The E6AP Binding Pocket of the HPV16 E6 Oncoprotein Provides a Docking Site for a Small Inhibitory Peptide Unrelated to E6AP, Indicating Druggability of E6

Abstract

The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.

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.…”

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

“…These observations indicate that the LxxLL motif structures the p53 binding cleft on E6, thereby rendering E6 competent for interaction with p53. Interestingly, we have shown that an in vitro selected peptide, targeting the LxxLL pocket of HPV16 E6, induces recruitment of p53 to E6 22, 23 . Consequently, cellular proteins other than E6AP, which bind to the LxxLL pocket, might also promote the E6-p53 interaction.…”

“…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.…”

Novel structural approaches concerning HPV proteins: Insight into targeted therapies for cervical cancer (Review)

“…The conclusion was that an inhibitory ligand can bind to E6 protein at its E6AP binding pocket, restore p53 expression and induce apoptosis in HPV16 positive cells. This important finding opens new perspectives for drug development against HPV (51). In a subsequent study, more details were provided concerning the specific interactions of E6, E6AP and p53.…”