Elsevier

Antiviral Research

Volume 72, Issue 1, October 2006, Pages 49-59
Antiviral Research

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

https://doi.org/10.1016/j.antiviral.2006.03.014Get rights and content

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.

Introduction

Papillomaviruses are small double-stranded DNA viruses that infect epithelial tissues and cause cutaneous, mucosal and anogenital warts. Genital human papillomavirus (HPV) DNA is detected in 5–20% of persons between the ages of 14 and 50 years and in 10–40% of sexually active women between the ages of 16 and 25 years in the USA (Phelps et al., 1998). Genital warts are highly transmissible and affect all races and socioeconomic groups. Although not a reportable condition, the Division of STD/HIV Prevention of the Centers for Disease Control estimates that there are 750,000 new cases of genital warts each year and 1.5 million persons under treatment. New cases represent only 10% of the ∼7 million individuals in whom HPV causes clinically detectable warts.

There are more than 100 genotypes of HPV, a subset of which are associated with the development of malignant lesions and classified as “high-risk” for their ability to promote cancer. DNA from high-risk HPV has been found in over 95% of cervical cancer cases (Munoz et al., 2003). Approximately, 50% of all cervical cancers contain HPV-16. Other viral genomes, HPV-18, HPV-31 and HPV-45, together comprise another 20% of cervical cancers (Munoz et al., 2003). The low-risk viruses, such as HPV-6 and HPV-11, are found in genital warts but are rarely associated with cervical cancer.

Papillomavirus infection is thought to begin with invasion of the basal epithelium. In undifferentiated basal cells, the viral genome is maintained extra-chromosomally at low copy number. Since the viral protein coding capacity is small, the virus hijacks cellular factors in order to replicate. As daughter cells begin to differentiate and become non-permissive for DNA synthesis, the virus induces the G1- to S-phase transition to initiate synthesis of viral DNA and expresses early viral genes to prevent cellular stress responses such as p53 activation (McMurray et al., 2001). Unscheduled cellular proliferation such as that caused by viral infection is a signal for cell death via apoptosis. The pro-apoptotic protein p53 is central to this cellular defense mechanism by up-regulating expression of apoptotic proteins in response to cellular stress.

The high-risk papillomaviruses have evolved a mechanism to block the p53 response. Papillomaviruses encode eight major proteins with additional products resulting from alternatively spliced mRNAs. The HPV-16 E6 protein complexes with the cellular factor E6AP (E6-associated protein) and forms an ubiquitin ligase that specifically binds to and targets p53 for ubiquitin-mediated degradation (Huibregtse et al., 1991, Scheffner et al., 1994, Scheffner et al., 1993). E6AP does not bind p53 in the absence of E6. HPV genomes encoding E6 mutants that are unable to degrade p53 cannot replicate in skin keratinocytes (Park and Androphy, 2002, Thomas et al., 1999).

In addition to targeting p53 for degradation, the E6 protein plays other roles in promoting viral replication (Underwood et al., 2000). E6 also disrupts cell cycle checkpoints to promote cellular proliferation (Kaufmann et al., 1997, Malanchi et al., 2002, Thompson et al., 1997). Cells expressing E6 have increased telomerase activity that delays cellular senescence (Klingelhutz et al., 1996, Stoppler et al., 1997). E6-induced transcription of the catalytic component of telomerase (hTERT) appears to involve E6AP, which may induce degradation of a repressive factor at its promoter (Gewin et al., 2004, Liu et al., 2005). The HPV E6 proteins also alter the transcriptional pattern of a variety of cellular and viral promoters, which seems to be in large part mediated by its interaction with E6AP (Kelley et al., 2005). E6 thus represents an excellent target for development of antiviral agents.

HPV E6 proteins contain about 150 amino acid residues and two “zinc finger” subdomains (Fig. 1) and function through interaction with cellular factors in addition to E6AP including E6BP/ERC-55, E6TP1, ADA3, IRF-3, Bak, MCM-7, Blk, paxillin, CBP/p300, hDlg and other PDZ domain containing proteins (Fehrmann and Laimins, 2003, Scheffner and Whitaker, 2003, Thomas and Chiang, 2005). For many of the factors, the essential core of the binding region has been delineated and contains a consensus sequence of LxxϕLsh, where L is leucine, s the small amino acid (glycine or alanine), ϕ the hydrophobic residue (usually leucine) and h usually aspartate, asparagine, glutamate, or glutamine and xx is a dipeptide where one of the residues is Asp, Glu, Asn, or Gln (Fig. 1A). The structure of several peptides containing this “charged leucine” E6-binding motif have been determined in the absence of E6 (Be et al., 2001, Chen et al., 1998). This domain forms an alpha helix, with the leucines forming a hydrophobic surface on one face of the helix and the charged amino acids on the opposite face (Fig. 1B). Replacement of any leucine in the binding motif by alanine disrupts binding to E6, as evidenced by the inability of GST-E6 to pull-down binding proteins (Be et al., 2001, Bohl et al., 2000, Chen et al., 1998). Polar residues that reside on the helix opposite the hydrophobic surface contribute to binding, as mutations in the related E6BP or paxillin proteins show loss in binding (Bohl et al., 2000, Chen et al., 1998).

Although the three-dimensional features of E6-binding motif containing peptides and proteins are known, the binding determinants of E6 are not well characterized (Nomine et al., 2006). Some investigators have found that an N-terminal portion of HPV-16 E6 is sufficient for binding, while others have clearly shown single-point mutants in the C-terminal half can disrupt binding to α-helical partners (Lagrange et al., 2005, Liu et al., 1999b, Nguyen et al., 2002). Several point mutants in E6 directly disrupt binding to E6AP (Liu et al., 1999a, Zimmermann et al., 1999). L37S, L50G and Y54D are in the top of the first zinc finger, while Q107R, L110Q, H118D, F125V, I128T, G130V, W132R, G134V are in the second zinc finger. A model of the structure of E6 predicts that all of these residues are buried, suggesting that each mutation disrupts E6 structure instead of modifying the surface properties of the protein (Nomine et al., 2006). Because there is evidence that each zinc finger of E6 represents a separately folded domain (Nomine et al., 2003) and since the functional site of many proteins typically lie at the interface of two domains, the α-helical partner E6AP protein may bind into a pocket formed by both zinc fingers of E6. Such a model is supported by the E6AP-binding mutations in E6 that occur in both zinc fingers, but not in the N- or C-termini of the protein or in the region connecting the zinc fingers (Fig. 1B). Peptides containing the charged leucine helical motif inhibit the interaction between E6 and both E6AP and E6BP, as well as the ability of E6 to promote the degradation of p53 (Bohl et al., 2000, Butz et al., 2000, Elston et al., 1998, Huibregtse et al., 1993, Liu et al., 2004, Sterlinko Grm and Banks, 2004). We hypothesize that non-peptidic compounds can be selected that resemble the structure and functional features of inhibitory peptides to compete with E6AP for binding to HPV-16 E6. Such low molecular weight inhibitors would serve as leads for antiviral agents and help understand the biology of E6.

There are no specific medical treatments targeting papillomavirus-induced diseases. Therapies for cutaneous and genital warts and advanced cases of cervical dysplasia involve destruction or removal of the infected tissue by cytotoxic agents or by surgery (Beutner and Ferenczy, 1997). Prophylactic HPV vaccines have shown promising results in clinical trials (Koutsky et al., 2002, Villa et al., 2005), although there are major challenges to widespread use of a vaccine (Schiller and Davies, 2004). Moreover, a prophylactic HPV vaccine would offer no benefit for the millions of people already infected. Therapeutic nucleic acids that target viral reading frames are also being developed (Alam et al., 2005, DiPaolo and Alvarez-Salas, 2004, Storey et al., 1991). An effective antiviral agent could be used therapeutically to treat papillomavirus infection and would decrease the likelihood of progression to invasive cervical cancer and the spread of virus. In this project, we have discovered a series of lead inhibitors of papillomavirus E6 protein using structure-based approaches.

Section snippets

Calculation of location spheres for pharmacophore points

The pharmacophore model for binding the high-risk papillomavirus E6 protein comprises three lipophilic points, two hydrogen-bonding points, and a space that excludes the presence of atoms (an exclusion sphere). The locations of these points were derived from two peptide structures that bind E6. The analysis used the best structure of the E6AP peptide and the best structure of the E6BP peptide determined by NMR methods in the absence of E6 protein and the known mutagenesis data (Be et al., 2001,

Definition of the E6-binding pharmacophore

Conversion of the 3D NMR structures of E6 inhibitory peptides to 3D queries was needed for database searches that could uncover small, non-peptidic molecules that bind to and inhibit the E6 protein. The points in space assumed necessary for activity were derived by considering the surface of the E6-binding partners available for contact with E6 is primarily composed of side-chain atoms of the polypeptide. The three-dimensional structure determination of E6AP and E6BP peptides provided a

Discussion

The papillomavirus E6 protein is required for viral infection and progression to cancer and therefore is a logical target for antiviral therapy. In this paper, the three-dimensional structures of the E6-binding sequences were coupled with knowledge of the important groups for interaction with E6 to create a pharmacophore for binding to E6. Compounds selected on the basis of resembling the pharmacophore inhibited the activity of E6. Several of these compounds appear to represent excellent

Acknowledgments

This work was supported by NIH grant R01 AI38001 to J.D.B. and E.J.A. J.J.C. was supported in part by the Cancer Research Foundation of America and NIH grant R03 CA92746.

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