Identification of inhibitors to papillomavirus type 16 E6 protein based on three-dimensional structures of interacting proteins

Abstract

Human papillomaviruses (HPV) cause cutaneous and genital warts. A subset of HPV types is associated with a high-risk for progression to malignancy. The E6 protein from the high-risk HPV types represents an attractive target for intervention because of its roles in viral propagation and cellular transformation. E6 functions in part by interaction with human cellular proteins, several of which possess a helical E6-binding motif. The role for each amino acid in this motif for binding E6 has been tested through structure determination and site-directed mutagenesis. These structural and molecular biological approaches defined the spatial geometry of functional groups necessary for binding to E6. This E6-binding information (the E6-binding pharmacophore) was transferred into a three-dimensional query format suitable for computational screening of large chemical databases. Compounds were identified and tested using in vitro and cell culture-based assays. Several compounds selectively inhibited E6 interaction with the E6-binding protein E6AP and interfered with the ability of E6 to promote p53 degradation. Such compounds provide leads for the development of new pharmacologic agents to treat papillomavirus infections and their associated cancers.

Figure 1

E6-binding structures in sequence and three-dimensional space. (A) Representative amino acid sequences shown to be necessary and sufficient for binding to HPV-16 E6. Sequence comparison among several of the E6-interacting proteins including E6AP, reveals a consensus sequence, LxxϕLsh, where L indicates leucine residues, ϕ is a hydrophobic residue, h is an amino acid residue with a side-chain capable of accepting hydrogen bonds (Asp, Glu, Asn, or Gln), s represents a small amino acid residue (Gly or Ala) and xx is a dipeptide where one of the residues is Asp, Glu, Asn, or Gln. (B) Representative structure of the E6-binding motif (Be et al., 2001) shown interacting with a schematic representing the two zinc fingers of E6. Side-chain positions of the amino acid residues essential in the motif for binding E6 are indicated by spheres. Published single-amino acid mutations of E6 that disrupt binding are in square boxes whereas mutations that retain binding are circled (Liu et al., 1999a; Nomine et al., 2006; Zimmermann et al., 1999). The E6-binding motif appears to bind to a structure formed between the two zinc finger-like structures of E6.

Figure 2

Pharmacophore description. The pharmacophores for binding the high-risk papillomavirus E6 protein comprises 3 lipophilic points (lip.), 2 hydrogen-bonding points (CO2H), and one exclusion sphere. The locations of these points were derived from two peptide structures that bind E6 (A, E6AP; and B, E6BP). The radii of the location spheres are calculated from a molecular dynamics simulation of the peptide structures.

Figure 3

Hits from searching the NCI database using the E6-binding pharmacophore. The compounds are: 1, NSC83143 (CAS 23870-53-3); 2, NSC117907 (CAS 55811-23-9); 3, NSC135098; and 4, NSC216029.

Figure 4

Hits from searching the Sigma-Aldrich database using the E6-binding pharmacophore. The compounds, all from Aldrich, are: 5, s327301; 6, 207721 (CAS 4680-78-8); 7, r218634; 8, r225975; 9, r278319; and 10, s204102.

Figure 5

Representative binding curves for (A) Compound 2, and (B) Compound 9. Experimental data for binding to E6 are depicted by open circles and by closed squares for anti-FLAG. The best fits of the data are indicated by the lines through the data points.

Figure 6

Inhibition by Compound 2 of E6-mediated degradation of p53 in cultured cells. NIKS-E6 cells were treated with MMC and then the compound at 500 μM. Total cell extracts were prepared. (A) p53 levels were determined by Western blot analysis. Tubulin was used as a loading control. (B) Quantitation of p53 levels in NIKS-E6 cells after treatment with Compound 2 The p53 levels in DMSO-treated cells were normalized to 1. Data represent the mean and standard deviation of seven experiments.