HIV-1 Integrase Structural Biology

描述（由申请人提供）：像所有逆转录病毒一样，HIV-1必须通过逆转录将cDNA拷贝整合到细胞染色体中才能复制。HIV-1整合是由必需的病毒酶整合酶催化的。这使得整合酶成为一个有吸引力的药物靶点，而抗整合酶化合物正在美国的临床试验中。合理的药物设计得益于可视化的三维酶结构，许多HIV-1整合酶催化核心区域的结构已经得到解决。虽然也解决了两个结构域n端/核心和核心/c端结构，但没有完整的整合酶蛋白结构，这在很大程度上可归因于蛋白质溶解度差。在细胞中，整合酶可能与正常的人类蛋白质相互作用，整合酶与转录共激活物晶状体上皮衍生生长因子（LEDGF）之间的相互作用形成了这种应用的基础。我们最近利用核磁共振光谱解决了LEDGF中整合酶结合域（IBD）的三维结构，并在此展示了与HIV-1整合酶二聚体核心域结合的IBD的晶体结构。基于这些结果，我们建议利用LEDGF IBD形成新的LEDGF- in复合物，并求解全长整合酶蛋白的三维结构。我们的新结构揭示了核心区域二聚体界面上的一个口袋，该口袋在络合物形成时被LEDGF热点残基占据。由于整合酶突变会破坏与LEDGF的相互作用，从而杀死HIV-1，因此我们假设，与核心区域结合并阻止LEDGF结合的化合物可能类似地削弱HIV-1。先前的结果显示，某些整合酶抑制剂结合在这个口袋或附近，但这些化合物不抑制ledgf -整合酶的相互作用。一种新的分析系统将被设计用于选择ledgf整合酶相互作用的抑制剂。这些实验的结果将显著有助于HIV-1整合酶功能抑制剂的设计，因为完整酶的三维结构将被阐明，并将开发一种可以选择HIV-1复制的新型抑制剂的检测方法。

DESCRIPTION (provided by applicant): Like all retroviruses, HIV-1 must integrate the cDNA copy made by reverse transcription into a cell chromosome in order to replicate. HIV-1 integration is catalyzed by the essential viral enzyme integrase. This makes the integrase an attractive drug target, and anti-integrase compounds are in US clinical trials. Rational drug design benefits from visualizing three-dimensional enzyme structures, and numerous structures of the HIV-1 integrase catalytic core domain have been solved. Although two domain N-terminal/core and core/C-terminal structures are also solved, there is no structure for the intact integrase protein, which in large part can be attributed to poor protein solubility. In cells, integrase is likely to interact with normal human proteins, and the interaction between integrase and the transcriptional co-activator lens epithelium-derived growth factor (LEDGF) forms the basis for this application. We recently solved the three-dimensional structure of the integrase-binding domain (IBD) in LEDGF using NMR spectroscopy and herein present the crystal structure of the IBD bound to the dimeric core domain of HIV-1 integrase. Building from these results, we propose to utilize the LEDGF IBD to form novel LEDGF-IN complexes and to solve the three-dimensional structure of the full-length integrase protein. Our novel structure revealed a pocket at the core domain dimer interface that is occupied by LEDGF hot spot residues upon complex formation. Because integrase mutations that ablate the interaction with LEDGF kill HIV-1, we hypothesize that compounds that bind to this region of the core and preclude LEDGF binding might similarly cripple HIV-1. Previous results revealed that certain integrase inhibitors bind at or near this pocket, but those compounds did not inhibit the LEDGF-integrase interaction. A novel assay system will be designed to select for inhibitors of the LEDGF-integrase interaction. The results of these experiments will significantly aid the design of inhibitors of HIV-1 integrase function, as the three-dimensional structure of the intact enzyme will be elucidated and an assay that could select for novel inhibitors of HIV-1 replication will be developed.