Optimization HIV Inhibition by Allosteric Integrase Inhibitors

摘要 艾滋病毒的全球负担仍然严重，对新的抗病毒药剂的耐药性稳步发展， 新疗法的不断发展。雷特格韦是FDA批准的第一种整合酶（IN）抑制剂， 酶的活性位点和阻断DNA链转移。存在雷特格韦的情况下HIV的演变 然而，耐药突变体，激发了对IN的潜在额外靶点的研究。IN结合宿主细胞 蛋白质PSIP 1/LEDGF/p75（此后称为LEDGF），其对于有效的IN功能，包括靶向 HIV通过系链机制整合到活性转录单位。我们和其他人发现， IN上的LEDGF结合位点可以被小分子抑制剂（变构整合酶抑制剂，或 ALLINI），现在可以获得靶向该位点的高活性抑制剂。令人惊讶的是，这些抑制 分子并不强烈地作用于复制周期的整合步骤，而是干扰 出芽后病毒核心的适当成熟，暗示IN参与组装。我们假设一个详细的 对ALLINI功能和逃逸的理解将允许开发更有效的抑制剂。我们因此 使与ALLINI GSK 1264结合的全长HIV-1 IN结晶，并分析抑制剂界面。这 代表了第一次X射线质量的晶体已经产生，含有全长HIV IN。的 结构显示GSK 1264结合到催化结构域的二聚体界面，并且也位于该界面处。 界面是来自相邻IN二聚体的C末端结构域（CTD）。在晶格中，IN形成开放的 通过这种相互作用介导的聚合物。对几种ALLINI的进一步研究表明， 降低的敏感性通常会改变位于或靠近通道介导界面的氨基酸， 突变通常编码减少多聚化的取代。我们认为ALLINI抑制颗粒 通过新鉴定的CTD催化结构域刺激IN的不适当聚合而成熟 接口.在初步的数据中，我们表明这些逃逸突变体更适合晶体学分析， 研究比野生型IN，使可能的积极的新研究的IN结构和抑制。在特定 目的1，我们将获得高分辨率结构的IN变体在ALLINI的存在下， 具体目标2，我们将通过产生HIV病毒来验证我们对ALLINI作用机制的假设 携带IN中的突变，旨在破坏CTD-催化结构域界面并评估它们的能力， 在细胞培养中的复制和对ALLINI的敏感性。在具体目标3中，我们将利用新的 有关IN性质的信息，用于结晶和确定IN/DNA复合物的第一结构。

Abstract The global burden of HIV remains severe, and resistance develops steadily to new antiviral agents, requiring ongoing development of new therapies. Raltegravir, the first FDA-approved inhibitor of integrase (IN), binds the enzyme active site and blocks DNA strand transfer. Evolution of HIV in the presence of raltegravir elicits resistance mutants, however, motivating studies of potential additional targets on IN. IN binds the host cell protein PSIP1/LEDGF/p75 (henceforth LEDGF), which is important for efficient IN function, including targeting HIV integration to active transcription units via a tethering mechanism. We and others have found that the LEDGF binding site on IN can be bound by small molecule inhibitors (allosteric integrase inhibitors, or ALLINIs), and now highly active inhibitors targeting this site are available. Surprisingly, inhibition by these molecules does not act strongly on the integration step of the replication cycle, but instead interferes with proper maturation of the viral core after budding, implicating IN in assembly. We hypothesized that a detailed understanding of ALLINI function and escape will allow development of more potent inhibitors. We thus crystallized full-length HIV-1 IN bound to the ALLINI GSK1264, and analyzed the inhibitor interface. This represents the first time X-ray quality crystals have been generated that contained full length HIV IN. The structure shows GSK1264 bound to the dimer-interface of the catalytic domain, and also positioned at this interface is a C-terminal domain (CTD) from an adjacent IN dimer. In the crystal lattice, IN forms an open polymer mediated by this interaction. Further studies of several ALLINIs show that HIV escape mutants with reduced sensitivity commonly alter amino acids at or near the inhibitor-mediated interface, and that HIV escape mutations often encode substitutions that reduce multimerization. We propose that ALLINIs inhibit particle maturation by stimulating inappropriate polymerization of IN through the newly identified CTD-catalytic-domain interface. In preliminary data, we show that these escape mutants are far more amenable to crystallographic studies than wild-type IN, making possible aggressive new studies of IN structure and inhibition. In Specific Aim 1, we will obtain high resolution structures of IN variants in the presence of ALLINIs In Specific Aim 2, we will test our hypothesis of the mechanism of ALLINI action by generating HIV viruses carrying mutations in IN designed to disrupt the CTD-catalytic domain interface and assess their capacity for replication in cell culture and sensitivity to ALLINIs. In Specific Aim 3, we will we will take advantage of new information on IN properties to crystallize and determine the first structures of IN/DNA complexes.