Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

Abstract

The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.

Fig 1. The unique topography of the α-helix binding groove of HPV-16 E6 is essential in maintaining strong polar contacts with the E6 binding motif.

Introduction

There is no effective medical therapy for women and men infected with human papillomavirus (HPV). Persistent infection with specific HPV types carries a high risk of progression from pre-malignant to invasive and metastatic cervical, anogenital and oropharyngeal cancers [12]. The prototype HPV associated with “high-risk” of neoplastic transformation is HPV-16, which accounts for ~50% of all cervical cancers across the world [34]. The HPV E6 protein is essential for viral replication and instrumental in bypassing host cell defenses and preventing apoptosis [56]. The high risk E6 protein binds to the HECT domain ubiquitin ligase, E6AP/UBE3A and this complex is responsible for ubiquitinylation of the p53 protein, a major suppressor of tumorigenesis, resulting in its degradation by the cellular proteasome [78]. This effect can be reversed in HPV-driven tumor cells by reduced expression of HPV E6, which reactivates p53 expression and leads to senescence or apoptosis [911]. Additional cellular factors interact with HPV E6 and may be targeted for degradation [1213]. These significant activities make E6 a compelling target for the treatment of HPV-associated infections.

Peptide ligands for the E6 hydrophobic groove that were derived from the alpha-helical LXXLL motif of E6AP have been characterized previously. While comparatively low in binding affinities, these peptides are able to disturb E6/E6AP interaction [1415]. A chimeric protein that contains the LXXLL motif and the PDZ motif displays much higher binding affinity when compared to the LXXLL motif alone [1617]. Interestingly, a novel small peptide unrelated to the E6AP binding motif, also inhibits E6 function by blocking the interaction of E6 with E6AP [1819]. Additional peptide-based protein-protein interaction inhibitors (PPI) have achieved some partial successes [2021].

The LXXLL containing alpha-helix binds in a hydrophobic pocket of E6 [22]. In this regard, the specific interface of the HPV-16 E6 protein with E6AP and its other LXXLL binding partners presents significant opportunities for targeting with small molecules (Fig 1). Despite the large overall surface area (902–1005 Å) for the groove, we and others have previously identified flavonoid derived compounds as E6 inhibitory compounds [2327]. A limited structure-activity study identified a tetrazole-substituted benzopyranone analog that antagonized HPV-16 E6 in vitro and had effective IC50 values in the lower micromolar range [25]. Initial molecular modeling studies showed that these compounds could bind within the hydrophobic groove of the E6 protein that has been shown to contact E6AP [25]. The motivation for the current study was to understand the contribution of the 2’-6’ substitution groups on the benzopyranone scaffold and to determine what features of E6 are involved. We observed increased inhibitory activity of small molecules with charged groups at position 6 as well as a higher activity for compounds with non-polar substituents at the 2 position. Based on these results, a subset of analogs was selected to probe E6 binding interactions. Molecular dynamics simulations of the HPV-16 E6 protein implicated a high degree of flexibility of charged residues along the helix groove that could dominate small molecule interactions. A focused set of mutations at these amino acids revealed important roles in defining the molecular interactions of the E6 hydrophobic groove and offered insights for future structure-guided ligand design.

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