Recent advances in the search for antiviral agents against human papillomaviruses

Abstract

Infection by human papillomavirus (HPV) is extremely common and associated with the development of benign warts or malignant lesions of the skin and mucosa. Infection by a high-risk (oncogenic) anogenital HPV type, most often through sexual contacts, is the starting point of virtually all cases of cervical cancers and the majority of anal cancers. The same viral types are also increasingly being linked with a subset of head-and-neck and non-melanoma skin cancers. Although prophylactic vaccines are now available to protect against the four types most commonly found in cervical and anal cancers (HPV16 and HPV18) and anogenital warts (HPV6 and HPV11), these neither protect against all genital HPVs nor are of therapeutic utility for already infected patients. Thus, the need for antiviral agents to treat HPV-associated diseases remains great, but none currently exist. This article reviews the recent progress made towards the development of antiviral agents to treat HPV infections, from target identification and validation to the discovery of lead compounds with therapeutic potential. Emphasis has been placed on novel low-molecular-weight compounds that antagonize HPV proteins or, alternatively, inhibit cellular proteins which have been usurped by papillomaviruses and are mediating their pathogenic effects.

Figure 1. HPV life cycle and structure of the viral genome

(A) Schematic representation of the human papillomavirus (HPV) life cycle within a differentiating epithelium. The different epithelial strata are indicated on the left. The diagram at the centre represents a prototypical infected keratinocyte undergoing terminal differentiation and harbouring viral episomes within its nucleus. Specific viral life cycle events occurring in each stratum are summarized on the right. (B) The genome of HPV16 is diagrammed in linear form. The coding regions of the early and late viral proteins are indicated by open boxes. Proteins that have been validated as potential antiviral targets are dark grey. The L1 protein, which has been shown to be a valid target for a microbicide, is light grey. The long-control region (LCR), which contains the transcriptional enhancer and promoter regions as well as the origin of DNA replication, is indicated by a two-way arrow.

Figure 2. HPV early proteins as antiviral targets

The HPV early proteins E1, E2, E6 and E7 that have been validated as potential antiviral targets are represented by boxes. The locations of specific functional domains discussed in this review are indicated and their activities listed underneath. Because the length of each protein can vary slightly between viral types, an approximate length in amino acids (aa) is indicated at the right of each protein. DBD, DNA-binding domain; H, hinge region; HDAC, histone deacetylase; NES, nuclear export sequence; NLS, nuclear localization sequence; TAD, transactivation domain; Zn, zinc finger.

Figure 3. Initiation of HPV DNA replication and inhibitors thereof

(A) Schematic representation of the minimal origin of viral DNA replication. Binding sites (BS) for E1 and E2 are indicated by white and black boxes, respectively. The location of an essential AT-rich region (AT) is indicated by a grey box. The nucleotide sequence of part of the HPV18 origin is given underneath. The left portion of this sequence contains a representative E2-binding site (ACCgN₄cGGT) and is the target of E2 polyamides. The right portion of the sequence contains the four E1 binding sites, shown by arrows, which are the target of E1 polyamides. (B) Schematic representation of the initiation of papillomavirus DNA replication. DNA replication is initiated by the recruitment of E1, by E2, to the viral origin. This step is blocked by the indandione class of inhibitors which bind to the E2 transactivation domain and prevent its interaction with E1. Assembly of a replication-competent double hexamer proceeds through the initial assembly of two E1 dimers on the two pairs of inverted E1 binding sites present in the origin. Conversion of this tetrameric E1 intermediate to an active double hexameric helicase requires additional E1 molecules as well as ATP to promote their oligomerization. E1 also interacts with host cell replication factors such as the polymerase α-primase (Pol α) to promote viral DNA replication. Biphenylsulphonacetic acid and other E1 ATPase inhibitors abrogate the helicase activity of E1 responsible for unwinding the origin and the DNA ahead of the two replication forks. All inhibitors are indicated in boxes.

Figure 4. Effects of E7 and E6 on the Rb and p53 pathways and inhibitors thereof

Schematic diagram of how (A) E7 and (B) E6 promote cellular proliferation and inhibit apoptosis, respectively, by targeting the Rb and p53 pathways. The processes of cellular proliferation and apoptosis are indicated by downwards arrows on the left of the figure. E2F and Rb, two major effectors of cellular proliferation and apoptosis, are shown next to these arrows. (A) summarizes the key activities of E7 necessary for unscheduled E2F activation, namely its ability to promote hyperphosphorylation of Rb by inhibiting the cylcin (Cln)-dependent kinase (Cdk)2 inhibitors p21 and p27 and stimulating the activity of Cdk2, to promote the proteasomal degradation of pRb and to associate with histone deacetylases (HDAC). (B) summarizes how E6 of high-risk HPV types promotes the ubiquitination and proteasomal degradation of p53 by associating with the cellular ubiqiutin (Ub) ligase E6-associated protein (E6AP). Specific inhibitors of the Rb pathway and of the E6–E6AP protein interaction are indicated in boxes. P-PCI, purine-derived pharmacological Cdk inhibitors.

Figure 5. E1–E2 interaction inhibitors

(A) Structures of the indandione inhibitors 1, 2, 3 and 4, described in the text. These compounds inhibit the assembly of the HPV11 E1–E2–origin ternary complex in vitro with 50% inhibitory concentrations of 7.8, 0.35, 0.02 and 0.18 μm, respectively. (B) Surface representation of the crystal structure of the HPV11 E2 transactivation domain (TAD; Protein Data Bank accession number: 1R6K). (C) Portion of the crystal structure of the HPV11 E2 TAD in complex with compound 4 (PDB accession number: 1R6N) highlighting how the inhibitor (in red) binds on the surface of E2. In both (B) and (C), key residues of the inhibitor-binding pocket discussed in the text are coloured.

Figure 6. E1 ATPase inhibitors

(A) Structures of the initial biphenylsulphonacetic acid inhibitor (screening hit) and of the most potent optimized compound. These compounds inhibit the ATPase activity of human papillomavirus 6 (HPV6) E1 in vitro with 50% inhibitory concentrations of 2 mM and 4 nM, respectively. (B) Side view of the crystal structure of the hexameric C-terminal helicase domain of bovine papillomavirus (BPV1) E1 bound to ADP (light blue; PDB accession number 2GXA). The monomers are differentially coloured. The right side of the panel shows an enlarged view of the ATP-binding pocket. The conserved lysine of the Walker A motif is coloured purple. The amino acid shown to be important for the binding of the biphenylsulphonic acid inhibitors is coloured yellow.

Figure 7. E6–E6AP interaction inhibitors

(A) NMR structure of the E6-associated protein (E6AP; PDB accession number: IEQX). The positions of the three leucine residues important for binding to E6 as well as those of the two conserved charged residues are indicated by arrows. (B) Structure of the most potent E6–E6AP inhibitor (IC50 of ~17 μM in vitro). (C) Structure of the E6–E6AP inhibitor active in a cell-based p53-degradation assay. This compound leads to a more than 4-fold increase in the levels of p53 when used at a concentration of 500 μM.

Figure 8. Structure of carrageenan

Carrageenan is a polymer of the indicated structure. The average number of repeated units is ~ 500 [189, 192].