Design and Synthesis of HIV Integrase as Potential Anti-AIDS Drugs

HIV整合酶的设计与合成作为潜在的抗艾滋病药物

• 批准号: 10702293
• 负责人: TERRENCE BURKE
• 金额: $ 136.28万
• 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/10702293
• 关键词: Acquired Immunodeficiency Syndrome; Adenosine; Anti-HIV Agents; Binding; Biological Assay; Cell Culture Techniques; Clinical; Collaborations; Complex; Cryoelectron Microscopy; DNA; Data; Drug resistance; Drug resistance pathway; Enzymes; Exhibits; FDA approved; Future; Generations; Geometry; Goals; HIV; HIV Integrase; HIV therapy; HIV-1; HIV-1 integrase; Hydration status; In Vitro; Individual; Infection; Institutes; Integrase; Integrase Inhibitors; Laboratories; Metals; Molecular Conformation; Mutate; Mutation; Naphthyridines; National Institute of Diabetes and Digestive and Kidney Diseases; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Play; Positioning Attribute; Process; Proteins; Reaction; Resistance; Reverse Transcriptase Inhibitors; Role; Salvage Therapy; Side; Structural Biologist; Structure; Variant; Viral; Virus; Water; Work; anti-viral efficacy; base; clinically relevant; cofactor; design; improved; inhibitor; mutant; next generation; novel; novel therapeutics; nucleoside analog; preclinical evaluation; resistance mechanism; response; treatment response; viral DNA

FDA-approved HIV-1 IN inhibitors belong to a class of drugs called "integrase strand transfer inhibitors" (INSTIs), due to their ability to preferentially block the enzymes strand transfer (ST) reaction as related to the enzymes 3-processing (3-P) reaction. The current recommended front-line therapy for HIV-1 infected patients is an INSTI, either Dolutegravir (DTG) or Bictegravir (BIC), in combination with two nucleoside analog reverse transcriptase inhibitors. Both DTG and BIC potently inhibit most of the first generation INSTI-resistant IN mutants. Although little resistance has been selected by either BIC or DTG in treatment-naive patients, patients who have preexisting first-generation INSTI-resistant mutants and have switched to a salvage therapy featuring DTG respond poorly, emphasizing the importance of developing new and improved IN inhibitors. This adds impetus to a continuing need to develop next-generation agents that can retain high antiviral efficacy against emerging strains of INSTI-resistant virus. Utilizing my laboratorys design and synthetic capabilities, we have teamed with pharmacologists (Dr. Yves Pommier, NCI) and virologists (Drs. Hughes and Eric Freed, NCI) to develop a new genre of INSTIs. We have examined our best inhibitors side by side with the clinically relevant INSTIs using a single round infection assay against panel of new IN-resistant mutants that were selected in vitro with DTG, BIC, and CAB. Of these three INSTIs, BIC and our compounds had the broadest efficacy and were superior to DTG. In further collaborations with structural biologists (Dr. Robert Craigie, NIDDK, Dr. Dmitry Lyumkis, the Salk Institute, Dr. Cherepanov, the Francis Crick Institute, UK) we have performed studies to better understand the interactions of INSTIs with intasomes (multimeric integrase with DNA substrate and metal cofactor) and to clarify the roles that mutations play in downregulating these interactions. Cryo-electron microscopy (Cryo-EM) has played a key role in these efforts. Cryo-EM structures of our best INSTIs bound to HIV-1 intasomes revealed a complex and dynamic network of water molecules surrounding bound INSTIs, with many of these waters appearing to be conserved and occupying similar positions in the unliganded and INSTI-bound structures. However, some waters are displaced or shifted as a consequence of binding of our INSTI; others are found only when INSTIs are bound, suggesting that the conformational changes induced by the binding stabilize their position. We concluded that within the "substrate envelope" (the region defined by the binding of host and viral DNA), differences in geometry of the catalytic pockets, their overall volume, the nearby patterns of hydration, among other features, all matter for understanding INSTI interactions. Most recently we have partnered with Dr. Lyumkis to employ cryo-EM to determine how INSTIs interact with INSTI-resistant intasome mutants and elucidate the mechanisms by which resistance to these drugs emerges. The focus of these efforts is to provide a mechanistic understanding of both why and how select viral resistant variants that arise in response to the clinically used DTG as well as our best in-house compound, which is currently under pre-clinical evaluation by the NCI. This collaboration is identifying and analyzing novel mechanisms and pathways of drug resistance that arise in response to treatment with 2nd generation drugs, highlighting both primary and compensatory mutations, and providing strategies to predict future variants. Our work will elucidate the structural basis for mechanisms underlying the superior potency of novel compounds against resistant mutant forms of IN. There are four primary pathways through which IN resistance occurs in response to therapy with the potent INSTI DTG, which involve these changes: Q148H/K/R, N155H, G118R, and R263K. Substitutions at one of these positions usually arise first, both in patients and in cell culture and can cause a major loss of INSTI potency. There are 20 additional positions where a residue can be mutated to give rise to more complex IN mutants. This collectively amounts to hundreds of possible combinations. The Hughes laboratory has determined antiviral EC50 values against viral constructs having the triple mutant E138K/G140A/Q148K and found that our INSTI 4d (XZ426) has an EC50 that is 20-fold lower than that of DTG. To understand the basis of this increased potency, Dr. Craigie has prepared HIV intasomes bearing these three triple mutations. Dr. Lyumkis has determined structures of Dr. Craigies triple mutant intasomes bound to either to DTG or to our current best INSTI. Although the binding modes of both INSTIs and the configuration of individual protein residues are similar, the terminal adenosine of vDNA exhibits a stacked configuration in the context of our INSTI, but an unstacked configuration in the context of DTG. These data suggest that adenosine stacking is a real phenomenon that specifically enhances the binding of our naphthyridine-based INSTIs which may contribute to the improved ability of our INSTI to retain antiviral efficacy against this (and perhaps other) mutant(s).

FDA 批准的 HIV-1 IN 抑制剂属于一类称为“整合酶链转移抑制剂”(INSTI) 的药物，因为它们能够优先阻断与酶 3-加工 (3-P) 反应相关的酶链转移 (ST) 反应。目前推荐的 HIV-1 感染患者一线治疗是 INSTI，即 Dolutegravir (DTG) 或 Bictegravir (BIC)，与两种核苷类似物逆转录酶抑制剂联合使用。 DTG 和 BIC 均能有效抑制大多数第一代 INSTI 抗性 IN 突变体。尽管 BIC 或 DTG 在初治患者中几乎没有产生耐药性，但先前存在第一代 INSTI 耐药突变体并转而采用 DTG 挽救疗法的患者反应较差，这强调了开发新的和改进的 IN 抑制剂的重要性。这进一步推动了对开发下一代药物的持续需求，这些药物可以对新出现的 INSTI 耐药病毒株保持高抗病毒功效。利用我的实验室设计和合成能力，我们与药理学家（Yves Pommier 博士，NCI）和病毒学家（Hughes 博士和 Eric Freed，NCI）合作开发了一种新型 INSTI。我们使用单轮感染试验，针对在体外通过 DTG、BIC 和 CAB 选择的一组新的 IN 抗性突变体，对我们最好的抑制剂与临床相关的 INSTI 进行了检查。在这三种 INSTI 中，BIC 和我们的化合物具有最广泛的功效，并且优于 DTG。在与结构生物学家（NIDDK 的 Robert Craigie 博士、索尔克研究所的 Dmitry Lyumkis 博士、英国 Francis Crick 研究所的 Cherepanov 博士）的进一步合作中，我们进行了研究，以更好地了解 INSTI 与 intasomes（具有 DNA 底物和金属辅因子的多聚体整合酶）的相互作用，并阐明突变在下调这些相互作用中所起的作用。冷冻电子显微镜（Cryo-EM）在这些努力中发挥了关键作用。 我们最好的 INSTI 与 HIV-1 嵌体结合的冷冻电镜结构揭示了结合的 INSTI 周围的复杂且动态的水分子网络，其中许多水似乎是保守的，并且在未配体和 INSTI 结合的结构中占据相似的位置。 然而，一些水域由于我们的 INSTI 的约束而被取代或转移；其他的仅在 INSTI 结合时才被发现，这表明结合引起的构象变化稳定了它们的位置。我们得出的结论是，在“底物包膜”（由宿主和病毒 DNA 结合定义的区域）内，催化袋几何形状的差异、其整体体积、附近的水合模式以及其他特征，对于理解 INSTI 相互作用都很重要。最近，我们与 Lyumkis 博士合作，采用冷冻电镜来确定 INSTI 如何与 INSTI 抗性嵌体突变体相互作用，并阐明对这些药物产生耐药性的机制。这些工作的重点是从机制上理解为什么以及如何选择针对临床使用的 DTG 以及我们最好的内部化合物而出现的病毒抗性变异，该化合物目前正在接受 NCI 的临床前评估。这项合作正在识别和分析因第二代药物治疗而出现的耐药性的新机制和途径，突出原发性突变和补偿性突变，并提供预测未来变异的策略。我们的工作将阐明新型化合物对抗 IN 耐药突变形式的卓越效力机制的结构基础。强效 INSTI DTG 治疗导致 IN 抵抗发生的主要途径有四种，其中涉及这些变化：Q148H/K/R、N155H、G118R 和 R263K。在患者和细胞培养物中，这些位置之一的取代通常首先出现，并且可能导致 INSTI 效力的重大损失。还有 20 个额外位置，其中残基可以突变以产生更复杂的 IN 突变体。这总共相当于数百种可能的组合。 Hughes 实验室已确定针对具有三重突变体 E138K/G140A/Q148K 的病毒构建体的抗病毒 EC50 值，并发现我们的 INSTI 4d (XZ426) 的 EC50 比 DTG 低 20 倍。为了了解这种效力增强的基础，Craigie 博士制备了带有这三种三重突变的 HIV 嵌体。 Lyumkis 博士已经确定了 Craigies 博士三突变体整合体的结构，该整合体与 DTG 或我们目前最好的 INSTI 结合。尽管两个 INSTI 的结合模式和单个蛋白质残基的构型相似，但 vDNA 的末端腺苷在我们的 INSTI 中表现出堆叠构型，但在 DTG 中表现出非堆叠构型。这些数据表明，腺苷堆积是一种真实的现象，可以特异性增强我们的基于萘啶的 INSTI 的结合，这可能有助于提高我们的 INSTI 保留针对这种（可能还有其他）突变体的抗病毒功效的能力。