Novel Stimulators of HIV-1 Integrase for Use in Combination Microbicide Regimens

描述(申请人提供)：该项目的长期目标是开发干扰艾滋病毒复制和传播的方法，这将对预防艾滋病毒/艾滋病的传播产生重大影响。任何成功的预防策略都必须在感染确定之前奏效，而已确定的逆转录病毒感染的标志是整合。病毒整合酶在体内至少催化两种内切酶反应：为整合准备病毒DNA末端的特异性划痕和将病毒DNA非特异性插入细胞DNA。整合酶也有很强的非特异性核酸内切酶活性，可以在体外切割任何DNA序列，这种活性可以被某些小化合物显著刺激。这些事实表明了一种新的(具有讽刺意味的)抗病毒策略，其中整合酶受到刺激，在整合之前破坏病毒DNA(对细胞DNA的任何破坏仅限于新感染的细胞，并阻止感染)。因此，这项建议的目标是确定HIV-1整合酶非特异性切割活性的有效刺激因素，并将其中至少一种药物推向临床测试的边缘。根据已知的先例和初步数据，中心假设是整合酶的非特异性内切酶活性可以被刺激，用于一种新的抗病毒策略，该策略可以成为安全有效的联合杀微生物剂方案的一部分。在R21阶段，Aim 1将优化整合酶介导的非特异性DNA划痕的高通量分析；Aim 2将在宾夕法尼亚州立大学药物开发和发现核心筛选50,000种化学物质，寻找刺激HIV-1整合酶非特异性划痕DNA的额外药物(通过适当的二次分析来验证阳性HITS)；以及Aim 3将使用定量抗病毒和细胞毒性研究，根据整合酶刺激物(IS)化合物的治疗指数优先排序，同时反馈给有机化学家，他们将设计和合成合理的先导化合物类似物，以便以迭代方式进行测试。在R33阶段，Aim 4将在扩大毒性研究中测试每个候选化合物的安全性，包括宫颈阴道毒性的小鼠模型；Aim 5将测试针对不同亚型HIV-1的抗病毒活性范围以及在NOD-SCID-Hu小鼠HIV-1感染模型中的抗病毒活性。这些数据还将反馈到发现途径，以制造IS化合物的新衍生品。最后，由于理想的杀微生物剂方案是将在细胞外工作以阻止病毒进入的药物与在细胞内工作以终止任何进入细胞的病毒感染的药物相结合，因此Aim 6将评估包括对HIV-1具有协同活性的化合物的微生物剂组合，为将至少一种候选药物转移到未来的临床试验做准备。

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop ways to interfere with HIV replication and transmission, which would have major implications for preventing the spread of HIV/AIDS. Any successful preventive strategy must work before infection becomes established, and the hallmark of an established retroviral infection is integration. The viral integrase enzyme catalyzes at least two endonuclease reactions in vivo: specific nicking to prepare the ends of viral DNA for integration and nonspecific insertion of the viral DNA into cellular DNA. Integrase also has a potent nonspecific endonuclease activity that can nick any DNA sequence in vitro, and this activity is dramatically stimulated by certain small compounds. These facts suggest a novel (and ironic) antiviral strategy in which integrase is stimulated to destroy viral DNA before integration (with any damage to cellular DNA being limited to newly infected cells and also blocking infection). Thus, the objectives of this proposal are to identify potent stimulators of the nonspecific nicking activity of HIV-1 integrase and to bring at least one of these agents to the verge of clinical testing. The central hypothesis, based on known precedents and preliminary data, is that integrase's nonspecific endonuclease activity can be stimulated for a new antiviral strategy that can be part of a safe and effective combination microbicide regimen. In the R21 phase, Aim 1 will optimize a high-throughput assay for integrase-mediated nonspecific DNA nicking; Aim 2 will screen 50,000 chemicals in the Penn State Drug Development and Discovery Core for additional agents that stimulate HIV-1 integrase to nick DNA nonspecifically (with appropriate secondary assays to validate positive hits); and Aim 3 will use quantitative antiviral and cell toxicity studies to prioritize integrase stimulator (IS) compounds based on their therapeutic indices, all the while feeding back to organic chemists who will design and synthesize rational analogues of lead compounds for testing in an iterative fashion. In the R33 phase, Aim 4 will test the safety of each candidate IS compound in expanded toxicity studies, including a mouse model of cervicovaginal toxicity; and Aim 5 will test the range of antiviral activity against different subtypes of HIV-1 and in a NOD-SCID-hu mouse model of HIV-1 infection. These data will also feed back to the discovery pathway to make new derivatives of IS compounds. Finally, because the ideal microbicide regimen would combine agents that work outside cells to impede virus entry with agents that work inside cells to abort infection for any viruses that do gain entry, Aim 6 will evaluate microbicide combinations that include IS compounds for cooperative activity against HIV-1, in preparation for moving at least one drug candidate into future clinical trials.