Small-Molecule Covalent E6 Antagonists for Treatment of HPV Infection

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.

Grant Number: 5R01CA252715-05
NIH Institute/Center: NIH
Principal Investigator: ELLIOT ANDROPHY