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

fda批准的HIV-1 IN抑制剂属于一类被称为“整合酶链转移抑制剂”（insis）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感染患者的一线治疗是INSTI， Dolutegravir （DTG）或Bictegravir (BIC)，联合两种核苷类似物逆转录酶抑制剂。DTG和BIC都能有效抑制大多数第一代抗ini的IN突变体。尽管BIC或DTG在未接受治疗的患者中几乎没有选择耐药性，但先前存在第一代ini耐药突变体并转向以DTG为特征的挽救性治疗的患者反应不佳，这强调了开发新的和改进的in抑制剂的重要性。这进一步推动了开发下一代药物的持续需要，这些药物可以对新出现的insi耐药病毒株保持高抗病毒功效。利用我实验室的设计和合成能力，我们与药理学家（NCI的Yves Pommier博士）和病毒学家（NCI的Yves Pommier博士）合作。Hughes和Eric Freed， NCI)开发了一种新的研究所类型。我们将我们最好的抑制剂与临床相关的inss一起使用单轮感染试验，对一组新的in耐药突变体进行检测，这些突变体是在体外用DTG、BIC和CAB选择的。其中，BIC和我们的化合物药效最广，且优于DTG。在与结构生物学家（Robert Craigie博士，NIDDK， Dmitry Lyumkis博士，Salk研究所，Cherepanov博士，Francis Crick研究所，英国）的进一步合作中，我们进行了研究，以更好地了解iniss与内粒体（与DNA底物和金属辅助因子的多聚整合酶）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。低温电子显微镜（Cryo-EM）在这些努力中发挥了关键作用。结合HIV-1内体的最佳inis的冷冻电镜结构揭示了一个复杂的、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inis结合结构中占据相似的位置。然而，由于我们的INSTI的结合，一些水被移位或移动；其他的只有在结合时才发现，这表明由结合引起的构象变化稳定了它们的位置。我们的结论是，在“底物包膜”（由宿主和病毒DNA结合定义的区域）中，催化口袋的几何形状、它们的总体体积、水合作用的附近模式以及其他特征的差异，都对理解INSTI相互作用很重要。最近，我们与Lyumkis博士合作，使用冷冻电镜来确定iniss如何与iniss耐药的侵入体突变体相互作用，并阐明对这些药物产生耐药性的机制。这些努力的重点是提供对临床使用的DTG和我们最好的内部化合物（目前正在NCI进行临床前评估）产生的病毒耐药变异的原因和如何选择的机制理解。这项合作旨在确定和分析第二代药物治疗产生的耐药性的新机制和途径，强调原发性和代偿性突变，并提供预测未来变异的策略。我们的工作将阐明新化合物对抗耐药突变型IN的优越效力的机制的结构基础。在对强效INSTI DTG治疗的反应中，发生IN耐药的主要途径有四种：Q148H/K/R、N155H、G118R和R263K。无论是在患者体内还是在细胞培养中，这些位置中的一个通常首先发生替换，并可能导致INSTI效力的重大丧失。有20个额外的位置，残基可以突变以产生更复杂的IN突变。这总共有数百种可能的组合。Hughes实验室测定了具有三重突变体E138K/G140A/Q148K的病毒构建物的抗病毒EC50值，发现我们的INSTI 4d （XZ426）的EC50值比DTG低20倍。为了了解这种增强效力的基础，Craigie博士准备了携带这三种三重突变的HIV内体。莱姆基斯博士已经确定了克雷格博士的三突变内粒体的结构要么与DTG结合要么与我们目前最好的INSTI结合。尽管这两种INSTI的结合模式和单个蛋白质残基的结构相似，但在我们的INSTI环境中，vDNA的末端腺苷呈现堆叠构型，而在DTG环境中则呈现非堆叠构型。这些数据表明腺苷堆积是一种真实的现象，它特异性地增强了我们基于萘嘧啶的INSTI的结合，这可能有助于提高我们的INSTI对这种（可能还有其他）突变体保持抗病毒功效的能力。

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).