Enhancing the Properties of HIV-1 Integrase and Determination of its Structure in Complex with DNA Substrates

 描述(申请人提供)：HIV-1整合酶(IN)是将病毒基因组插入宿主DNA的基本病毒蛋白，是有效的抗病毒靶标。最近，针对IN活性部位的抗艾滋病药物已经开发出来，现在正在临床使用。然而，正如经常观察到的其他药物靶标一样，对这些IN抑制剂具有抗药性的艾滋病毒株已经出现，这突显了不断需要增加我们对这种病毒蛋白的了解，以便设计新的抑制剂来增加我们对抗这种致命病毒的武器。这项申请建议单独研究HIV IN的分子结构，并结合病毒DNA底物进行研究。为了进行整合，IN蛋白必须在特定的排列中与连接体复合体中的病毒DNA多聚化。我们的理由是，了解肠小体如何形成的分子细节将揭示新的漏洞，可用于开发抑制剂。我们建议通过引入连续的靶向改变来改善IN的物理性质，这些改变将促进肠小体的形成和稳定性，同时保持整合所需的催化活性。这些变化将被结合在一起，以优化肠酶体的形成，一旦分离，肠酶体将受到高分辨率和低分辨率方法的结构测定。从本申请中描述的实验中获得的结构信息将为开发针对整合酶的新型抗艾滋病药物奠定基础。

 DESCRIPTION (provided by applicant): HIV-1 Integrase (IN) is the essential viral protein that inserts the viral genome into host DNA, and is a validated antiviral target. Most recently anti-AIDS drugs that target the active site of IN have been developed and are now in clinical use. However, as is often observed with other drug targets, HIV strains that are resistant to these IN inhibitors have emerged, highlighting the constant need to increase our knowledge of this viral protein so that new inhibitors may be designed to add to our arsenal against this deadly virus. This application proposes to study the molecular structure of HIV IN alone and in combination with viral DNA substrates. To perform integration, IN proteins must multimerize in a particular arrangement with viral DNA in the intasome complex. We reason that understanding the molecular details of how the intasome is formed will reveal new vulnerabilities that can be exploited for developing inhibitors. We propose to improve the physical properties of IN for biophysical studies by introducing sequential targeted changes that will promote intasome formation and stability, while maintaining the catalytic activity required for integration. These changes will be combined to optimize intasome formation and, once isolated, intasomes will be subjected to structure determination by both high and low resolution methods. The structural information gained from the experiments described in this application will lay the foundation for the development of a new class of anti-AIDS drugs that target integrase.