Optimization HIV Inhibition by Allosteric Integrase Inhibitors

摘要 全球艾滋病毒的负担仍然严重，对新的抗病毒药物的抗药性稳步发展，需要 正在开发新的治疗方法。FDA批准的第一个整合酶(IN)抑制剂Raltegravir与 酶的活性部位和阻断DNA链转移。Raltegravir存在下HIV的进化 然而，抗药性突变体激发了对IN潜在额外靶点的研究。In结合宿主细胞 蛋白质PSIP1/LEDGF/p75(以下简称LEDGF)，对包括靶向在内的有效IN功能非常重要 HIV通过拴系机制整合到激活的转录单位。我们和其他人发现， IN上的LEDGF结合部位可被小分子抑制剂(变构整合酶抑制剂或 ALLINI)，现在有针对这个部位的高活性抑制剂可用。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 病毒核心在萌发后适当成熟，意味着IN在组装中。我们假设一个详细的 了解Allini的功能和逃逸将有助于开发更有效的抑制剂。因此，我们 结晶了与Allini GSK1264结合的全长HIV-1 IN，并分析了抑制物界面。这 代表着第一次产生了含有全长HIV IN的X射线质量晶体。这个 结构显示GSK1264结合到催化结构域的二聚体-界面上，并且也定位在 接口是来自相邻IN二聚体的C-末端结构域(CTD)。在晶格中，In形成一个开放的 通过这种相互作用调节的聚合物。对几种等位基因的进一步研究表明，艾滋病毒逃逸突变株具有 敏感性降低通常会改变抑制物介导界面上或附近的氨基酸，从而使艾滋病毒逃逸 突变通常编码减少多聚化的替换。我们认为ALLINI抑制粒子 通过新发现的CTD催化结构域刺激IN的不适当聚合而成熟 界面。在初步数据中，我们表明这些逃逸突变体更容易受到结晶学的影响。 比野生型IN的研究，使对IN结构和抑制的积极新研究成为可能。具体而言 目标1，我们将获得在Allini存在的情况下IN变体的高分辨结构 具体目标2，我们将测试我们关于Allini通过产生HIV病毒而起作用的机制的假说 携带突变的IN旨在破坏CTD-催化结构域界面并评估其结合能力 细胞培养中的复制和对ALLINI的敏感性。在具体目标3，我们将我们将利用新的 关于IN特性的信息，以结晶和确定IN/DNA络合物的第一结构。

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.