Pharmacology of HIV Viral DNA Retroviral Integrases

整合酶（IN）由HIV前病毒的Pol基因编码，并且可以有效地表达为活性重组蛋白。我们的实验室开创了整合酶抑制剂研究领域（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），并获得了用于治疗开发的专利化合物。我们目前的研究集中在发现新的化学型整合酶抑制剂，以克服对雷特格韦的耐药性和靶向IN的新位点。我们已经发现了源自短Vpr肽的新化学型，其充当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，通过添加聚精氨酸尾巴赋予细胞摄取。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，发表了新型合成化学型作为IN链转移抑制剂（INSTI），包括邻苯二甲酰亚胺和喹啉酮衍生物并获得专利。为了进行这些实验，我们开发了一组重组IN蛋白，其携带在对雷特格韦、埃替格韦和度鲁特韦产生耐药性的患者中观察到的突变。使用我们的一组耐药IN突变体，我们表征了elvitegravir，dolutegravir和我们的新型抑制剂的分子药理学，并将它们与雷特格韦进行比较。我们已经表明，雷特格韦、埃替格韦、度鲁特韦和我们的新系列对链转移反应具有高度选择性，而对3 '-加工反应的效力低100倍以上，并且对整合酶介导的崩解反应几乎无活性。对链转移（由整合酶介导的3种反应之一）的选择性活性证明了临床开发的IN链转移抑制剂（INSTIs）的高度特异性。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在病毒DNA的3 '-加工后螯合酶催化位点中的二价金属来捕获IN-病毒DNA复合物，并与我们的共晶体结构和分子建模数据一致。我们已经表征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中的双突变体，这些突变体通常出现在雷特格韦耐药患者中。工作假设是，第二个突变作为功能的获得，以挽救IN的生化活性后，它已成为有缺陷的第一个突变的存在。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140 S-148 H对雷特格韦和埃替格韦都具有交叉耐药性，但对度鲁特韦和我们的一些新衍生物的耐药性要低得多。另一方面，143 Y突变体主要对雷特格韦耐药，对埃替格韦和度鲁特韦耐药最低。这些结果为在由于突变143 Y（但不是突变140 S-148 H）而对雷特格韦产生耐药性的患者中使用埃替格韦提供了依据。我们的研究结果支持度鲁特韦的价值，以克服耐药性雷特格韦和elvitegravir和促进患者的依从性。为了阐明IN抑制剂的效力和合理设计的结构基础，我们确定了与药物结合的野生型和突变型原型泡沫病毒（PFV）的晶体结构。这项工作是与位于伦敦的英国克莱尔霍尔癌症中心的彼得·切列帕诺夫博士合作完成的（不久将转移到克里克研究所）。结构上适应与耐药性相关的结构变化的能力似乎是一个理想的特征，可以用于我们的新INSTI的发展。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（也在NCI-Frederick实验室（HIV耐药性计划））以及伦敦的Peter Cherepanov博士长期合作的结果。

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.