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(INSTI)的药物，因为与酶3‘-加工(3’-P)反应相比，它们能够优先阻断酶的链转移(ST)反应。不幸的是，IN的突变形式的出现导致了对这些INSTI的临床耐药性。这增加了继续需要开发下一代制剂的动力，这些制剂能够保持对新出现的INSTI耐药病毒株的高抗病毒效果。利用我的实验室的设计和合成能力，我们与药理学家(NCI的伊夫·波米尔博士)、病毒学家(休斯博士，NCI)和结构生物学家(NIDDK的Robert Craigie博士、Dmitry Lyumkis博士、Salk研究所的Dmitry Lyumkis博士和英国Francis Crick研究所的Cherepanov博士)合作，更好地了解INSTI与内含体(具有DNA底物和金属辅因子的多聚体整合酶)的相互作用以及突变在下调这些相互作用中所起的作用。低温EM在这些努力中发挥了关键作用。我们最好的INSTI与内质粒结合的冷冻-EM结构揭示了一个围绕结合的INSTI的复杂而动态的水分子网络，其中许多水似乎是保守的，并且在未连接和INSTI结合的结构中占据相似的位置。然而，由于INSTI的结合，一些水被移动或移动；另一些只有在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，催化口袋的几何形状、其总体积、附近的水化模式等方面的差异，以及其他特征，都对理解INSTI相互作用很重要。我们还与伦敦帝国理工学院Goedele Maerten博士的实验室合作，检查我们针对人类T细胞白血病病毒1型(HTLV-1)选择的多种IN抑制剂，我们的一些合成结构显示出针对这一目标的显著效力。我们最好的INST在单轮复制检测中表现出比目前FDA批准的药物更好的抗病毒特性，该检测使用了100多种耐药突变形式的整合酶。为了生成我们最好的INSITs的关键药代动力学(PK)数据，我们与NCI发明开发计划(IDP)合作，获得了早期PK数据，包括对啮齿动物的研究。

FDA-approved inhibitors of HIV-1 IN belong to a class of drugs called " integrase strand transfer inhibitors" (INSTIs), due to their ability to preferentially block the enzyme's strand transfer (ST) reaction as compared to the enzymes 3'-processing (3'-P) reaction. Unfortunately, mutant forms of IN arise that lead to clinical resistance against these INSTIs. This adds impetus to a continuing need to develop next-generation agents that have the ability to retain high antiviral efficacy against emerging strains of INSTI-resistant virus. Utilizing my laboratory's design and synthetic capabilities, we have teamed with pharmacologists (Dr. Yves Pommier, NCI), virologists (Dr. Hughes, NCI) and structural biologists (Dr. Robert Craigie, NIDDK, Dr. Dmitry Lyumkis, the Salk Institute and Dr. Cherepanov, the Francis Crick Institute, UK) to better understand the interactions of INSTIs with intasomes (multimeric integrase with DNA substrate and metal cofactor) and the roles that mutations play in down-regulating these interactions. Cryo-EM has played a key role in these efforts. Cryo-EM structures of our best INSTIs bound to 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 the 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. We also have a collaboration in place with the Imperial College London laboratory of Dr. Goedele Maertens to examine a diverse selection of our IN inhibitors against human T-cell leukemia virus type 1 (HTLV-1) and a number of our synthetic constructs show significant potency against this target. Our best INSTs exhibit better antiviral profiles than current FDA-approved agents when assayed in single round replication assays that employ a panel of more than 100 drug-resistant mutant forms of integrase. In order to generate key pharmacokinetic (PK) data on our best INSITs, we have partnered with the NCI Invention Development Program (IDP) to obtain early-stage PK data, including studies in rodents.