ABSTRACT “High-risk” human papillomavirus (HPV) types such as 16 (HPV-16) are identified in the majority of HPV- associated pre-malignant and malignant pathologies of cervical, anogenital, and oropharyngeal epithelia. The E6 protein is essential for viral replication and cellular models of oncogenic transformation. We hypothesized that small molecules that bind to and form a covalent bond with E6 will antagonize its functions, including the ability to bind the ubiquitin ligase E6AP and recruitment of p53 for proteasomal degradation. Structure-based computational screening followed by design and synthesis of derivatives led to the identification of a series of small molecules that interact with and form a covalent bond to the HPV-16 E6 protein and inhibit both E6•E6AP association in vitro and E6-mediated p53 degradation in cells. Time- and concentration-dependent mass spectrometry and high resolution co-crystal structures of four small molecules bound to E6 confirmed this hypothesis. The objective of this grant application is to extend our discovery of novel E6 inhibitor chemotypes using computational, biochemical, crystallographic, pharmacologic and cell biological assays to increase potency and activity. In Aim 1, we combine predictive modeling algorithms with these X-ray structures to instruct modifications that engage additional residues at the E6•E6AP interface. In Aim 2, robust biochemical techniques will characterize the binding and reaction kinetics of these inhibitors. X-ray crystallography will be applied to resolve atomic coordinates of new compounds bound to HPV E6 and thereby guide the structure-based computational designs proposed in Aim 1. In Aim 3, we test the small-molecule E6 inhibitors for their specific ability to restore p53 levels, and induce apoptosis ord senescence using HPV-16 expressing cancer cell lines. Direct engagement of E6 in cells will be investigated and potential off-target cellular proteins will be identified. Our expectation is that 2-3 drug-like candidates will emerge that selectively inhibit HPV-16 E6 function and exhibit sub-micromolar IC50 activity and suitable pharmacologic properties to advance toward first in human clinical trials.