Identification and Characterization of Small Molecule Human Papillomavirus E6 Inhibitors

Abstract

Cervical cancer is the sixth most common cancer in women worldwide and the leading cause of women’s death in developing countries. Nearly all cervical cancers are associated with infection of the human papillomavirus (HPV). This sexually transmitted pathogen disrupts the cell cycle via two oncoproteins: E6 and E7. Cells respond to E7-mediated degradation of pRB by upregulating the p53 tumor suppressor pathway. However, E6 thwarts this response by binding to the cellular E6-Associating Protein (E6AP) and targeting p53 for degradation. These two virus-facilitated processes pave the way for cellular transformation. Prophylactic HPV vaccines are available, but individuals already infected with HPV lack drug-based therapeutic options. To fill this void, we sought to identify small molecule inhibitors of the E6–E6AP interaction. We designed an ELISA-based high throughput assay to rapidly screen compound libraries, and hits were confirmed in several orthogonal biochemical and cell-based assays. Over 88,000 compounds were screened; 30 had in vitro potencies in the mid-nanomolar to mid-micromolar range and were classified as validated hits. Seven of these hits inhibited p53 degradation in cell lines with HPV-integrated genomes. Two compounds of similar scaffold successfully blocked p53 degradation and inhibited cell proliferation in cells stably transfected with E6. Together, these studies suggest that small molecules can successfully block E6-dependent p53 degradation and restore p53 activity. The compounds identified here constitute attractive starting points for further medicinal chemistry efforts and development into beneficial therapeutics.

A unique surface on Pat1 C-terminal domain directly interacts with Dcp2 decapping enzyme and Xrn1 5′–3′ mRNA exonuclease in yeast

“…Such a motif is also crucial for the cellular ubiquitin ligase E6AP to interact with the viral oncoprotein E6 from human papilloma virus and then to trigger p53 degradation (46,47). As proapoptotic peptides and small molecules targeting LxxLL pocket of E6 oncoprotein have been recently tested (48)(49)(50), future studies aimed at identifying molecules precluding Dcp2 or Xrn1 HLMs to bind to this fungal-specific Pat1 region could be of great medical interest to develop antifungal drugs.…”

Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors

“…A significant enrichment was seen for active compounds, with 10% of the 554 compounds being active at Ͻ50 M against either wild-type MARV or wild-type EBOV. This represents a 100-fold enrichment over that seen when screening a random library of compounds for antivirals (40,41).…”

Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53

“…Recent studies employing pro-apoptotic peptides 22 as well as small molecules 27, 28 directed against the LxxLL pocket have provided experimental evidence that this pocket is druggable. The p53-binding cleft observed in the present structure may represent a second potential binding site for drugs.…”