Pharmacology of HIV Viral DNA Retroviral Integrases

HIV 病毒 DNA 逆转录病毒整合酶的药理学

• 批准号: 8937654
• 负责人: YVES POMMIER
• 金额: $ 63.96万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937654
• 关键词: Active Sites; Antiviral Agents; Arginine; Binding; Binding Sites; Biochemical; Biological Assay; Biology; CCR; Catalytic Domain; Characteristics; Chemical Structure; Chemicals; Clinical; Collaborations; Collection; Communities; Complex; DNA; Data; Development; Drug Binding Site; Drug resistance; Enzymatic Biochemistry; Enzymes; FDA approved; Family; Goals; HIV; HIV Drug Resistance Program; HIV Integrase; Healthcare; Integrase; Integrase Inhibitors; International; Laboratories; Lead; Legal patent; Licensing; London; Malignant Neoplasms; Mediating; Molecular; Molecular Biology; Molecular Mechanisms of Action; Molecular Models; Mutation; Nature; Patients; Peptide Hydrolases; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Process; Proteins; Proviruses; Publishing; RNA-Directed DNA Polymerase; Reaction; Recombinant Proteins; Recombinants; Research; Resistance; Resistance development; Schedule; Science; Series; Site; Specificity; Spumavirus; Structure; Tail; Viral; Virus; Work; base; compliance behavior; design; divalent metal; drug discovery; drug sensitivity; flexibility; gain of function; inhibitor/antagonist; interfacial; molecular modeling; mutant; novel; pol genes; prototype; research study; resistance mutation; symposium; therapeutic development; therapeutic target; uptake; viral DNA

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 and elvitegravir. Using our set of raltegravir- and elvitegravir-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 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 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 intasomes bound to the drugs. This work was done in collaboration with Dr. Peter Cherepanov at the Clare Hall Cancer UK Center in London. The abilityto 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.

整合酶 (IN) 由 HIV 原病毒的 Pol 基因编码，可以有效表达为活性重组蛋白。我们实验室开创了整合酶抑制剂研究领域（PNAS 1993），发现了多个先导抑制剂家族（Nature Rev Drug Discovery 2005；Current Topics in Medicinal Chemistry 2009；Viruses 2010；Adv Pharmacol 2013），证明IN抑制剂可作为界面抑制剂（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型化学型整合酶抑制剂，以克服对拉替拉韦的耐药性并靶向 IN 的新位点。我们发现了源自短 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，通过添加聚精氨酸尾部以赋予细胞摄取，它们可以作为抗病毒药物。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，发布了新型合成化学型作为 IN 链转移抑制剂 (INSTI) 并获得了专利，其中包括邻苯二甲酰亚胺和喹啉衍生物。为了进行这些实验，我们开发了一组重组 IN 蛋白，这些蛋白带有在对拉替拉韦和艾维拉韦产生耐药性的患者中观察到的突变。使用我们的一组拉替拉韦和埃替拉韦耐药 IN 突变体，我们表征了埃替拉韦、多替拉韦和我们的新型抑制剂的分子药理学，并将它们与拉替拉韦进行比较。我们已经证明，拉替拉韦、埃替拉韦、多替拉韦和我们的新系列对链转移反应具有高度选择性，而对 3' 加工反应的效力要低 100 倍以上，并且对整合酶介导的崩解反应几乎没有活性。针对链转移（整合酶介导的 3 个反应之一）的选择性活性证明了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）一致，即链转移抑制剂通过在病毒 DNA 3' 处理后与我们的共晶螯合酶催化位点中的二价金属来捕获 IN-病毒 DNA 复合物。结构和分子建模数据。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。我们已将这些研究扩展到整合酶柔性环中的双突变体，这种突变体通常出现在拉替拉韦耐药患者中。工作假设是，在因第一个突变的存在而导致 IN 的生化活性出现缺陷后，第二个突变起到了功能增益的作用，以挽救 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 对拉替拉韦和埃替拉韦具有交叉耐药性，但对多鲁特韦和我们的一些新衍生物的交叉耐药性要小得多。另一方面，143Y突变体主要对拉替拉韦具有耐药性，对埃替拉韦和多替拉韦具有最低程度的耐药性。这些结果为因突变 143Y 而对拉替拉韦产生耐药性的患者使用埃替拉韦提供了依据（但突变 140S-148H 则不然）。我们的结果支持多替拉韦克服拉替拉韦和艾维拉韦耐药性并促进患者依从性的价值。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原型泡沫病毒嵌体的晶体结构。这项工作是与伦敦克莱尔霍尔癌症英国中心的 Peter Cherepanov 博士合作完成的。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性，可用于开发我们的新 INSTI。我们的研究是我们与 Terrence Burke 博士（化学生物学实验室，CCR-NCI）、NCI-Frederick 实验室的 Stephen Hughes 博士（HIV 耐药项目）以及 Dr. Stephen Hughes 长期合作的结果。彼得·切列帕诺夫在伦敦。