Covalent Ligands that Target the Human Papillomavirus E6 Oncoprotein

The high-risk human papillomavirus (HPV) early protein 6 (E6) viral oncoprotein is a key player in HPV-mediated cancers and is the major driver of cervical, anal, and a substantial portion of head and neck cancers. HPV E6 recruits the cellular ubiquitin E3-ligase E6-associated protein (E6AP), also termed UBE3A, and this E6/E6AP complex in turn recruits the tumor suppressor p53, resulting in the ubiquitin-mediated degradation of p53. The cysteine-rich E6 protein presents an attractive target for chemical intervention through covalent, cysteine-reactive chemistries. While previous studies have uncovered peptidic covalent inhibitors of E6, we sought to determine whether we could identify nonpeptidic functional binders. We screened a library of cysteine-reactive covalent ligands and identified a pyrazoline hit, EN218, bearing a chloroacetamide cysteine-reactive warhead. Evaluation of pyrazoline analogs led to the development of an improved molecule, (S)-WX48A, that stereoselectively reacted with C58 on E6 to inhibit E6-mediated p53 ubiquitination. While this molecule is still in an early stage and requires further medicinal chemistry optimization to exert on-target cellular activity, our study demonstrates that E6 can be directly and stereoselectively targeted with a covalent nonpeptidic small-molecule and provides a foundation for developing more drug-like E6 inhibitors for HPV therapy.