E2 displacement assay for identifying inhibitors of HPV

Details E2 displacement assay for identifying inhibitors of HPV E2 displacement assay for identifying inhibitors of HPV

2003-02-05

2004-11-30

Riley, Jezia (Department: 1637)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S006120, C514S001000, C514S152000, C514S231500, C514S351000, C536S023100, C548S127000

Reexamination Certificate

active

06825176

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to an assay for identifying inhibitors of the papilloma virus (PV), particularly human papilloma virus (HPV). In particular, the present invention provides a novel probe in a competitive assay to identify HPV inhibitors. More particularly, the present invention relates to the synthesis and use of a probe that binds with specificity to the transactivation domain (TAD) of HPV E2 to form a complex therewith, and which is capable of being displaced by inhibitors of HPV.
BACKGROUND OF THE INVENTION
Papillomaviruses are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been identified in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types of papillomavirus that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases such as cervical carcinomas.
There are more than 25 HPV types that are implicated in anogenital diseases, these are grouped into “low risk” and “high risk” types. The low risk types include HPV type 6 and type 11 and induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States, 1% of the sexually active population has genital warts of which 90% is attributed to HPV-6 and HPV-11.
The high risk types are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and 18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide.
The life cycle of PV is closely coupled to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. Unlike normal cells, the cellular DNA replication machinery is maintained as the cell undergoes vertical differentiation. As the infected cells undergo progressive differentiation the viral genome copy number and viral gene expression in turn increase, with the eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles.
The coding strands for each of the papillomavirus contain approximately ten designated translational open reading frames (ORFs) that have been classified as either early ORFs or late ORFs based on their location in the genome. E1 to E8 are expressed early in the viral replication cycle, and two late genes (L1 and L2) encode the major and minor capsid proteins respectively. The E1 and E2 gene products function in viral DNA replication, whereas E5, E6 and E7 are expressed in connection with host cell proliferation. The L1 and L2 gene products are involved in virion structure. The function of the E3 and E8 gene products is uncertain at present.
Studies of HPV have shown that proteins E1 and E2 are the only two viral proteins that are necessary for viral DNA replication in vitro and in vivo, in addition to the host DNA replication machinery. This requirement is similar to that of bovine papillomavirus type 1 (BPV-1). Indeed, there is a high degree of similarity between E1 and E2 proteins and the ori-sequences of all papillomaviruses (PV) regardless of the viral species and type. Evidence emanating from studies of BPV-1 have shown that E1 possesses ATPase and helicase activities that are required in viral DNA replication.
The E2 protein is a transcriptional activator that binds to E1 protein and forms a complex that binds specifically to the ori sequence (Mohr et al., 1990, Science 250:1694-1699). It is believed that E2 enhances binding of E1 to the BPV origin of replication (Seo et al., 1993, Proc. Natl. Acad. Sci., 90:2865-2869). In HPV, Lui et al. suggested that E2 stabilizes E1 binding to the ori (1995, J. Biol. Chem., 270(45):27283-27291). The HPV-16 transactivation domain (TAD) of E2 has been described in J. E. Burns et al., 1998 (Acta Cryst. D54, 1471-1474) and amino acids 1-190 were found to be required and sufficient for E1 binding (Yasugi et al., 1997, J. Virol. 71, 891-899).
To thwart this disease, a chemical entity that would interfere with or inhibit viral DNA replication is therefore desirable. Previously described methods to evaluate inhibitors of the E1-E2 interaction (U.S. Pat. No. 5,925,516 and Titolo et al. 1999, J. Virol. 73, 5282-5293) have relied on the production of full-length E1 and E2 proteins. HPV E2 and especially E1 have been difficult to obtain in sufficient quantity and purity for effective drug screening (White et al., 2001, J. Biol. Chem., 276(25), 22426-22438; Rocque et al., 2000, Protein, Expression Purif. 18, 148-159). Furthermore, one common assay for this interaction involves measuring the cooperative binding of E1 and E2 to double-stranded DNA referred to herein as the E2-dependent E1 DNA binding assay (Titolo et al. 1999, J. Virol. 73, 5282-5293). This method is highly sensitive to salt concentration and pH, as is well known to be true in general for protein-DNA interactions. Furthermore, protein-DNA interactions are sensitive to inhibition by nonspecific DNA intercalators (Lai et al., 1992, Proc Natl. Acad. Sci. USA, 89(15):6958-62).
One family of chemical entities that inhibit HPV replication is disclosed in WO 02/50082 published Jun. 27, 2002. The mechanism of action of these inhibitors was elucidated and they were found to inhibit the E1-E2 interaction by binding to the E2 TAD. We have therefore rationalized that, used as probes, these could be displaced by test compounds that also inhibit or disrupt the E1:E2 interaction, an interaction that is critical for the complex to bind to DNA and proceed with viral replication. Validation of this rationale could be obtained by testing the inhibitors identified in the present assay with a well known E2-dependent E1-DNA binding assay.
The present invention therefore provides a probe and a novel displacement assay for screening for potential inhibitors of papilloma viral replication. Advantageously, this displacement assay of the present invention is easy to use and inexpensive and amenable to adjustments in salt concentration or pH levels. This type of assay is also amenable to a high sensitivity and a high throughput format, and uses a protein that has a low molecular weight, which is easy to purify.
It is a further advantage of the present invention to provide a probe that binds to the transactivation domain of HPV E2 with a high affinity, and which is displaced by inhibitors of HPV.
The present description refers to a number of documents, the content of which is herein incorporated by reference.
SUMMARY OF THE INVENTION
In a first embodiment, the invention provides a probe of formula (I) or its enantiomers or diastereoisomers thereof:
wherein:
A is a 5- or 6-membered homocyclic ring, or a 5- or 6-membered heterocyclic ring containing 1 or more heteroatoms selected from N, O and S;
X is H and W is OH; or X and W together form a carbonyl group or an epoxide;
R
1
is H; or one or two substituents independently selected from the group consisting of: hydroxy, halo, lower alkyl, lower alkoxy, lower thioalkyl, haloalkyl (e.

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