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

整合酶结合蛋白作为抑制 HIV-1 组装的药物靶点

• 批准号: 9072152
• 负责人: GANJAM V KALPANA
• 金额: $ 16.7万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9072152
• 关键词: Acquired Immunodeficiency Syndrome; Anti-Retroviral Agents; Award; Binding; Binding Proteins; Biochemical; Biological Assay; C-terminal; Cell Nucleus; Cell membrane; Chemicals; Complex; Computer Simulation; Computing Methodologies; Cytoplasm; Defect; Development; Dominant-Negative Mutation; Drug Design; Drug Targeting; Epigenetic Process; Event; FDA approved; Frequencies; Funding; Genetic Screening; Genetic Transcription; Goals; HIV; HIV-1; HIV-1 integrase; Hand; Human; In Vitro; Infection; Integrase; Laboratories; Lead; Libraries; Malignant Childhood Neoplasm; Malignant Neoplasms; Maps; Mediating; Membrane; Methods; Modeling; Molecular Cloning; Mutate; Mutation; Nuclear Export; Parents; Pharmaceutical Preparations; Preclinical Drug Evaluation; Production; Property; Protein p53; Proteins; Rhabdoid Tumor; Role; SMARCB1 gene; SWI/SNF Family Complex; Staging; Structure; Testing; Therapeutic; Time; Transcriptional Regulation; Tumor Suppressor Proteins; Viral; Virion; base; cancer genome; chromatin remodeling; combat; drug development; epigenetic regulation; in vivo; insight; membrane assembly; mutant; new therapeutic target; novel; novel strategies; pandemic disease; particle; peptidomimetics; pol Gene Products; protein complex; protein structure; public health relevance; three dimensional structure; tool; trafficking; tumorigenesis; yeast two hybrid system

 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.

 描述（由申请人提供）：本申请是对已授予的R 01“1 R 01 GM 112520 -01，整合酶结合蛋白作为抑制HIV-1组装的药物靶标的修订。“这次修订的原因是INI 1新的基于NMR的解决方案结构的可用性。NMR结构的可用性增强了我们实现R 01资助目标的能力，其目标如下。目标一是了解INI 1影响组装的机制;目标二是了解INI 1的核输出特性;目标三是： 使用计算建模定义INI 1的最小IN结合结构域与IN的相互作用，并鉴定IN-INI 1相互作用中涉及的界面残基;和（ii）使用Alpha Screen筛选破坏IN-INI 1相互作用的药物/肽模拟物。然而，基于NMR的INI 1或IN-INI 1相互作用的结构测定不是R 01目标的一部分。新提议的补充研究将加强我们实现目标一和目标三的能力，如下所述。 INI 1是一种整合酶结合蛋白，已显示其影响HIV-1复制的多个阶段，包括LTR介导的转录、组装和颗粒产生和整合。此外，INI 1是一种表观遗传调节因子，是SWI/SNF复合物的一个组成部分，参与染色质重塑。它是在大量人类癌症中突变的肿瘤抑制因子。尽管它在癌症、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INI 1的结构尚不清楚，主要是由于无法获得大量的纯蛋白或无法结晶。 W最近已经成功地纯化和获得了INI 1的反式显性负突变体（也称为S6（Rpt 1）或INI 1183 -265）的NMR结构。INI 1183 -265的解决方案结构迅速增强了我们理解IN-INI 1相互作用的结构基础的能力，并丰富了我们实现R 01目标中提出的目标的能力，特别是目标III。有了INI 1183 -265结构，补充申请的目标如下。在补充目标I中，我们将使用基于NMR的化学位移扰动（CSP）映射来识别IN-INI 1复合物内的界面残基，并使用标准NMR方法来确定INI 1183 -265 - IN复合物的完整结构。在补充目标II中，我们将通过产生IN和INI 1的界面残基的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，特别是组装和颗粒产生。这些研究将首次了解IN-INI 1相互作用的结构基础及其对HIV-1复制的影响，并为药物开发铺平道路，以破坏这些相互作用。