An increase in the understanding of the molecular events affected by the HPV E6 and E7 oncoproteins will provide novel information for identifying new drug targets. Furthermore, solving structures of these oncoproteins with their cellular targets can aid in structure-based drug design efforts. Small molecules can become promising in inhibiting key viral processes in infected cells and become useful therapeutically. A combination of small molecules that target various affected pathways, could lead to a better "cocktail" that would destroy infected cells more effectively. Of course, the hope is that such cocktails do not have side effects.
Our identification of small molecule inhibitors against the HPV E6 and E7 oncoproteins provides a starting point for designing more potent inhibitors that can be useful to treat HPV- mediated pathologies. Of course, work still needs to be done to determine how stable our compounds are in cells, and how well they are distributed. Additional screens against other cellular pathways in HPV positive cells, may also result in a more potent and effective drug combination. Such small molecule drugs, in combination with the vaccines available, may help reduce the burdens of HPV-induced carcinomas.
APPENDIX A
Structure-Based Identification of Human Papillomavirus E7 Inhibitors Using an In Silico Approach
A.1 Introduction
As described in Chapter 2, E7 can disrupt complexes formed between pRb and E2F, even in the absence of cell-cycle regulatory mechanisms, such as pRb phosphorylation.
Therefore, in order to prevent uncontrolled cell-cycle progression, it would be useful to find small molecules that bind to and inhibit the function of E7. As described in Chapter 2, a solution high- throughput screen was done that led to the identification of a class of small molecules, called thiadiazolidinediones, that bind to pRb to prevent E7 binding and E2F displacement. We also sought other methods, in parallel, to identify E7 inhibitors, again with an ability to prevent E2F displacement from pRb. More specifically, we decided to perform an in silico screen to identify inhibitors that bind directly to E7. Interestingly, Roughley et al. had performed a combination of virtual screening and solution screening against Hsp90, which resulted in potent compounds that reached clinical trials (Roughley et al., 2011). Therefore, combining our results from solution and virtual screening may reveal more potent methods of inhibition against E7.
Virtual screening has been used successfully by a number of other groups to identify small molecule inhibitors against enzymes, such as p300/CBP histone acetyltransferase, BRAF kinase, CK2 kinase, and thymidylate synthase-dihydrofolate reductase (Bowers et al., 2010; Dasgupta et al., 2009; Luo et al., 2008; Martucci et al., 2009; Roughley et al., 2011;
Vangrevelinghe et al., 2003; Vinh et al., 2012). In each case, the inhibitors bound the active site and had IC50 or Ki values ranging from the low nanomolar to the tens of micromolar range.
Generally, it is more challenging to target protein-protein interactions than to target enzymes with small molecules. A number of virtual screens have been carried out with some success by other groups to inhibit protein-DNA or protein-protein interactions. Still, results indicate that IC50 values for disrupting protein-DNA or protein-protein interactions are generally higher than those for disrupting enzymatic activity. Inhibitors that bind to Epstein Barr Virus EBNA1 and prevent it from binding DNA were identified by using the programs DOCK, Xscore, SLIDE, and Autodock (Li et al., 2010). These inhibitors were shown to have inhibition in the 20-
500µM concentration range in in vitro assays and were able to inhibit activity in cell-based assays. A different virtual screening approach, using the FLAP software, was used to identify inhibitors that disrupt binding of the PB1 and PA subunits of the influenza A virus polymerase (Muratore et al., 2012). In this case, the IC50 values for disrupting the protein-protein interaction were in the range of 25µM to 200µM. One of the compounds from this screen was found to act as a potent replication inhibitor. The results from these virtual screens indicate that it is possible to disrupt protein-protein interactions with small molecules identified through in silico approaches, thereby providing a motivation to identify inhibitors that bind HPV E7 and prevent its interaction with pRb or E2F.
In each virtual screening case, a crystal structure of the enzyme with a substrate, or the protein-protein or protein-DNA complex, had been solved and the structure of the protein was used as a receptor to search for small molecules. In our case, virtual screening was facilitated by the fact that the X-ray crystal structure of the CR3 domain of HPV 1A E7 had been determined (Liu et al., 2006a). This domain is important because it is thought to destabilize the interaction between pRb and E2F and contribute to its disruption. The X-ray crystal structure of the E7 CR3 domain revealed that the CR3 domain forms an obligate zinc homodimer with two conserved surface patches. Mutation of residues within these patches showed that one patch is required for pRb binding and the other is required for E2F binding (Figure A.1) (Liu et al., 2006a).
Since there are no co-crystal structures of the CR3 domain of E7 with either pRb or E2F, the mutational studies that identified the E2F and pRb binding patches were important. These patches on E7 could act as hot spots and could be good targets for small molecules that may prevent protein-protein interactions. Since the pRb binding patch is shallow, it did not seem to be a good target for small molecule inhibitors. The E2F binding patch, on the other hand, forms a pronounced groove which could be a good drug target (Figure A.1). Of note, residues Arg-60 and Leu-61 in the E2F binding region are highly conserved among the E7 proteins. Furthermore, a sequence alignment of E7 proteins showed a high degree of sequence conservation within the CR3 region with greater than 40% identity and 60% homology between proteins, suggesting that
all E7-CR3 regions adopt the same dimeric structure. Consequently, we believed that using the E2F binding pocket in the CR3 domain from HPV 1A E7 would be a good target for small molecule inhibitors against various HPV types.
Figure A.1. Structure of the CR3 domain of HPV1A-E7. Highlighted in red are patches shown through mutagenesis to disrupt either E2F or pRb binding. Dark and light green represents highly and partially conserved residues, respectively. Residues that were mutated for analysis are highlighted. This figure has been adapted from (Liu et al., 2006a).