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).

抑制剂中FDA批准的HIV-1属于称为“整合酶链转移抑制剂”（Instis）的一类药物，因为它们优先阻断了与酶3处理（3-P）反应有关的酶链转移（ST）反应（ST）反应。目前针对HIV-1感染患者的当前建议的前线治疗是DoluteGravir（DTG）或Bictegravir（BIC）的一个Insti，以及两个核苷类似物逆转录酶抑制剂。 DTG和BIC都在突变体中有效抑制了大多数第一代抗性。尽管BIC或DTG在接受治疗的患者中几乎没有抗药性，但先前存在的第一代耐药性突变体并已转化为DTG反应较差的打捞疗法的患者，强调了在抑制剂中开发新和改善的重要性。这增加了发展下一代药物的持续需求，该代理可以保留针对耐药病毒的新兴菌株的高抗病毒功效。利用我的实验室设计和合成能力，我们与药理学家（NCI Yves Pommier博士）和病毒学家（Hughes博士和Eric Freed，NCI）合作，开发了一种新的Instis类型。我们已经使用单个圆形感染测定法对临床相关的研究所进行了对最佳抑制剂，并针对新的耐耐药突变体的小组进行了单一的感染测定，这些突变体是在体外与DTG，BIC和CAB一起选择的。在这三个学院中，BIC和我们的化合物具有最广泛的功效，并且优于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.冷冻电子显微镜（Cryo-EM）在这些努力中发挥了关键作用。 与HIV-1诱导群结合的最佳学院的冷冻EM结构揭示了围绕结合学院的水分子的复杂而动态的网络，其中许多水似乎是保守的，并且在非配置和结合的结构中占据了相似的位置。 但是，由于我们的研究结合，某些水被移位或转移。仅当研究所结合时才发现其他，这表明结合诱导的构象变化稳定了其位置。我们得出的结论是，在“底物包膜”（由宿主和病毒DNA的结合定义的区域）中，催化口袋的几何形状差异，它们的整体体积，附近的水合模式以及其他特征，所有特征都可以理解Insti相互作用。最近，我们与Lyumkis博士合作采用了冷冻EM来确定Instis如何与Insti-Insti-Inter抗性突变体相互作用，并阐明了对这些药物的耐药性出现的机制。这些努力的重点是对响应于临床使用的DTG以及我们最好的内部化合物而产生的选择和抗性变体的机理理解，以及目前由NCI进行的临床前评估。这种合作正在识别和分析对第二代药物治疗而产生的新型机制和耐药性的途径，强调原发性突变和补偿性突变，并提供了预测未来变体的策略。我们的工作将阐明新型化合物反对抗性突变体形式的优势效力的机制的结构基础。有四个主要途径在抗药性中响应有效的Insti DTG的治疗，涉及这些变化：Q148h/k/r，N155H，G118R和R263K。这些位置之一的替代通常首先出现，无论是在患者还是在细胞培养中，可能会导致效力的重大损失。还有20个位置可以突变残留物在突变体中产生更复杂的位置。这共同等于数百种可能的组合。休斯实验室已经确定了具有三重突变体E138K/G140A/Q148K的病毒构建体的抗病毒EC50值，发现我们的Insti 4D（XZ426）的EC50比DTG低20倍。为了了解这种提高的效力的基础，克雷吉博士准备了带有这三个三重突变的HIV诱导症。 Lyumkis博士确定了Craigies Dr. Triple突变体插入的结构，与DTG或我们当前的最佳Insti约束。尽管Instis的结合模式和单个蛋白质残基的配置都是相似的，但VDNA的末端腺苷在我们Insti的上下文中表现出堆叠的构型，但在DTG的背景下是未添加的配置。这些数据表明，腺苷堆叠是一种真正的现象，它专门增强了我们基于萘胺的研究的结合，这可能有助于提高我们Insti对此（甚至其他）突变体的抗病毒疗效的能力。