Small molecule targeting viral nucleotidyltransferases

在鉴定AHT之后，最初的努力集中在制备天然产物甘露醇的衍生物作为改进的HIV RNA酶H抑制剂。这种化学型通过在RNase H活性位点螯合二价金属来起作用。未能增强效力很可能是由于不能衍生化7元环庚三烯酚酮环，因为X射线晶体学表明甘露醇修饰朝向溶剂，即没有产生新的蛋白质接触。与布鲁克林学院的Ryan Murelli博士发起了一项合作，该小组对从头合成AHT感兴趣。这项合作导致了70种新型AHT的制备，这些AHT在环酚酮环的多个位置被取代。因此，我们已经确定了新的AHT，在生物化学试验中对HIV-1 RNase H具有活性，IC 50为50 nM。虽然在抑制HIV复制方面具有活性，但治疗指数为10，这表明存在一些脱靶效应，并且正在计划将光亲和性连接到我们最有希望的命中模块以识别其他靶标。与此同时，我们的兴趣已经扩展到临床重要病毒的相关NTA酶是否受到AHT抑制。一个靶点是HBV DNA聚合酶的RNase H活性，我们的合作者已经证明甘露醇确实抑制HBV复制。疱疹病毒编码几种对复制至关重要的NT酶。此外，α-（HSV）β-（HCMV）和γ-疱疹病毒（KSHV）的NT酶显示出显著的保守程度，表明可能开发出“泛疱疹”抑制剂。然而，由于KSHV是一种潜伏性感染，因此靶向关键病毒酶的小分子仅在从潜伏期重新激活的病毒的情况下有效。基于ZIA BC 101493项目的发现，我们相信我们现在手头上有一种诱导KSHV裂解再活化的分子，从而支持开发“踢杀”策略的可能性。作为朝着这个方向迈出的第一步，我们与北卡罗来纳大学查佩尔山的开花达马尼亚博士发起了合作，他将针对KSHV测试我们的AHT库。在稍后的阶段，其中最有效的将与我们的延迟激活器结合使用。由于疱疹病毒编码一种以上的NTase，我们的基础研究策略涉及确定病毒的目标。为此，并基于它们的结构相似性，我们已经检查了重组HSV-1 pUL 15 C（处理多联体病毒基因组的末端酶分子机器的组分）和pUL 12（具有外切酶和内切酶活性的磷蛋白，对复制至关重要）的AHT抑制。由于目前用于检测疱疹病毒核酸酶的方法涉及琼脂糖凝胶电泳，我们开发了一种“用户友好”的高通量双探针荧光测定法，其包括在末端具有供体/猝灭剂对的DNA发夹。此外，由于不能排除低水平的核酸酶污染，我们开发了一种互补的热变性试验（HTS-thermofluor），该试验评估了小分子配体存在下蛋白质稳定性的变化。酶促测定鉴定了在低纳摩尔范围内抑制pUL 15 C的AHT，这与配体稳定对抗热变性的能力非常强相关。然而，尽管生物学测试鉴定了几种高效HSV-1和HSV-2抑制剂，但与其生物化学活性几乎呈负相关，表明末端酶不是生物学靶标。随后，我们从康涅狄格大学的Sandra Weller小组获得了重组HSV pUL 12，并使用我们的双探针荧光测定法显示，这种酶也对AHT抑制敏感。令人欣慰的是，生物活动与生物化学活动密切相关。提示HSV pUL 12是生物学靶标。我们的工作最初集中在HSV酶上，主要是基于它们易于纯化。然而，已经证明pUL 12作为生物靶标，我们计划纯化和表征KSHV等价物pUL 37。虽然用AHT靶向KSHV pUL 37作为我们提出的“踢-杀”策略的一部分，但还应该指出的是，（i）AHT抑制HSV-1和HSV-2复制的效率几乎是阿昔洛韦的100倍，（ii）AHT对阿昔洛韦耐药性HSV-1和HSV-2有活性，以及（iii）我们的合作者无法选择耐药性HSV变体。我们对AHT介导的疱疹病毒复制抑制的研究为药物“再利用”的概念提供了一个很好的例子。基础研究也在继续，目标是获得HSV pUL 12和/或KSHV pUL 37的高分辨率晶体结构。最后，使用固定化pUL 37的新策略将被用于通过亲和捕获来鉴定除了靶向活性位点的配体之外的配体，其优点在于该策略可以应用于天然产物提取物并且可以独立于酶测定来进行。

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.