Integrase Binding Proteins as Drug Targets to Inhibit HIV-1 Assembly

 描述(由申请人提供)：本申请是对授予的R01“1R01GM112520-01，整合酶结合蛋白作为抑制HIV-1组装的药物靶标”的修订。进行这一修订的原因是INI1的新的基于核磁共振的解决方案结构的可用性。核磁共振结构的可用性增强了我们实现基金R01目标的能力，其目标如下。目标一是了解INI1影响组装的机制；目标二是了解INI1的核出口性质；目标三是：(1) 使用计算模型定义INI1的最小IN结合域与IN的相互作用，并确定IN-INI1相互作用中涉及的界面残基；以及(Ii)使用Alpha Screen筛选破坏IN-INI1相互作用的药物/肽类药物。然而，基于核磁共振的INI1或IN-INI1相互作用的结构确定不是R01目标的一部分。新提议的补充研究将提高我们实现目标一和目标三的能力，如下所述。INI1是一种整合酶结合蛋白，已被证明影响HIV-1复制的多个阶段，包括LTR介导的转录、组装和颗粒生产和整合。此外，INI1是一种表观遗传调节因子，也是参与染色质重塑的SWI/SNF复合体的组成部分。它是一种肿瘤抑制基因，在大量人类癌症中发生突变。尽管INI1在癌症、表观遗传和转录调控以及HIV-1复制中具有广泛的重要性，但其结构尚不清楚，主要是由于无法获得大量纯蛋白质或无法结晶。W最近成功地纯化和获得了INI1的跨显性负突变，也被称为S6(Rpt1)或INI1183-265的核磁共振结构。拥有INI1183-265的溶液结构迅速提高了我们理解IN-INI1相互作用的结构基础的能力，并丰富了我们实现R01目标，特别是目标III中提出的目标的能力。在补充目标I中，我们将使用基于核磁共振的化学位移微扰(CSP)映射来确定IN-INI1络合物中的界面残基，并使用标准的核磁共振方法来确定INI1183-265-IN络合物的完整结构。在补充AIM II中，我们将通过产生IN和INI1的界面残基突变来进行基于结构的功能研究，并测试这些突变体对HIV-1复制的影响，特别是组装和颗粒生产。这些研究将首次使人们能够了解IN-INI1相互作用的结构基础及其对HIV-1复制的影响，并为药物开发破坏这些相互作用铺平道路。

 DESCRIPTION (provided by applicant): This application is a revision to the awarded R01 "1R01GM112520-01, Integrase Binding Proteins as Drug Targets to Inhibit HIV-1 Assembly." The reason for this revision is the availability of new NMR based solution structure for INI1. Availability of NMR structure enhances our ability to perform the goals of funded R01, whose Aims are as follows. The Aim I is to understand the mechanism by which INI1 influences assembly; Aim II is to understand the nuclear export properties of INI1; and the Aim III is to: (i) define the interaction of minimal IN binding domain of INI1 with IN using computational modeling and identify the interface residues involved in IN-INI1 interactions; and (ii) to screen for drugs/peptidomimetics that disrupt IN-INI1 interaction using Alpha Screen. However, NMR based structural determination of INI1 or IN-INI1 interactions are not part of the goals of the R01. The newly proposed supplemental studies will enhance our ability to perform the goals of Aims I and III, as described below. INI1 is an integrase binding protein that has been shown to influence multiple stages of HIV-1 replication including LTR-mediated transcription, assembly and particle production and integration. In addition, INI1 is an epigenetic regulator and a component of SWI/SNF complex involved in chromatin remodeling. It is a tumor suppressor mutated in large number of human cancers. Despite its wide spread importance in cancer, epigenetic and transcriptional regulation and HIV-1 replication, the structure of INI1 is unknown, largely due to inability to obtain large quantities of pure protein or inability to crystallize. W have been recently successful in purifying and obtaining NMR structure of the transdominant negative mutant of INI1, also termed, S6(Rpt1) or INI1183-265. Having solution structure of INI1183-265 rapidly enhances our ability to understand the structural basis of IN-INI1 interactions and enriches our ability to attain the goals proposed in the aims of R01, especially aim III. With INI1183-265 structure in hand, the goals for the supplemental application are as follows. In Supplemental Aim I we will use NMR-based chemical shift perturbation (CSP) mapping to identify the interface residues within IN-INI1 complexes and use standard NMR methods to determine the complete structure of the INI1183-265 - IN complex. In Supplemental Aim II, we will carry out structure-based functional studies by generating mutations of interface residues of IN and INI1 and test the effect of these mutants on HIV-1 replication, especially assembly and particle production. These studies will allow, for the first time, to gain understanding of the structural basis of IN-INI1 interactions, their effect on HIV-1 replication, and pave the way for drug development to disrupt these interactions.