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

描述（由申请人提供）：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。任何成功的预防战略都必须在感染确定之前发挥作用，而确定的逆转录病毒感染的标志是整合。病毒整合酶在体内催化至少两种核酸内切酶反应：为整合准备病毒DNA末端的特异性缺口和将病毒DNA非特异性插入细胞DNA。整合酶还具有强大的非特异性内切酶活性，可以在体外切割任何DNA序列，并且这种活性受到某些小化合物的显著刺激。这些事实提示了一种新颖（且具有讽刺意味）的抗病毒策略，即在整合之前刺激整合酶破坏病毒DNA（对细胞DNA的任何损伤仅限于新感染的细胞，并阻断感染）。因此，本提案的目标是确定HIV-1整合酶的非特异性切口活性的有效刺激物，并将这些药物中的至少一种带到临床试验的边缘。基于已知的先例和初步数据的中心假设是，整合酶的非特异性核酸内切酶活性可以刺激一种新的抗病毒策略，这种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期，Aim 1将优化整合酶介导的非特异性DNA缺口的高通量检测；Aim 2将在宾夕法尼亚州立大学药物开发和发现中心筛选50,000种化学物质，以寻找刺激HIV-1整合酶非特异性地切割DNA的其他药物（通过适当的二次分析来验证阳性命中）；Aim 3将使用定量抗病毒和细胞毒性研究，根据整合酶刺激剂（IS）化合物的治疗指标对其进行优先排序，同时反馈给有机化学家，他们将以迭代的方式设计和合成先导化合物的合理类似物进行测试。在R33期，Aim 4将在扩大的毒性研究中测试每种候选IS化合物的安全性，包括小鼠宫颈阴道毒性模型；而Aim 5将在NOD-SCID-hu感染HIV-1小鼠模型中测试针对不同亚型HIV-1的抗病毒活性范围。这些数据也将反馈到发现途径，以制造新的IS化合物衍生物。最后，由于理想的杀微生物剂方案将结合细胞外阻止病毒进入的药物和细胞内阻止任何进入的病毒感染的药物，Aim 6将评估包括IS化合物的杀微生物剂组合对HIV-1的协同活性，为至少一种候选药物进入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.