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

 性状（由申请方提供）：HIV-1整合酶（IN）是将病毒基因组插入宿主DNA的必需病毒蛋白，是经验证的抗病毒靶标。最近，靶向IN活性部位的抗艾滋病药物已经开发出来，目前已投入临床使用。然而，正如在其他药物靶点中经常观察到的那样，已经出现了对这些IN抑制剂具有耐药性的HIV毒株，这突出了我们不断需要增加对这种病毒蛋白的了解，以便可以设计新的抑制剂来增加我们对抗这种致命病毒的武器库。本申请提出研究HIV IN单独和与病毒DNA底物组合的分子结构。为了进行整合，IN蛋白必须在整合体复合物中与病毒DNA以特定排列多聚化。我们的理由是，了解整合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变化来改善IN的物理性质，这些变化将促进整合体的形成和稳定性，同时保持整合所需的催化活性。这些变化将被组合以优化整合体形成，并且一旦分离，整合体将通过高分辨率和低分辨率方法进行结构测定。从本申请中描述的实验中获得的结构信息将为开发靶向整合酶的一类新的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.