Pharmacology of HIV Viral DNA Retroviral Integrases

整合酶（IN）由HIV原病毒Pol基因编码，是一种高效表达的重组活性蛋白。我们的实验室是整合酶抑制剂研究领域的先驱（PNAS 1993），发现了几个先导抑制剂家族（Nature Rev Drug Discovery 2005; Current Topics in Medicinal Chemistry 2009; Viruses 2010; Adv Pharmacol 2013），证明了in抑制剂作为界面抑制剂（Nature Rev Drug Discovery 2012），以及用于治疗开发的专利化合物。我们目前的研究重点是发现新的化学型整合酶抑制剂，以克服对雷替格拉韦的耐药性和靶向新的IN位点。我们已经发现了来自短Vpr肽的新型化学型，它们作为3'加工和链转移抑制剂。我们已经证明它们可以作为抗病毒药物，通过添加一个多精氨酸尾部来授予细胞摄取。长期目标是构建这些Vpr肽的非肽衍生物。我们还与药物化学实验室（CCR， NCI）的Terrence Burke博士合作，发表了新的合成化学型，如IN链转移抑制剂（iniss），包括酞酰亚胺和喹啉基衍生物，并获得了专利。为了进行这些实验，我们开发了一组重组IN蛋白，这些蛋白携带在对雷替格拉韦、依替格拉韦和多替格拉韦产生耐药性的患者中观察到的突变。利用我们的一组耐药IN突变体，我们表征了elvitegravity， dolutegravir和我们的新型抑制剂的分子药理学，并将它们与raltegravir进行比较。我们已经证明，雷替重力韦、艾替重力韦、多替重力韦和我们的新系列药物对链转移反应具有高度选择性，而对3'加工反应的效力要低100倍以上，对整合酶介导的分解反应几乎没有活性。对链转移（整合酶介导的3种反应之一）的选择性活性表明临床开发的IN链转移抑制剂（iniss）具有非常高的特异性。这与我们的药理学假设（Nature Drug Discovery 2012）一致，即链转移抑制剂通过螯合病毒DNA 3'加工后酶催化位点上的二价金属来捕获in -病毒DNA复合物，并与我们的共晶结构和分子模型数据一致。我们已经描述了赋予临床耐药性的IN突变体的生化酶活性和药物敏感性。我们已经将这些研究扩展到整合酶柔性环的双突变体，这些突变体通常出现在替地韦耐药患者中。目前的假设是，第二次突变作为功能的获得，以挽救因第一次突变而出现缺陷的IN的生化活性。目的之一是了解这种互补的分子机制以及柔性环、病毒和宿主dna以及抑制剂之间的结构联系。我们发现，柔性环双突变体140S-148H对雷替重力韦和韦替重力韦都有交叉抗性，但对多替重力韦和我们的一些新衍生物的抗性要低得多。另一方面，143Y突变体主要对雷替重力韦耐药，对利替重力韦和多替重力韦耐药最少。这些结果为由于143Y突变（而不是140S-148H突变）而对雷替格拉韦产生耐药性的患者使用韦替格拉韦提供了理论依据。我们的研究结果支持多替重力韦在克服对雷替重力韦和韦替重力韦的耐药性和促进患者依从性方面的价值。为了阐明IN抑制剂效力和合理设计的结构基础，我们测定了野生型和突变型原型泡沫病毒（PFV）与药物结合的内体的晶体结构。这项工作是在伦敦克莱尔霍尔癌症英国中心（不久将转移到克里克研究所）与Peter Cherepanov博士合作完成的。结构上适应与耐药相关的结构变化的能力似乎是一种可取的特性，可以用于我们的新inis的开发。我们的研究是我们与Terrence Burke博士（化学生物学实验室，CCR-NCI）、Stephen Hughes博士（同样在NCI-Frederick实验室（HIV耐药性项目））和Peter Cherepanov博士在伦敦长期合作的结果。

Integrase (IN) is encoded by the Pol gene from the HIV provirus and can be efficiently expressed as an active recombinant protein. Our laboratory has pioneered the integrase inhibitors research field (PNAS 1993), discovered several families of lead inhibitors (Nature Rev Drug Discovery 2005; Current Topics in Medicinal Chemistry 2009; Viruses 2010; Adv Pharmacol 2013), demonstrated that IN inhibitors act as interfacial inhibitors (Nature Rev Drug Discovery 2012), and patented compounds for therapeutic development. Our current studies are focused on the discovery of novel chemotype integrase inhibitors to overcome resistance to raltegravir and target novel sites of IN. We have discovered novel chemotypes derived from short Vpr peptides, which act as 3'-processing and strand transfer inhibitors. We have shown they could serve as antivirals by adding a poly-arginine tail to confer cellular uptake. A long-term goal is to build non-peptidic derivatives of those Vpr peptides. We have also published and patented novel synthetic chemotypes as IN strand transfer inhibitors (INSTIs) including phtalimide and quinolinonyl derivatives in collaborations with Dr. Terrence Burke, Laboratory of Medicinal Chemistry (CCR, NCI). To perform these experiments, we have developed a panel of recombinant IN proteins bearing the mutations observed in patients that develop resistance to raltegravir, elvitegravir and dolutegravir. Using our set of resistant IN mutants, we have characterized the molecular pharmacology of elvitegravir, dolutegravir and our novel inhibitors, comparing them to raltegravir. We have shown that raltegravir, elvitegravir, dolutegravir and our novel series are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3'-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed IN strand transfer inhibitors (INSTIs). It is consistent with our pharmacological hypothesis (Nature Drug Discovery 2012) that the strand transfer inhibitors trap the IN-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3'-processing of the viral DNA and with our co-crystal structure and molecular modeling data. We have characterized the biochemical enzymatic activities and drug sensitivities of the IN mutants that confer clinical drug resistance. We have expanded these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. The working hypothesis is that the second mutation acts as gain of function to rescue the biochemical activity of IN after it had become defective by the presence of the first mutation. One of aims is to understand the molecular mechanisms of such complementation and the structural connections between the flexible loop, the viral and host DNAs, and the inhibitors. We found that the flexible loop double-mutant 140S-148H is cross-resistant to both raltegravir and elvitegravir but much less to dolutegravir and to some of our new derivatives. On the other hand, the 143Y mutant is primarily resistant to raltegravir and minimally resistant to elvitegravir and dolutegravir. These results provide a rationale for using elvitegravir in patients that develop resistance to raltegravir due to mutation 143Y (but not in the case of mutations 140S-148H). Our results support the value of dolutegravir to overcome resistance to raltegravir and elvitegravir and facilitate patient compliance. To elucidate the structural basis for the potency and rational design of IN inhibitors, we determined crystal structures of wild-type and mutant prototype foamy virus (PFV) intasomes bound to drugs. This work has been done in collaboration with Dr. Peter Cherepanov at the Clare Hall Cancer UK Center in London (soon moving to the Crick Institute). The ability to structurally adapt to the structural changes associated with drug resistance appears to be a desirable characteristic that could be used in the development of our new INSTIs. Our studies are the result of our long-term collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI), with Dr. Stephen Hughes, also at the NCI-Frederick Laboratory (HIV Drug Resistance Program), and with Dr. Peter Cherepanov in London.