Following identification of AHTs, initial efforts have focused on preparing derivatives of the natural product manicol as improved HIV RNase H inhibitors. This chemotype functions by chelating divalent metal at the RNase H active site. The failure to enhance potency most likely arose from the inability to derivatize the 7-membered tropolone ring, as X-ray crystallography indicated the manicol modifications there oriented towards solvent, i.e. made no new protein contacts. A collaboration was initiated with Dr. Ryan Murelli, Brooklyn College, whose group was interested in de novo AHT synthesis. This collaboration has resulted in the preparation of 70 novel AHTs substituted at multiple positions of the tropolone ring. As a result, we have identified novel AHTs that are active against HIV-1 RNase H in biochemical assays with an IC50 of 50 nM. Although active in inhibiting HIV replication, the therapeutic indices are 10, suggesting some off-target effects, and plans are underway to attach a photo-affinity to our most promising hit module to identify other targets. At the same time, our interests have extended to whether related NTAses of clinically-significant viruses are subject to AHT inhibition. One target is the RNase H activity of HBV DNA polymerase, where our collaborator has demonstrated that manicol indeed inhibits HBV replication. Herpesviruses encode several NTases that are critical for replication. Moreover, the NTases of alpha- (HSV) beta- (HCMV) and gamma-herpesviruses (KSHV) show a remarkable degree of conservation, suggesting a pan-herpes inhibitor might be developed. However, since KSHV is a latent infection, small molecules targeted to critical viral enzymes are only effective in the context of a virus that is reactivated from latency. Based on the findings of project ZIA BC 101493, we believe we now have a molecule on hand that induces KSHV lytic reactivation, thereby supporting the possibility of developing a kick-and-kill strategy. As a first step in this direction, a collaboration has been initiated with Dr. Blossom Damania, UNC Chapel Hill, who will test our library of AHTs against KSHV. At a later stage, the most potent of these will be used in combination with our latency activator. Since herpesviruses encode more than one NTase, our basic research strategy has involved determining the viral target. To this end, and based on their structural similarity, we have examined AHT inhibition of recombinant HSV-1 pUL15C (a component of the terminase molecular machine that processes the concatameric viral genome) and pUL12 (a phosphoprotein with both exo- and endonuclease activity and is critical for replication). Since current methods for examining herpesvirus nucleases involved agarose gel electrophoresis, we developed a user-friendly high throughput dual-probe fluorescence assay comprising a DNA hairpin with a donor/quencher pair at the termini. Also, since low-level nuclease contamination could not be ruled out, we developed a complimentary thermal denaturation assay (HTS-thermofluor) which assesses changes on protein stability in the presence of a small molecule ligand. Enzymatic assays identified AHTs that inhibited pUL15C in the low nanomolar range, which correlated very strongly with the ability of the ligand to stabilize against thermal denaturation. However, although biological testing identified several highly potent HSV-1 and HSV-2 inhibitors, there was an almost inverse correlation with their biochemical activity, suggesting terminase was not the biological target. Subsequently, we received recombinant HSV pUL12 from the group of Sandra Weller at the University of Connecticut and showed, using our dual probe fluorescence assay, that this enzyme was also susceptible to AHT inhibition. Gratifyingly, biological activity tracks well with biochemical activity. Suggesting HSV pUL12 is the biological target. Our work has initially focused on HSV enzymes, based primarily on their ease of purification. However, having demonstrated pUL12 as the biological target, we plan to purify and characterize the KSHV equivalent, pUL37. While targeting KSHV pUL37 with AHTs as part of our proposed kick-and-kill strategy, it should also be pointed out that (i) AHTs inhibit HSV-1 and HSV-2 replication almost 100-fold more efficiently than acyclovir, (ii) AHTs are active against acyclovir-resistant HSV-1 and HSV-2 and (iii) our collaborators have been unable to select a drug-resistant HSV variant. Our studies with AHT-mediated inhibition of herpesvirus replication provides a good example of the concept of drug re-purposing. Basic research studies are also continuing with the goal of obtaining a high-resolution crystal structure of HSV pUL12 and/or KSHV pUL37. Finally, a novel strategy using immobilized pUL37 will be used to identify ligands other than those targeting the active site by affinity capture, that advantage of which is that this strategy can be applied to natural product extracts and can be performed independent of an enzymatic assay.
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