Small Molecule E6 Inhibitors to Treat Dysplasia Caused by HPV Infections

点击翻译按钮获取中文摘要

Human papillomaviruses (HPV) cause exceedingly common infections of cutaneous and mucosal epithelia. Infection with specific “high risk” HPV genotypes can progress to pre- malignant lesions called dysplasias, which over a period of years, can eventuate in invasive and metastatic epithelial malignancies. Our therapeutic goal is to treat early stage infections of the cervix, genitalia, and anus that afflict millions of women and men before these progress to invasive cancers. Kovina Therapeutics strategy is to eliminate HPV infection by selectively targeting the E6 protein and preventing interactions with its cellular binding partners. E6 is necessary for viral genome replication and maintenance and is always expressed in HPV-associated dysplasia and malignancies. We performed in silico screening of chemical libraries for small molecules that would resemble the interface of E6 with several of its cellular partners. Our assays focused on the protein-protein interaction of E6 with the ubiquitin ligase E6AP, which mediates ubiquitination and proteasome mediated destruction of the tumor suppressor protein p53. Using this model, we designed and synthesized compounds that 1) bind to HPV-16 E6 and 2) are armed with a ‘warhead’ to make a covalent bond with a specific cysteine in HPV-16 E6 binding pocket for E6AP. The approach to identify molecules equipped with reactive warheads that mediate covalent and irreversible binding to cysteine is exemplified in several new clinically available drugs that target proteins previously considered undruggable. We discovered several compounds covalently bound to E6 via this cysteine residue, block E6 interaction with E6AP, restore P53 function, and induce death of HPV-16 expressing cells. Multiple biochemical analyses including X-ray crystallography prove these inhibitors occupy the targeted E6 pocket and form a single adduct with this cysteine. Iterative optimization guided by the co-crystal data enabled design and synthesis of >80 novel compounds and has resulted chemotypes with increased activity. This one-year Phase I SBIR proposal plans to screen our existing E6 inhibitor collection to determine their biological activities in cell-based assays and pharmacokinetic properties that would meet FDA guidelines for a topical agent. We will initiate drug development studies including formulation for topical application and early stage pharmacokinetic and toxicology characterization. The success of Phase I study will validate this antiviral strategy and position us to proceed to apply for a Phase II SBIR to perform IND-enabling studies.