or in tandem, e.g., head to head tandems, or head to tail or tail to tail or as multiple copies.
The successful expression of the transgene can be detected by any of several means well known to those skilled in the art. Non-limiting examples include Northern blot, in situ hybridization of mRNA analysis, Western blot analysis, immunohistochemistry, and FACS analysis of protein expression.
In a further aspect, the application features non-human animal cells containing a POSH transgene, preferentially a human POSH transgene. For example, the animal cell (e.g., somatic cell or germ cell (i.e. egg or sperm)) can be obtained from the transgenic animal. Transgenic somatic cells or cell lines can be used, for example, in drug screening assays. Transgenic germ cells, on the other hand, can be used in generating transgenic progeny, as described above.
The transgenic animals can be used in in vivo assays to identify viral therapeutics. For example, the animals can be used in assays to identify compounds which reduce or inhibit any phase of the viral life cycle, e.g., expression of one or more viral genes, activity of one or more viral proteins, glycosylation of one or more viral proteins, processing of one or more viral proteins, viral replication, assembly of virions, and/or budding of infectious virions.
In an exemplary embodiment, the assay comprises administering a test compound to a transgenic animal of the application infected with a virus including RNA viruses, DNA viruses, retroidvirus and/or envelope viruses, and comparing a phenotypic change in the animal relative to a transgenic animal which has not received the test compound. For example, where the animal is infected with HIV, the phenotypic change can be the amelioration in an AIDS related complex (ARC), cataracts, inflammatory lesions in the central nervous system (CNV), a mild kidney sclerotic lesion, or a skin lesion, such as psoratic dermatitis, hyperkerstotic lesions, Kaposi's sarcoma or cachexia. The effect of a compound on inhibition of Kaposi's sarcoma can be determined, as described, e.g., in PCT/US97/11202 (WO97/49373) by Gallo et al. These and other HIV related symptoms or phenotypes are further described in Leonard et al. (1988) Science 242:1665.
In another embodiment, the phenotypic change is release/budding of virus particles. In yet another embodiment, the phenotypic change is the number of CD4+ T cells or the ratio of CD4+ T cells versus CD8+ T cells. In HIV infected humans as well as in HIV transgenic mice, analysis of lymph nodes indicate that the number of CD4+ T cells decreases and the number of CD8+ T cells increases. Numbers of CD4+ and CD8+ T cells can be determined, for example, by indirect immunofluorescence and flow cytometry, as described, e.g., in Santoro et al., supra.
Alternatively, a phenotypic change, e.g., a change in the expression level of an HIV gene can be monitored. The HIV RNA can be selected from the group consisting of gag mRNA, gag-pro-pol mRNA, vif mRNA, vpr mRNA, tat mRNA, rev mRNA, vpu/env mRNA, nef mRNA, and vpx mRNA. The HIV protein can be selected from the group consisting of Pr55 Gag and fragments thereof (p17 MA, p24 CA, p7 NC, p1, p9, p6, and p2), Pr160 Gag-Pro-Pol, and fragments thereof (p10 PR, p51 RT, p66 RT, p32 IN), p23 Vif, p15 Vpr, p14 Tat, p19 Rev, p16 Vpu, gPr 160 Env or fragments thereof (gp120 SU and gp41TM), p27 Nef, and p14 Vpx. The level of any of these mRNAs or proteins can be determined in cells from a tissue sample, such as a skin biopsy, as described in, e.g., PCT/US97/11202 (WO97/49373) by Gallo et al. Quantitation of HIV mRNA and protein is further described elsewhere herein and also in, e.g., Dickie et al. (1996) AIDS Res. Human Retroviruses 12:1103. In a preferred embodiment, the level of gp120 on the surface of PBMC is determined. This can be done, as described in the examples, e.g., by immunofluorescence on PBMC obtained from the animals.
A further phenotypic change is the production level or rate of viral particles in the serum and/or tissue of the animal. This can be determined, e.g., by determining reverse transcriptase (RT activity) or viral load as described elsewhere herein as well as in PCT/US97/11202 (WO97/49373) by Gallo et al., such as by determining p24 antigen.
Yet another phenotypic change, which can indicate HIV infection or AIDS progression is the production of inflammatory cytokines such as IL-6, IL-8 and TNF-.alpha.; thus, efficacy of a compound as an anti-HIV therapeutic can be assessed by ELISA tests for the reduction of serum levels of any or all of these cytokines.
Cells from the transgenic animals of the application can be established in culture and immortalized to establish cell lines. For example, immortalized cell lines can be established from the livers of transgenic rats, as described in Bulera et al. (1997) Hepatology 25: 1192. Cell lines from other types of cells can be established according to methods known in the art.”
In one cell-based assay, cells expressing a POSH transgene can be infected with a virus of interest and incubated in the presence a test compound or a control compound. The production of viral particles is then compared. This assay system thus provides a means of identifying molecular antagonists which, for example, function by interfering with viral release/budding.
Cell based assays can also be used to identify compounds which modulate expression of a viral gene, modulate translation of a viral mRNA, or which modulate the stability of a viral mRNA or protein. Accordingly, a cell which is infected with a virus of interest can be incubated with a test compound and the amount of the viral protein produced in the cell medium can be measured and compared to that produced from a cell which has not been contacted with the test compound. The specificity of the compound for regulating the expression of the particular virus gene can be confirmed by various control analyses, e.g., measuring the expression of one or more control genes. This type of cellular assay can be particularly useful for determining the efficacy of antisense molecules or ribozymes.
6. Drug Screening Assays
The application not only provides compounds which inhibit the ligase activity of a polypeptide and compounds which inhibit viral budding, but in certain aspects, the present application also provides assays for identifying yet additional agents that either interfere with or promote the activity of a ubiquitin ligase, particularly a POSH polypeptide.
In certain embodiments, the application provides assays to identify, optimize or otherwise assess agents that increase or decrease a ubiquitin-related activity of a ubiquitin ligase, such as a POSH polypeptide. Ubiquitin-related activities of POSH polypeptides may include the self-ubiquitination activity of a POSH polypeptide, generally involving the transfer of ubiquitin from an E2 enzyme to the POSH polypeptide, and the ubiquitination of a target protein, generally involving the transfer of a ubiquitin from a POSH polypeptide to the target protein. In certain embodiments, a POSH activity is mediated, at least in part, by a POSH RING domain.
In certain embodiments, an assay comprises forming a mixture comprising a POSH polypeptide, an E2 polypeptide and a source of ubiquitin (which may be the E2 polypeptide pre-complexed with ubiquitin). Optionally the mixture comprises an E1 polypeptide and optionally the mixture comprises a target polypeptide. Additional components of the mixture may be selected to provide conditions consistent with the ubiquitination of the POSH polypeptide. One or more of a variety of parameters may be detected, such as POSH-ubiquitin conjugates, E2-ubiquitin thioesters, free ubiquitin and target polypeptide-ubiquitin complexes. The term “detect” is used herein to include a determination of the presence or absence of the subject of detection (e.g., POSH-ubiqutin, E2-ubiquitin, etc.), a quantitative measure of the amount of the subject of detection, or a mathematical calculation of the presence, absence or amount of the subject of detection, based on the detection of other parameters. The term “detect” includes the situation wherein the subject of detection is determined to be absent or below the level of sensitivity. Detection may comprise detection of a label (e.g., fluorescent label, radioisotope label, and other described below), resolution and identification by size (e.g., SDS-PAGE, mass spectroscopy), purification and detection, and other methods that, in view of this specification, will be available to one of skill in the art. For instance, radioisotope labeling may be measured by scintillation counting, or by densitometry after exposure to a photographic emulsion, or by using a device such as a Phosphorimager. Likewise, densitometry may be used to measure bound ubiquitin following a reaction with an enzyme label substrate that produces an opaque product when an enzyme label is used. In a preferred embodiment, an assay comprises detecting the POSH-ubiquitin conjugate.
In certain embodiments, an assay comprises forming a mixture comprising a POSH polypeptide, a target polypeptide and a source of ubiquitin (which may be the POSH polypeptide pre-complexed with ubiquitin). Optionally the mixture comprises an E1 and/or E2 polypeptide and optionally the mixture comprises an E2-ubiquitin thioester. Additional components of the mixture may be selected to provide conditions consistent with the ubiquitination of the target polypeptide. One or more of a variety of parameters may be detected, such as POSH-ubiquitin conjugates and target polypeptide-ubiquitin conjugates. In a preferred embodiment, an assay comprises detecting the target polypeptide-ubiquitin conjugate. In another preferred embodiment, an assay comprises detecting the POSH-ubiquitin conjugate.
An assay described above may be used in a screening assay to identify agents that modulate a ubiquitin-related activity of a POSH polypeptide. A screening assay will generally involve adding a test agent to one of the above assays, or any other assay designed to assess a ubiquitin-related activity of a POSH polypeptidee. The parameter(s) detected in a screening assay may be compared to a suitable reference. A suitable reference may be an assay run previously, in parallel or later that omits the test agent. A suitable reference may also be an average of previous measurements in the absence of the test agent. In general the components of a screening assay mixture may be added in any order consistent with the overall activity to be assessed, but certain variations may be preferred. For example, in certain embodiments, it may be desirable to pre-incubate the test agent and the E3 (e.g., the POSH polypeptide), followed by removing the test agent and addition of other components to complete the assay. In this manner, the effects of the agent solely on the POSH polypeptide may be assessed. In certain preferred embodiments, a screening assay for an antiviral agent employs a target polypeptide comprising an L domain, and preferably an HIV L domain.
In certain embodiments, an assay is performed in a high-throughput format. For example, one of the components of a mixture may be affixed to a solid substrate and one or more of the other components is labeled. For example, the POSH polypeptide may be affixed to a surface, such as a 96-well plate, and the ubiquitin is in solution and labeled. An E2 and E1 are also in solution, and the POSH-ubiquitin conjugate formation may be measured by washing the solid surface to remove uncomplexed labeled ubiquitin and detecting the ubiquitin that remains bound. Other variations may be used. For example, the amount of ubiquitin in solution may be detected. In certain embodiments, the formation of ubiquitin complexes may be measured by an interactive technique, such as FRET, wherein a ubiquitin is labeled with a first label and the desired complex partner (e.g., POSH polypeptide or target polypeptide) is labeled with a second label, wherein the first and second label interact when they come into close proximity to produce an altered signal. In FRET, the first and second labels are fluorophores. FRET is described in greater detail below. The formation of polyubiquitin complexes may be performed by mixing two or more pools of differentially labeled ubiquitin that interact upon formation of a polyubiqutin (see, e.g., US Patent Publication 20020042083). High-throughput may be achieved by performing an interactive assay, such as FRET, in solution as well. In addition, if a polypeptide in the mixture, such as the POSH polypeptide or target polypeptide, is readily purifiable (e.g., with a specific antibody or via a tag such as biotin, FLAG, polyhistidine, etc.), the reaction may be performed in solution and the tagged polypeptide rapidly isolated, along with any polypeptides, such as ubiquitin, that are associated with the tagged polypeptide. Proteins may also be resolved by SDS-PAGE for detection.
In certain embodiments, the ubiquitin is labeled, either directly or indirectly. This typically allows for easy and rapid detection and measurement of ligated ubiquitin, making the assay useful for high-throughput screening applications. As descrived above, certain embodiments may employ one or more tagged or labeled proteins. A “tag” is meant to include moieties that facilitate rapid isolation of the tagged polypeptide. A tag may be used to facilitate attachment of a polypeptide to a surface. A “label” is meant to include moieties that facilitate rapid detection of the labeled polypeptide. Certain moieties may be used both as a label and a tag (e.g., epitope tags that are readily purified and detected with a well-characterized antibody). Biotinylation of polypeptides is well known, for example, a large number of biotinylation agents are known, including amine-reactive and thiol-reactive agents, for the biotinylation of proteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated by reference. A biotinylated substrate can be attached to a biotinylated component via avidin or streptavidin. Similarly, a large number of haptenylation reagents are also known.
An “E1” is a ubiquitin activating enzyme. In a preferred embodiment, E1 is capable of transferring ubiquitin to an E2. In a preferred embodiment, E1 forms a high energy thiolester bond with ubiquitin, thereby “activating” the ubiquitin. An “E2” is a ubiquitin carrier enzyme (also known as a ubiquitin conjugating enzyme). In a preferred embodiment, ubiquitin is transferred from E1 to E2. In a preferred embodiment, the transfer results in a thiolester bond formed between E2 and ubiquitin. In a preferred embodiment, E2 is capable of transferring ubiquitin to a POSH polypeptide.
In an alternative embodiment, a POSH polypeptide, E2 or target polypeptide is bound to a bead, optionally with the assistance of a tag. Following ligation, the beads may be separated from the unbound ubiquitin and the bound ubiquitin measured. In a preferred embodiment, POSH polypeptide is bound to beads and the composition used includes labeled ubiquitin. In this embodiment, the beads with bound ubiquitin may be separated using a fluorescence-activated cell sorting (FACS) machine. Methods for such use are described in U.S. patent application Ser. No. 09/047,119, which is hereby incorporated in its entirety. The amount of bound ubiquitin can then be measured.
In certain preferred embodiments, agents that inhibit (or promote) the ubiquitin ligase activity of a ubiquitin ligase, such as POSH, may be identified using a homogenous time resolved fluorescence assay. Such an assay may employ a GST fused to the RING subunit of a ubiquitin ligase (e.g. POSH). Two flurophore-conjugated detection reagents are also used: anti-GSTXL665 and europium cryptate labeled ubiqutin. The ubiqutination of the ubiquitin ligase by ubiqutin cryptate and binding of the anti-GST tagged XL665 brings the fluorophores into close proximity allowing FRET reaction to occur. Accordingly, FRET serves as a measurement of ubiquitination of the ubiquitin ligase. An enzymatic system may be set up to provide activated ubiquitin, e.g., by providing ubiquitin, an E1 and an E2. Compounds that will not allow the FRET reaction are considered to be inhibitors.
In a screening assay, the effect of a test agent may be assessed by, for example, assessing the effect of the test agent on kinetics, steady-state and/or endpoint of the reaction.
The components of the various assay mixtures provided herein may be combined in varying amounts. In a preferred embodiment, ubiquitin (or E2 complexed ubiquitin) is combined at a final concentration of from 5 to 200 ng per 100 microliter reaction solution. Optionally E1 is used at a final concentration of from 1 to 50 ng per 100 microliter reaction solution. Optionally E2 is combined at a final concentration of 10 to 100 ng per 100 microliter reaction solution, more preferably 10-50 ng per 100 microliter reaction solution. In a preferred embodiment, POSH polypeptide is combined at a final concentration of from 1 ng to 500 ng per 100 microliter reaction solution.
Generally, an assay mixture is prepared so as to favor ubiquitin ligase activity and/or ubiquitination acitivty. Generally, this will be physiological conditions, such as 50-200 mM salt (e.g., NaCl, KCl), pH of between 5 and 9, and preferably between 6 and 8. Such conditions may be optimized through trial and error. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40 degrees C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.5 and 1.5 hours will be sufficient. A variety of other reagents may be included in the compositions. These include reagents like salts, solvents, buffers, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal ubiquitination enzyme activity and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The compositions will also preferably include adenosine tri-phosphate (ATP). The mixture of components may be added in any order that promotes ubiquitin ligase activity or optimizes identification of candidate modulator effects. In a preferred embodiment, ubiquitin is provided in a reaction buffer solution, followed by addition of the ubiquitination enzymes. In an alternate preferred embodiment, ubiquitin is provided in a reaction buffer solution, a candidate modulator is then added, followed by addition of the ubiquitination enzymes.
In general, a test agent that decreases a POSH ubiquitin-related activity may be used to inhibit POSH function in vivo, while a test agent that increases a POSH ubiquitin-related activity may be used to stimulate POSH function in vivo. Test agent may be modified for use in vivo, e.g., by addition of a hydrophobic moiety, such as an ester.
An additional POSH-AP may be added to a POSH ubiquitination assay to assess the effect of the POSH-AP on POSH-mediated ubiquitination and/or to assess whether the POSH-AP is a target for POSH-mediated ubiquitination.
In certain embodiments, a ubiquitination assay as described above for POSH can similarly be conducted for a Cbl-b, a PEM-3-like, a SIAH1, or a TTC3 polypeptide. In certain embodiments, the application provides assays to identify, optimize or otherwise assess agents that increase or decrease a ubiquitin-related activity of a Cbl-b, a PEM-3-like, a SIAH1, or a TTC3 polypeptide. Ubiquitin-related activities of Cbl-b, PEM-3-like, SIAH1, or TTC3 polypeptides may include the self-ubiquitination activity of a Cbl-b, PEM-3-like, SIAH1, or TTC3 polypeptide, generally involving the transfer of ubiquitin from an E2 enzyme to the respective Cbl-b, PEM-3-like, SIAH1, or TTC3 polypeptide, and the ubiquitination of a target protein, e.g., the p85 subunit of P13K, e.g, synaptophysin, generally involving the transfer of a ubiquitin from a Cbl-b, PEM-3-like, SIAH1, or TTC3 polypeptide to the target protein, e.g, the p85 subunit of P13K, e.g., synaptophysin. In certain embodiments, a Cbl-b, a PEM-3-like, a SIAH1, or a TTC3 activity is mediated, at least in part, by a RING domain of a Cbl-b, a PEM-3-like, a SIAH1, or a TTC3 polypeptide, respectively.
Certain embodiments of the application relate to assays for identifying agents that bind to a POSH, optionally a particular domain of POSH such as an SH3 or RING domain or a particular domain of a Cbl-b, such as an SH2 or RING domain of Cbl-b or a RING domain of SIAH1 or TTC3. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. The purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions and design of test agents. In one embodiment, an assay detects agents which inhibit interaction of one or more subject POSH polypeptides with a POSH-AP. In another embodiment, the assay detects agents which modulate the intrinsic biological activity of a POSH polypeptide or POSH complex, such as an enzymatic activity, binding to other cellular components, cellular compartmentalization, and the like.
In one aspect, the application provides methods and compositions for the identification of compositions that interfere with the function of ubiquitin ligase polypeptides. In another aspect, the application provides methods and compositions for the identification of compositions that interfere with the function of the RING domain of a polypeptide, e.g., the RING domain of a POSH or a Cbl-b. In an additional embodiment, the application provides methods and compositions for the identification of agents that interact with the RING domain of a polypeptide, e.g., the RING domain of a POSH or a Cbl-b.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays of the present application which are performed in cell-free systems, such as may be developed with purified or semi-purified proteins or with lysates, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target.
In a further embodiment, agents that bind to a ubiquitin ligase may be identified by using an immobilized ubiquitin ligase. In an illustrative embodiment, a fusion protein can be provided which adds a domain that permits the protein to be bound to an insoluble matrix. For example, GST-POSH fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with a potential labeled binding agent and incubated under conditions conducive to binding. Following incubation, the beads are washed to remove any unbound agent, and the matrix bead-bound label determined directly, or in the supernatant after the bound agent is dissociated.
As described above, Fluorescence Resonance Energy Transfer (FRET)-based assays may also be used to evaluate the formation of ubiquitin-ubiquitin ligase conjugates. Fluorescent molecules having the proper emission and excitation spectra that are brought into close proximity with one another can exhibit FRET. The fluorescent molecules are chosen such that the emission spectrum of one of the molecules (the donor molecule) overlaps with the excitation spectrum of the other molecule (the acceptor molecule). The donor molecule is excited by light of appropriate intensity within the donor's excitation spectrum. The donor then emits the absorbed energy as fluorescent light. The fluorescent energy it produces is quenched by the acceptor molecule. FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor, reduction in the lifetime of its excited state, and/or re-emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor. When the fluorescent proteins physically separate, FRET effects are diminished or eliminated. (U.S. Pat. No. 5,981,200).
For example, a cyan fluorescent protein is excited by light at roughly 425-450 nm wavelength and emits light in the range of 450-500 nm. Yellow fluorescent protein is excited by light at roughly 500-525 nm and emits light at 525-500 nm. If these two proteins are placed in solution, the cyan and yellow fluorescence may be separately visualized. However, if these two proteins are forced into close proximity with each other, the fluorescent properties will be altered by FRET. The bluish light emitted by CFP will be absorbed by YFP and re-emitted as yellow light. This means that when the proteins are stimulated with light at wavelength 450 nm, the cyan emitted light is greatly reduced and the yellow light, which is not normally stimulated at this wavelength, is greatly increased. FRET is typically monitored by measuring the spectrum of emitted light in response to stimulation with light in the excitation range of the donor and calculating a ratio between the donor-emitted light and the acceptor-emitted light. When the donor: acceptor emission ratio is high, FRET is not occurring and the two fluorescent proteins are not in close proximity. When the donor: acceptor emission ratio is low, FRET is occurring and the two fluorescent proteins are in close proximity. In this manner, the interaction between a first and second polypeptide may be measured.
The occurrence of FRET also causes the fluorescence lifetime of the donor fluorescent moiety to decrease. This change in fluorescence lifetime can be measured using a technique termed fluorescence lifetime imaging technology (FLIM) (Verveer et al. (2000) Science 290: 1567-1570; Squire et al. (1999) J. Microsc. 193: 36; Verveer et al. (2000) Biophys. J. 78: 2127). Global analysis techniques for analyzing FLIM data have been developed. These algorithms use the understanding that the donor fluorescent moiety exists in only a limited number of states each with a distinct fluorescence lifetime. Quantitative maps of each state can be generated on a pixel-by-pixel basis.
Suitable fluorescent labels are, in view of this specification, well known in the art. Examples are provided below, but suitable fluorescent labels not specifically discussed are also available to those of skill in the art. Fluorescent labeling may be accomplished by expressing a polypeptide as a fusion protein with a fluorescent protein, for example fluorescent proteins isolated from jellyfish, corals and other coelenterates. Exemplary fluorescent proteins include the many variants of the green fluorescent protein (GFP) of Aequoria Victoria. Variants may be brighter, dimmer, or have different excitation and/or emission spectra. Certain variants are altered such that they no longer appear green, and may appear blue, cyan, yellow or red (termed BFP, CFP, YFP and RFP, respectively). Fluorescent proteins may be stably attached to polypeptides through a variety of covalent and noncovalent linkages, including, for example, peptide bonds (eg. expression as a fusion protein), chemical cross-linking and biotin-streptavidin coupling. For examples of fluorescent proteins, see U.S. Pat. Nos. 5,625,048; 5,777,079; 6,066,476; 6,124,128; Prasher et al. (1992) Gene, 111:229-233; Heim et al. (1994) Proc. Natl. Acad. Sci., USA, 91:12501-04; Ward et al. (1982) Photochem. Photobiol., 35:803-808; Levine et al. (1982) Comp. Biochem. Physiol., 72B:77-85; Tersikh et al. (2000) Science 290: 1585-88.
Other exemplary fluorescent moieties well known in the art include derivatives of fluorescein, benzoxadioazole, coumarin, eosin, Lucifer Yellow, pyridyloxazole and rhodamine. These and many other exemplary fluorescent moieties may be found in the Handbook of Fluorescent Probes and Research Chemicals (2000, Molecular Probes, Inc.), along with methodologies for modifying polypeptides with such moieties. Exemplary proteins that fluoresce when combined with a fluorescent moiety include, yellow fluorescent protein from Vibrio fischeri (Baldwin et al. (1990) Biochemistry 29:5509-15), peridinin-chlorophyll a binding protein from the dinoflagellate Symbiodinium sp. (Morris et al. (1994) Plant Molecular Biology 24:673:77) and phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin (Wilbanks et al. (1993) J. Biol. Chem. 268:1226-35). These proteins require flavins, peridinin-chlorophyll a and various phycobilins, respectively, as fluorescent co-factors.
FRET-based assays may be used in cell-based assays and in cell-free assays. FRET-based assays are amenable to high-throughput screening methods including Fluorescence Activated Cell Sorting and fluorescent scanning of microtiter arrays.
In general, where the screening assay is a binding assay (whether protein-protein binding, agent-protein binding, etc.), one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g., magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
A variety of other reagents may be included in a screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce nonspecific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4° and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening.
7. Methods and Compositions for Treatment of Viral Disorders
In a further aspect, the application provides compounds for treatment of viral disorders, and particularly disorders caused by retroid viruses, RNA viruses and/or envelope viruses, including but not limited to retroviruses, rhabdoviruses, lentiviruses, and filoviruses. Certain therapeutics of the application may function by disrupting the biological activity of a POSH polypeptide or POSH complex in viral maturation. Certain therapeutics of the application may function by disrupting the activity of Cbl-b. Certain therapeutics of the application may function by disrupting the activity of PEM-3-like. Certain therapeutics of the application may function as anti-viral agents by inhibiting the RING domain of a polypeptide, for example, by interacting with the RING domain of a polypeptide. In certain embodiments, therapeutics of the invention interact with the RING domain of a POSH or a POSH-AP (e.g., Cbl-b, SIAH1, TTC3). In a further embodiment, therapeutics of the invention may function as anti-viral agents by inhibiting the ligase activity of a polypeptide, for example, by inhibiting the ubiquitin ligase activity of POSH or a POSH-AP (e.g., Cbl-b, SIAH1, TTC3). In additional embodiments, therapeutics of the invention function as anti-viral agents by inhibiting viral maturation (observed as viral budding). Exemplary therapeutics of the application are presented in Table 7.
In view of the teachings herein, one of skill in the art will understand that the methods and compositions of the application are applicable to a wide range of viruses such as for example retroid viruses, RNA viruses, and envelope viruses. In a preferred embodiment, the present application is applicable to retroid viruses. In a more preferred embodiment, the present application is further applicable to retroviruses (retroviridae). In another more preferred embodiment, the present application is applicable to lentivirus, including primate lentivirus group. In a most preferred embodiment, the present application is applicable to Human Immunodeficiency virus (HIV), Human Immunodeficiency virus type-1 (HIV-1), Hepatitis B Virus (HBV) and Human T-cell Leukemia Virus (HTLV).
While not intended to be limiting, relevant retroviruses include: C-type retrovirus which causes lymphosarcoma in Northern Pike, the C-type retrovirus which infects mink, the caprine lentivirus which infects sheep, the Equine Infectious Anemia Virus (EIAV), the C-type retrovirus which infects pigs, the Avian Leukosis Sarcoma Virus (ALSV), the Feline Leukemia Virus (FeLV), the Feline Aids Virus, the Bovine Leukemia Virus (BLV), the Simian Leukemia Virus (SLV), the Simian Immuno-deficiency Virus (SIV), the Human T-cell Leukemia Virus type-I (HTLV-I), the Human T-cell Leukemia Virus type-II (HTLV-II), Human Immunodeficiency virus type-2 (HIV-2) and Human Immunodeficiency virus type-1 (HIV-1).
The method and compositions of the present application are further applicable to RNA viruses, including ssRNA negative-strand viruses and ssRNA positive-strand viruses. The ssRNA positive-strand viruses include Hepatitis C Virus (HCV). In a preferred embodiment, the present application is applicable to mononegavirales, including filoviruses. Filoviruses further include Ebola viruses and Marburg viruses.
Other RNA viruses include picornaviruses such as enterovirus, poliovirus, coxsackievirus and hepatitis A virus, the caliciviruses, including Norwalk-like viruses, the rhabdoviruses, including rabies virus, the togaviruses including alphaviruses, Semliki Forest virus, denguevirus, yellow fever virus and rubella virus, the orthomyxoviruses, including Type A, B, and C influenza viruses, the bunyaviruses, including the Rift Valley fever virus and the hantavirus, the filoviruses such as Ebola virus and Marburg virus, and the paramyxoviruses, including mumps virus and measles virus. Additional viruses that may be treated include herpes viruses.
7. Methods and Compositions for Treatment of Neurological Disorders
In a further aspect, the application provides compounds useful for the treatment of neurological disorders. Treatment or prevention of a neurological disorder includes inhibition of the progression of a neurological disorder.
Certain therapeutics of the application may function by disrupting the biological activity of a POSH polypeptide (e.g., the ubiquitin ligase activity of a POSH polypeptide) or POSH complex involved in the processing of amyloid polypeptides. In certain embodiments, an agent useful in the treatment or prevention of a neurological disorder interferes with the ubiquitin ligase activity of POSH (e.g., POSH ubiquitination of a target protein such as HERPUD1). Certain therapeutics of the application may function by inhibiting aberrant protein processing associated with a neurodegenerative disorder, such as for example, the processing of amyloid beta precursor protein (APP) associated with Alzheimer's disease. Exemplary therapeutics of the application are presented in Table 7.
In certain embodiments, therapeutics of the application inhibit the transport of APP in a cell by inhibiting the ubiquitin ligase activity of a polypeptide. In further embodiments, therapeutics of the application inhibit amyloid polypeptide production in a cell by inhibiting the ubiquitin ligase activity of a polypeptide, such as, for example, by inhibiting the ubiquitin ligase activity of POSH.
In certain embodiments, agents of the application interfere with the trafficking of a protein through the secretory pathway. In other embodiments, agents disclosed herein inhibit or promote POSH and POSH-AP mediated cellular processes such as protein processing in the secretory pathway, for example, processing of amyloid polypeptides.
Neurological disorders include CNS disorders, such as Alzheimer's disease, cerebral vascular disease, and schizophrenia. Neurological disorders further include disorders associated with increased levels of plasma homocysteine, increased levels of amyloid beta production, or aberrant presenilin acitivity. Examples of neurological disorders are Parkinson's disease, Huntington's disease, Pick's disease, Niemann-Pick's disease, prion-associated diseases (e.g., Mad Cow disease), depression, and schizophrenia.
8. Methods and Compositions for Treatment of Cancer Diseases
In a further aspect, the application provides compounds useful for the treatment or prevention of cancer diseases. In certain embodiments, a therapeutic of the application inhibits cell proliferation by inhibiting the ubiquitin ligase activity of a polypeptide. In certain embodiments, the polypeptide is a POSH polypeptide.
Certain therapeutics of the application may function by disrupting the biological activity of a POSH polypeptide or POSH complex involved in cell proliferation. In certain embodiments, an agent useful in the treatment or prevention of a cancer disease interferes with the ubiquitin ligase activity of POSH. Exemplary therapeutics of the application are presented in Table 7.
The terms “cancer,” “tumor,” and “neoplasia” are used interchangeably herein. As used herein, a cancer (tumor or neoplasia) is characterized by one or more of the following properties: cell growth is not regulated by the normal biochemical and physical influences in the environment; anaplasia (e.g., lack of normal coordinated cell differentiation); and in some instances, metastasis. Cancer diseases include, for example, anal carcinoma, bladder carcinoma, breast carcinoma, cervix carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, endometrial carcinoma, hairy cell leukemia, head and neck carcinoma, lung (small cell) carcinoma, multiple myeloma, non-Hodgkin's lymphoma, follicular lymphoma, ovarian carcinoma, brain tumors, colorectal carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, lung (non-small cell carcinoma), melanoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, and soft tissue sarcoma. Additional cancer disorders can be found in, for example, Isselbacher et al. (1994) Harrison's Principles of Internal Medicine 1814-1877, herein incorporated by reference.
9. Effective Dose
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The Ld50 (The Dose Lethal To 50% Of The Population) And The Ed50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the application, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
10. Formulation and Use
Pharmaceutical compositions for use in accordance with the present application may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
An exemplary composition of the application comprises an RNAi mixed with a delivery system, such as a liposome system, and optionally including an acceptable excipient. In a preferred embodiment, the composition is formulated for topical administration for, e.g., herpes virus infections.
For such therapy, the compounds of the application can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the application can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present application are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. in addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the oligomers of the application are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
For therapies involving the administration of nucleic acids, the oligomers of the application can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, intranodal, and subcutaneous for injection, the oligomers of the application can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligomers of the application are formulated into ointments, salves, gels, or creams as generally known in the art.
The application now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present application, and are not intended to limit the application.
1. Identification of Compounds
Evaluation of POSH-specific inhibitors was conducted as follows:
Step 1: identifying compounds that block POSH autoubiquitnation at 10 micromolar DMSO solution of the compound. Compounds that have shown inhibition rate of 80% and more were designated as good inhibitor.
Step 2: filtering compounds that block E1 and E2 ubiqutination activity in compound concentration of 1 micro molar
Step 3: establish IC 50 value measurements. Good inhibitors were designated as having IC 50 with in the range of 1 micromolar-100 nano molar
Step 4: specificity of POSH inhibitors was established by determine the ability of POSH inhibitors to inhibit ubiquitination (% A) of different known E3 ligase at concentration of 1 micro molar. We have designates as selective inhibitors compound that fail to inhibit 60% of ubiquitination (% A less than 60%) according to protocol as described for HTS screening assay
Compounds were further assessed for effects on maturation of HIV particles in a virus-like particle assay.
The amount of budding in the presence of inhibitors at concentration of 3 micro molar was determined by using RETRO-TEK HIV-1 p24 Antigen ELISA kit. (ZeptoMetrix Corporation, ZMC catalog #: 0801111, Buffalo, N.Y., USA). an enzyme-linked immunoassay used to detect the amount of HIV-1 p24 released during budding process.
A compound was identified as an effective inhibitor when less than 69% budding, relative to control, was observed.
Examples of identified compounds are provided in the following table:
Experimental Protocols:
1. HTS Screening Assay Procedure
Microplates 10109D to 10114D compounds at 1 mM.
(Controls in column 1).
All compounds were perchase from ChemBridge corporation: ChemBridge corporation 16981 Via Tazon, Suite G San Diego, Calif. 92127
a. Microplates Labeling
Put labels to 6 PS U-bottom clear microplate.
Put labels to 18 black microplates (triplicats).
b. Microplates Preparation
GST-POSH, 33 nM
Enzymes solution ×4.
GST XL 665 in Rec. buffer
EDTA 0.5 M pH=8
Reconstitution buffer
Check pH=7
Sigma DMSO D-8418
Titrated ATP 0.1 M Amount
Checked with pH stick paper.
DTT1M
Ovalbumin 10%
Ubiquitin 1 mg/ml
Results:
The following table presents the IC50 for each compound with respect to the ubiquitin ligase activity of POSH.
2. Budding results:
The following table shows the effects of each compound on HIV maturation (assessed as budding) in a VLP assay.
2. In-Vitro Assay of Human Posh Self-Ubiquitination
Recombinant hPOSH was incubated with ATP in the presence of E1, E2 and ubiquitin as indicated in each lane. Following incubation at 37° C. for 30 minutes, reactions were terminated by addition of SDS-PAGE sample buffer. The samples were subsequently resolved on a 10% polyacrylamide gel. The separated samples were then transferred to nitrocellulose and subjected to immunoblot analysis with an anti ubiquitin polyclonal antibody. The position of migration of molecular weight markers is indicated on the right.
Poly-Ub: Ub-hPOSHconjugates, detected as high molecular weight adducts only in reactions containing E1, E2 and ubiquitin. hPOSH-176 and hPOSH-178 are a short and a longer derivatives (respectively) of bacterially expressed hPOSH; C, control E3
preliminary steps in high-throughput screen
Objective
1. Test Ub detection with in a Ub chain as function of an E3 (HRD1) and POSH auto-Ubiquitination.
2. Test Boston Biochem reagents.
Materials
1. E1 recombinant from bacculovirus
2. E2 Ubch5c from bacteria
3. Ubiquitin
4. POSH #178 (1-361) gst fusion-purified but degraded
5. POSH # 176 (1-269) gst fusion-purified but degraded
6. hsHRD1 soluble ring containing region
5. Bufferx12 (Tris 7.6 40 mM, DTT 1 mM, MgCl2 5 mM, ATP 2 uM)
6. Dilution buffer (Tris 7.6 40 mM, DTT 1 mM, ovalbumin 1 ug/ul) protocol
1. Incubate for 30 minutes at 37° C.
2. Run 12% SDS PAGE gel and transfer to nitrocellulose membrane
3. Incubate with anti-Ubiquitin antibody.
Results, shown in
3. Cbl-b affects VLP Production
Pulse-chase kinetics
A. Transfections
8. Next day, perform HIV trasfection (pNLenv-1 # 111), 11 μg/plate with the appropriate siRNA at a concentration of 100 nM.
B. Pulse-Chase
Chase/Stop Medium
Complete RPMI
10% FCS
10 mM cold methionine
5 mM HEPES pH7.5
Glutamine (1:100)
Pen/Strep \(1:100)
Prepare 50 ml aliquots and freeze at −20° C.
Prior to use, thaw, shake intensively and place on ice.
C. IP with anti-p24
7. At the end of incubation, transfer sup+beads to fresh tubes, spin down beads and wash twice with 1 ml high salt buffer, once with medium salt buffer and twice with low salt buffer (listed below).
Results are presented in
D. Check Cbl-b Levels by IP/IB.
Results are presented in
4. Cbl-b Affects the Release of VLP at Steady State
Plasmid no. 111: pNlenv-1.
Transfections:—
Steady state VLP Assay
A. Cell Extracts
Results are Presented in
Examples of Cbl-b sequences are presented below.
HeLa cells subject to siRNA knockdown of POSH expression showed a reversible decrease in cell growth.
Protocol:
Day 1: Plate two 6-well plates with HeLa SS6 cells (5×105) cells/well.
Day 2: Change medium to medium without antibiotics.
Day 3: Perform the same transfection as noted below.
Transfection: 10 nM RNAi: A2=RNAi #153; A3=RNAi#187 (see Example 2 for RNAi information)
Prepare mix of LF2000+OptiMEM: 3.25 ml optimem with 65 ul LF2000, incubate together for 5 min. Add to reactions as noted above. Incubate for 25 min. Add 500 ul to the corresponding well.
Day 4: Harvest one of each reaction for both protein extraction and RNA. (#153 day 4 and #187 day 4). For the other set of reactions: count and split each well into (5×105 cells) for transfection next day.
Day 5: Perform the same transfection as noted above.
Day 6: Harvest one plate for protein and RNA extraction and the other split to (5×105 cells) and (2.5×105 cells)
Day 8: Harvest and count and to the (5×105 cells) plate form day six.
Day 10: Harvest and count the (2.5×105 cells) plate from day 6.
Protein extract: for POSH expression evaluation
1. Wash cells twice with cold PBS on ice.
2. Add PBS and scrape cells off to eppendorf tubes.
3. Centrifuge at 40 deg. C. at 1800 rpm for 5 min.
4. Add 100 ul of RIPA Buffer+protease inhibitors (PI3K) (1:200)+EDTA (1:100) to the remaining cells on plate.
5. Incubate on ice for 15 minutes.
6. Centrifuge for 10 min in 40 deg. C. at maximum speed.
7. Proteins (15 ug/lane) were loaded onto a 10% tris glycine gel, resolved by electrophoresis, transferred to nitrocellulose membrane and probed with anti POSH antibody for 1 h (1:2000 dilution). Detected was by ECL.
8. Evaluations of protein levels were performed using an ImageQuant analyzer. Results are shown in
HeLa SS6 cells that express reduced levels of POSH(H153) and control cells expressing scrambled RNAi (H187) were transfected with a plasmid expressing amyloid precursor protein (APP) and presenilin 1 (PS1). Cells were metabolic labeled and protein extracts were immunoprecipitated with anti-amyloid beta specific antibody, which recognize an epitope common to APP, C199 and Aβ polypeptides. A labeled protein was specifically precipitated by the antibody in H187-transfected cells (see
Methods
Cloning of pIRES-APP-PS1
Cloning was performed in two steps: Presenilin 1 (PS1) was first cloned from human brain library into pIREs (pIREs-PS1). Then APP-695 was obtained from amplifying two image clones (3639599 and 5582406) and mixing their PCR products in an additional PCR reaction to yield full-length APP695 that was further ligated into pIREs-PS1 to generate pIREs-APP-PS1.
Transfection, Metabolic Labeling and Immunoisolation of Amyloid Beta (Aβ)
Hela SS6 cells expressing POSH-specific RNAi or scrambled RNAi (H153 and H187, respectively) were transfected with pIREs-APP-PS1 (24 μg) using lipofectamin 2000 reagent (Invitrogen, LTD). Twenty-four hours post-transfection, cells were metabolic labeled with 1 mCi of 35S-methionine at 37° C. for an additional twenty-four hours. Media was collected from cells and spun at 3000 rpm for 10 min to pellet cell debris. Protease inhibitors and 2 mM 1, 10-phenanthroline were added to the cleared cell media. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH7.8, 150 mM sodium chloride, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate and protease inhibitors). Cell media and lysate were immunoprecipitated with anti-Aβ (1-17) antibody (6E10) (Chemicon) or a non-relevant (NR) antibody. Precipitated proteins were separated on 16% Tris-Tricine gel. Gel was dried and bands detected by phosphoimager (Typhoon instrument, Amersham Biosciences, Corp.).
PEM-3-like reduction reduces reverse transcriptase (RT) activity in release virus-like-particles (VLP):
HeLa SS6 cell cultures (in triplicates) were transfected with siRNA targeting PEM-3-like or with a control siRNA. Following gene silencing by siRNA, cells were transfected with pNLeny1, encoding an envelope-deficient subviral Gag-Pol expression system (Schubert, U., Clouse, K. A., and Strebel, K. (1995). Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes. J Virol 69, 7699-7711) and RT activity in VLP released into the culture medium was determined (
Cell Culture and Transfections:
Hela SS6 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and 100 units/ml penicillin and 100 μg/ml streptomycin. For transfections, HeLa SS6 cells were grown to 50% confluency in DMEM containing 10% FCS without antibiotics. Cells were then transfected with the relevant double-stranded siRNA (50-100 nM) using lipofectamin 2000 (Invitrogen, Paisley, UK). On the day following the initial transfection, cells were split 1:3 in complete medium and co-transfected 24 hours later with HIV-1NLeny1 (2 μg per 6-well) (Schubert, U., Clouse, K. A., and Strebel, K. (1995). Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes. J Virol 69, 7699-7711) and a second portion of double-stranded siRNA.
Assays for Virus Release by RT Activity:
Virus and virus-like particle (VLP) release was determined one day after transfection with the pro-viral DNA as previously described (Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59:284-291; Fukumori, T., Akari, H., Yoshida, A., Fujita, M., Koyama, A. H., Kagawa, S., and Adachi, A. (2000). Regulation of cell cycle and apoptosis by human immunodeficiency virus type 1 Vpr. Microbes Infect 2, 1011-1017; Lenardo, M. J., Angleman, S. B., Bounkeua, V., Dimas, J., Duvall, M. G., Graubard, M. B., Hornung, F., Selkirk, M. C., Speirs, C. K., Trageser, C., et al. (2002). Cytopathic killing of peripheral blood CD4(+) T lymphocytes by human immunodeficiency virus type 1 appears necrotic rather than apoptotic and does not require env. J Virol 76, 5082-5093). The culture medium of virus-expressing cells was collected and centrifuged at 500×g for 10 minutes. The resulting supernatant was passed through a 0.45 μm-pore filter and the filtrate was centrifuged at 14,000×g for 2 hours at 4° C. The resulting supernatant was removed and the viral-pellet was re-suspended in cell solubilization buffer (50 mM Tris-HCl, pH7.8, 80 mM potassium chloride, 0.75 mM EDTA and 0.5% Triton X-100, 2.5 mM DTT and protease inhibitors). The corresponding cells were washed three times with phosphate-buffered saline (PBS) and then solubilized by incubation on ice for 15 minutes in cell solubilization buffer. The cell detergent extract was then centrifuged for 15 minutes at 14,000×g at 4° C. The sample of the cleared extract (normally 1:10 of the initial sample) were resolved on a 12.5% SDS-polyacrylamide gel, then transferred onto nitrocellulose paper and subjected to immunoblot analysis with rabbit anti-CA antibodies. The CA was detected after incubation with a secondary anti-rabbit antibody conjugated to Cy5 (Jackson Laboratories, West Grove, Pa.) and detected by fluorescence imaging (Typhoon instrument, Molecular Dynamics, Sunnyvale, Calif.). The Pr55 and CA were then quantified by densitometry. A colorimetric reverse transcriptase assay (Roche Diagnostics GmbH, Mannenheim, Germany) was used to measure reverse transcriptase activity in VLP extracts. RT activity was normalized to amount of Pr55 and CA produced in the cells.
Scanning Electron Microscopy:
HeLa cells were fixed for two hours in 0.1M phosphate buffer (PB) (pH 7.2) containing 2.5% glutaraldehyde and then washed three times with PB. The cells were then dehydrated by gradual increase of the ethanol concentration (25%, 75%, 95%, 100%). The samples at 100% ethanol were dried in a critical point dryer BIO-RAD (C.P.D750) and then coated with gold. Images were taken on a Jeol 5410 LV scanning electron microscope at 25 kV.
The production of infectious virus over a single cycle of HIV-1 replication, in the presence of normal or reduced levels of PEM-3-like was compared (
Examples of PEM-3-like sequences are presented below. Additional examples of PEM-3-like sequences are presented in the PCT patent application, International Application No. PCT/US04/16865, filed on May 28, 2004 and entitled “PEM-3-like Compositions and Related Methods Thereof”.
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
While specific embodiments of the subject applications have been discussed, the above specification is illustrative and not restrictive. Many variations of the applications will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the applications should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
This application is a continuation of PCT Application No. PCT/US2004/021900, filed Jul. 9, 2004, which claims the benefit of the filing date of U.S. Provisional Application Nos. 60/486,730, filed Jul. 11, 2003, 60/489,795, filed Jul. 11, 2003, and 60/549,896, filed Mar. 2, 2004. PCT Application No. PCT/US2004/021900 is also a continuation-in-part of PCT International Application Nos. PCT/US04/06619, filed Mar. 5, 2004, and a continuation-in-part of PCT/US04/10582, filed Apr. 5, 2004, and a continuation-in-part of PCT/US04/16865, filed May 28, 2004, PCT/US03/35712, filed Nov. 10, 2003 and PCT/US04/06308, filed Mar. 2, 2004. The specifications of all of the above-referenced applications are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60486730 | Jul 2003 | US | |
| 60489795 | Jul 2003 | US | |
| 60549896 | Mar 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US04/21900 | Jul 2004 | US |
| Child | 11330500 | Jan 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US04/06619 | Mar 2004 | US |
| Child | 11330500 | Jan 2006 | US |
| Parent | PCT/US04/10582 | Apr 2004 | US |
| Child | 11330500 | Jan 2006 | US |
| Parent | PCT/US04/16865 | May 2004 | US |
| Child | 11330500 | Jan 2006 | US |
| Parent | PCT/US03/35712 | Nov 2003 | US |
| Child | 11330500 | Jan 2006 | US |
| Parent | PCT/US04/06308 | Mar 2004 | US |
| Child | 11330500 | Jan 2006 | US |
