Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-08-25
2001-03-13
Minnifield, Nita (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S041000, C435S183000, C424S184100, C424S204100, C424S277100, C530S395000
Reexamination Certificate
active
06200746
ABSTRACT:
FIELD OF THE INVENTION
The invention provides methods to identify anti-viral agents, and more specifically, inhibitors of human papillomavirus E7 protein-induced increase in CDK2 kinase activity.
BACKGROUND
The human papillomaviruses (HPVs) are a family of more than 80 small (approximately 8 kb) DNA viruses that infect stratified squamous epithelia causing warts. Certain high-risk HPV strains, including HPV16, HPV18, and HPV31, have been implicated as the most important etiological agents in cervical cancer [zur Hausen, Biochim. Biophys. Acta 1288:F55-78 (1996)], which is consistent with the observation that E6 and E7 genes from the high risk HPVs are potent oncogenes. Oncogenic potential of E6 and E7 may arise from binding properties to host cell proteins. For example, E6 binds to the tumor-suppressor protein p53 leading to ubiquitin-dependent degradation of the protein [Scheffher, et al., Cell 63:1129-36 (1990)], and E7 binds and promotes degradation of the tumor-suppressor retinoblastoma protein (pRB) [Dyson, et al., Science 243:934-7 (1989); Jones, et al., Genes & Dev 11:2101-11 (1997)]. While E6 and E7 have other activities, their roles in the viral life cycle are not fully elucidated.
The HPV life cycle is regulated in a differentiation-dependent manner within stratified-squamous epithelia [Jones, et al., Genes & Dev 11:2101-11 (1997)]. The virus is maintained as an episome in the basal cell layer, which is the replicating cell population in stratified epithelia. With differentiation of the host cells into keratinocytes, the virus undergoes a burst of DNA replication. Following differentiation, keratinocytes exit the cell cycle and die during the normal course of epithelial stratification. These events are normally irreversible, but HPV E7 activity is sufficient to promote an unscheduled round of DNA synthesis in differentiated keratinocytes [Cheng, et al., Genes & Dev. 9:2335-49 (1995)]. The newly synthesized viral DNA is packaged in the upper viable layers of the epithelia, and sloughed into the environment in the dead, differentiated cells. The unscheduled DNA synthesis in differentiated cells is central to the HPV viral life cycle, and the E7 gene product has been implicated as a key viral protein in this event. The E7 gene product is a 98 amino acid protein that binds a number of regulatory proteins, including pRb and proteins in the cyclin-dependent kinase inhibitory protein (KIP) family, the function of which is critical for entry into S-phase entry of the cell cycle [Morgan, Ann. Rev. Cell Dev. Biol. 13:261-291 (1997)].
How E7 promotes progression into S phase has been the subject of intense research because of the importance of this event to the viral life cycle and HPV-related cancer. E7 can overcome negative cellular growth signals including, for example, those mediated by TGF-&bgr; [Pietenpol, et al., Cell 61:777-85 (1990)], loss of substrate adherence [Ruesch, et al., Virol. 250:19-29 (1998)], and serum deprivation [Pei, et al., Carcinogenesis 19:1481-6 (1998)]. This activity correlates, in part, with the ability of E7 to transform cells and bind pRb family members [Galloway, et al., Semin. Cancer Biol. 7:309-15 (1996)]. E7 binds other proteins, including, for example transcription factors such as TATA-binding proteins [Massimi, et al., Oncogene 12:2325-30 (1996)], and c-jun and c-fos family members [Antinore, et al., EMBO. J., 15:1950-60 (1996)].
Despite these binding activities, it is unclear which known function(s) of E7, if any, are key for the viral life cycle. Most notably, E7 binds pRb family members [Dyson, et al., Science 243:934-7 (1989); Ciccolini, et al., Oncogene 9:2633-8 (1994); Wu, et al., J. Virol. 67:2402-7 (1993)], p21 [Funk, et al., Genes & Dev 11:2090-100 (1997); Jones, et al., Genes & Dev 11:2101-11 (1997)], and p27 [Zerfass, et al., Oncogene 13:2323-30 (1996)], proteins that participate in the cyclin-dependent kinase phosphorylation pathway regulating cell cycle progression. The cyclin-dependent kinases regulate cell cycle progression by a variety of means [Morgan, Ann. Rev. Cell Dev. Biol. 13:261-291 (1997)], including inhibiting the ability of pRb to sequester E2F [Mulligan, et al., Trends Genet 14:223-9 (1998)], a protein that upregulates a variety of genes required for entry into S phase. E7 binding to p21 and p27, both of which inhibit CDK phosphorylation, results in a net increase in CDK2 activity. These inhibitor proteins have been implicated as key regulators of cell cycle progression that act, at least in part, via a common cyclin-dependent kinase inhibitory domain found in the amino terminus of these proteins [Polyak, et al., Cell 78:59-66 (1994); Chen, et al., Mol. Cell. Biol. 16:4673-82 (1996)]. E7 from viruses with low oncogenic potential lacks these binding activities, suggesting that interaction with one or more cellular proteins is important for neoplastic progression. Whether any of these properties are essential in the viral life cycle is unclear [Davies, et al., J. Virol., 67:2521-8 (1993); Funk, et al., Genes & Dev 11:2090-100 (1997)].
Thus there exists a need in the art to more fully determine mechanisms by which E7 is able promote viral replication and to develop methods to identify inhibitors of E7 specific activity. Inhibition of E7 can result in potent anti-viral activity and therefore, methods to identify inhibitors of E7-dependent activity are desirable.
SUMMARY OF THE INVENTION
The present invention provides methods for identifying anti-viral agents. In a preferred embodiment, methods of the invention identify agents that reduce or inhibit proliferation of human papillomaviruses.
The invention provides methods for identifying an inhibitor of E7-induced CDK2 kinase activity comprising the steps of: a) measuring CDK2 kinase activity on a CDK2 substrate in the presence of human papillomavirus (HPV) E7, or a CDK2 binding fragment thereof and in the presence and absence of a test compound, and b) identifying the test compound as an inhibitor of E7-induced CDK2 kinase activity when decreased phosphorylation of the CDK2 substrate is detected in the presence of the test compound compared to phosphorylation of the CDK2 substrate detected in the absence of the test compound. In one aspect, the methods of the invention include use of an E7 fragment that binds and activates CDK2, wherein the E7 fragment consists of a continuous amino terminal fragment of E7 beginning at amino acid residue 1 and terminating at a carboxy terminal residue selected from the group consisting of amino acid residues 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 ,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, and 97 as set out in SEQ ID NO. 1. In a preferred embodiment, methods of the invention comprising use of an E7 fragment selected from the group consisting of amino acid residues 1 to 48, amino acid residues 1 to 69, amino acid residues 1 to 87 as set out in SEQ ID NO: 1. Methods of the invention preferably include a CDK2 substrate selected from the group consisting of histone H1 and HPV protein E1.
The invention also provide methods for identifying an inhibitor of E7-induced CDK2 kinase activity comprising the steps of: a) measuring CDK2 kinase phosphorylation of a substrate; b) measuring increased CDK2 kinase phosphorylation of a substrate in the presence of human papillomavirus (H?V) E7, or a CDK2 binding fragment thereof, to determine E7-induced CDK2 kinase activity; c) measuring CDK2 kinase phosphorylation of a substrate in the presence of HPV E7, or a CDK2 binding fragment thereof and in the presence of a test inhibitor compound; and d) identifying the test compound as an inhibitor of E7-induced CDK2 kinase activity when the increased phosphorylation measured in step (b) is reduced in step (c) the presence of the test compound. In one aspect,
Fisher Christopher
He Wanxia
Baskar Padma
Darnley , Jr. James D.
Minnifield Nita
Pharmacia & Upjohn
Pharmacia & Upjohn Company
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