Oncogenic human papillomaviruses (HPV) such as HPV type 16 are the etiologic agent of 50-70% of cancers of the oropharynx. In the USA, the incidence of HPV-associated oropharyngeal cancers is increasing. This is partly because there is no established screening test for oral HPV. HPV-16 DNA is detected by PCR in 1-8% of oral swabs depending on the surveyed populations, with the highest frequencies in HIV+ individuals. Only a small fraction of these infections will progress to frank malignancy. Moreover, early lesions are difficult to locate and patients are largely asymptomatic until they have advanced tumors. Kovina Therapeutics is developing anti-viral therapeutics that selectively bind to the HPV-16 E6 oncoprotein and prevent interactions with its cellular targets. E6 is necessary for viral genome replication and maintenance and is always expressed in HPV-associated dysplasia and malignancies. We performed in silico structural screening of chemical libraries for molecules that fit the E6 docking site for several of its cellular partners. Our assays focused on the E6 protein-protein interaction with E6AP, which transfers ubiquitin onto the tumor suppressor protein p53 and leads to its destruction by the proteasome. We designed and synthesized compounds that 1) bind to HPV-16 E6 and 2) form a covalent bond with a specific cysteine in HPV-16 E6 at its protein-protein interface with E6AP as well as other cellular interacting proteins. The approach to identify molecules equipped with reactive ‘warheads’ that mediate covalent and irreversible binding to cysteine has been successful for several new clinically available drugs, and this field is rapidly progressing to target proteins that were previously considered undruggable. We show that these compounds covalently bind to HPV-16 E6, block E6 interaction with E6AP, restore P53 function, and drive the HPV infected cell to apoptosis. Multiple biochemical analyses including X-ray crystallography prove these inhibitors occupy this E6 pocket and form a single adduct with this cysteine. Iterative optimization guided by the co-crystal data resulted in the design and synthesis of >200 novel chemotypes that resulted in increased activity. This one-year Phase I SBIR proposal will select leads based on for pharmacokinetic characterization. The most promising E6 inhibitors will then be test in vivo proof of concept experiments using human oropharyngeal cancer derived cells in a tumor xenograft model. Successful results in these Phase I experiments will strongly position us for a Phase II SBIR grant to carry out IND-enabling studies.