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）和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来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。我们与明尼苏达大学药物设计中心的王正强博士和Robert Vince博士一起报道了新的insis化学型。与Seth Cohen博士（USCD化学与生物化学系）一起，我们探索了金属结合基团作为IN金属酶抑制剂的作用。为了进行这些实验，我们开发了一组重组蛋白，携带在对雷替格拉韦和依替格拉韦产生耐药性的患者中观察到的突变。利用我们的一组抗雷替重力突变体，我们表征了elvitegravavir、dolutegravavir和MK-0536的分子药理学，并将它们与雷替重力进行比较。我们已经证明，雷替重力韦和韦替重力韦对链转移反应都有很高的选择性，而对3？对整合酶介导的分解反应几乎无活性。对链转移（整合酶介导的3种反应之一）的选择性活性表明，临床开发的链转移抑制剂具有非常高的特异性。这与我们的药理学假设（Nature Drug Discovery 2012）是一致的，即链转移抑制剂通过螯合酶催化位点的二价金属来捕获in -病毒DNA复合物。-处理病毒DNA。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elvitegravir的价值，它与Quad药丸中的其他3种药物（可比司他提高elvitegravir的药代动力学和两种逆转录酶抑制剂，恩曲他滨和替诺福韦）联合使用。吉利德Quad药丸于2012年5月获得FDA咨询委员会的支持，被批准为每日一次的HIV单片治疗方案。dolutegravir。我们还在酶和结构水平上研究了多替格拉韦的分子作用，多替格拉韦是ViiV Healthcare的一种先导化合物（Hare et al. 2011）。我们的研究结果表明，多替格拉韦能有效抑制一组对雷替格拉韦耐药的HIV-1 IN变异体。为了阐明DTG增强抗抗替地韦病毒效力的结构基础，我们测定了与DTG结合的野生型和突变型泡沫病毒原型囊体的晶体结构。这项工作是与伦敦帝国理工学院的Peter Cherepanov博士合作完成的。dolutegravity的整体IN结合模式与三环羟基吡咯MK-2048 （Merck & Co.）惊人地相似。两种第二代inis在IN活性位点内占据相同的物理空间，并与催化核心结构域的β -4- α -2环接触。此外，结构表明，DTG表现出相当大的灵活性，特别是在连接金属螯合核心和氟苯基的连接体中，使其能够适应突变整合酶活性位点的结构变化。在结构上适应与耐药性有关的结构变化的能力似乎是一种可取的特征，可用于开发新的insi。我们还报道了MK-0536的生化和抗病毒特性(见M？tifiot 2011)。我们与Terrence Burke博士（化学生物学实验室，CCR-NCI）合作合成了这种化合物。MK-0536在实验室使用我们的重组突变整合酶小组进行评估，而抗病毒试验在Steven Hughes博士实验室（HIV耐药项目，CCR-NCI）进行。我们证明，与RAL一样，MK-0536对重组蛋白和病毒复制具有很强的抑制作用。它对携带三种主要抗重力突变（Y143R、N155H和较小程度的G140S-Q148H）的整合酶和G118R突变体也有效。与Steven Hughes博士（Johnson et al. 2012）合作，从最近的PFV结构中开发了整合酶的分子模型。这些模型使我们能够在突变整合酶的背景下合理化RAL和MK-0536之间的差异。我们将继续与Terrence Burke博士（CCR-NCI化学生物学实验室）的长期合作。我们最近为第二代inis开发了一个新的药效团模型。我们已经申请了专利，并且最近发表了我们最新的化合物系列（见Zhao et al. 2011）。与NCI-Frederick （HIV耐药项目）的Stephen Hughes博士一起，我们正在测试这些化合物的抗病毒活性；与伦敦帝国理工学院的Peter Cherepanov博士一起，我们最近获得了共晶体结构。我们的目标是优化我们的新化学系列，以获得有效的新型抑制剂，有效对抗临床使用的inis。

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) 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 site 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). We have reported novel INSTIs chemotypes with Dr. ZhengQuiang Wang and Dr. Robert Vince at the Center for Drug Design, University of Minnesota. And with Dr. Seth Cohen (Department of Chemistry and Biochemistry, USCD), we have probed the role of the metal-binding group as IN metalloenzyme inhibitors.To perform these experiments, we have developed a panel of recombinant IN proteins bearing the mutations observed in patients that develop resistance to raltegravir and elvitegravir. Using our set of raltegravir-resistant IN mutants, we have characterized the molecular pharmacology of elvitegravir, dolutegravir and MK-0536, comparing them to raltegravir. We have shown that both raltegravir and elvitegravir 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 of raltegravir and elvitegravir against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed strand transfer inhibitors. 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.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 elvitegravir, which is combined with 3 other drugs in the Quad pill (cobicistat to boost elvitegravir pharmacokinetics and two reverse transcriptase inhibitors, emtricitabine and tenofovir). The Gilead Quad pill received support of the FDA Advisory Committee in May 2012 for approval as once daily single tablet regimen for HIV. dolutegravir.We have also studied the molecular of action of dolutegravir, a lead compound from ViiV Healthcare, at the enzymatic and structural levels (Hare et al. 2011). Our results demonstrate that dolutegravir effectively inhibits a panel of HIV-1 IN variants resistant to raltegravir. To elucidate the structural basis for the increased potency of DTG against raltegravir-resistant INs, we determined crystal structures of wild type and mutant prototype foamy virus intasomes bound to the drug. This work was done in collaboration with Dr. Peter Cherepanov at the Imperial College in London(1). The overall IN binding mode of dolutegravir is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048 (Merck & Co.). Both second-generation INSTIs occupy the same physical space within the IN active site and make contacts with the beta-4-alpha-2 loop of the catalytic core domain. Furthermore, the structures indicate that DTG displays considerable flexibility, particularly in the linker connecting the metal chelating core and the fluorophenyl group, allowing it to adjust to the structural changes in the active sites of the mutant integrases. 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 new INSTIs.We also reported the biochemical and antiviral properties of MK-0536 (see M?tifiot 2011). We synthesized this compound in collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI). MK-0536 was evaluated in the laboratory using our panel of recombinant mutant integrases while the antiviral assays where conducted in Dr. Steven Hughes laboratory (HIV Drug Resistance Program, CCR-NCI). We demonstrated that, like RAL, MK-0536 is highly potent against recombinant IN and viral replication. It is also effective against integrases that carry the three main raltegravir-resistance mutations (Y143R, N155H and to a lesser extent G140S-Q148H) and against the G118R mutant. Molecular models of integrases developed from the recent PFV structures, were generated in collaboration with Dr. Steven Hughes (Johnson et al. 2012). These models allow us to rationalize the differences between RAL and MK-0536 in the context of mutant integrases.We are continuing our long-term collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI). We recently developed a new pharmacophore model for second generation INSTIs. A patent has been filed and we recently published our most recent series of compounds (see Zhao et al. 2011). With Dr. Stephen Hughes, also at the NCI-Frederick (HIV Drug Resistance Program), we are testing the antiviral activity of those compounds and with Dr. Peter Cherepanov at the Imperial College in London we have recently obtained co-crystal structures. Our aim is to optimize our novel chemical series to obtain potent novel inhibitors active against clinically used INSTIs.