Development and Characterization of Antiviral Drugs That

通过合作研究，我们已经确定含叠氮基的β-二酮酸衍生物（DKA）作为HIV-1复制的有效抑制剂。我们还分析了DKA对2-LTR环的频率以及2-LTR环连接处的序列的影响。这些结果表明，DKA的结构可以影响宿主核酸酶降解病毒DNA末端的程度和2-LTR环连接处缺失的频率。目前还没有合适的动物模型系统用于测试HIV-1 IN抑制剂。我们正在构建依赖于HIV-1 RT和IN复制的猴免疫缺陷病毒（SIV）-HIV-1杂交病毒（SHIV）（SHIV-RT-IN）。SHIV-RT-IN病毒应提供合适的动物模型系统，以评价含有抗IN抑制剂单独或与RT抑制剂联合的联合抗病毒治疗，并表征HIV-1 IN中赋予IN抑制剂耐药性的突变。与Kvaratskhelia博士的实验室合作，我们进行了质谱分析，以确定HIV-1 IN的核苷酸类似物抑制剂5 '-磷酸吡哆醛的HIV-1 IN结合位点。这些结果首次表明，小的抑制剂分子可以通过结合蛋白质C-末端来抑制IN活性。在合作研究中，我们正在探索新的方法来鉴定抑制HIV-1 IN的先导化合物。该项目的目的是在生物信息学和计算机辅助药物设计的最新技术的基础上发现HIV-1 IN的候选抑制剂。将在体外测试所有候选化合物抑制HIV-1整合酶的能力，并制备足量的最有效物质。有前途的先导化合物将在我们开发的基于细胞的测定中进行测试，以确定它们抑制HIV-1复制的能力，并确定抗病毒活性的分子靶点。结合这些研究，我们正在开发体内试验，以确定HIV-1 IN抑制剂的细胞内作用机制，并正在开发定量方法，以确定潜在的HIV-1 IN抑制剂对2-LTR环形成的影响。最后，我们正在开发新的体内试验，以确定新的先导化合物，抑制HIV-1 IN。

Through collaborative studies, we have identified azido-group containing beta-diketo acid derivatives (DKAs) as potent inhibitors of HIV-1 replication. We have also analyzed the effects of the DKAs on the frequency of 2-LTR circles as well as the sequences at the 2-LTR-circle junctions. These results suggest that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. A suitable animal model system for testing of HIV-1 IN inhibitors is currently not available. We are constructing simian immunodeficiency virus (SIV)-HIV-1 hybrid viruses (SHIVs) that are dependent on the HIV-1 RT and IN for replication (SHIV-RT-IN). The SHIV-RT-IN viruses should provide a suitable animal model system to evaluate combination antiviral therapies containing anti-IN inhibitors alone or in combination with RT inhibitors and to characterize mutations in HIV-1 IN that confer resistance to the IN inhibitors. In collaboration with Dr. Kvaratskhelia's laboratory, we have performed mass spectrometric analysis to identify the HIV-1 IN binding site for a nucleotide analog inhibitor of HIV-1 IN, pyridoxal 5'-phosphate. These results indicate for the first time that a small inhibitor molecule can inhibit IN activity by binding to the protein C-terminus. In collaborative studies, we are exploring novel methods to identify lead compounds that inhibit HIV-1 IN. The purpose of the project is to discover candidate inhibitors of HIV-1 IN on the basis of latest technologies in bioinformatics and computer-aided drug design. The capability of all candidate compounds to inhibit HIV-1 integrase will be tested in vitro, and the most potent substances will be prepared in sufficient amounts. The promising lead compounds will be tested in the cell-based assays that we have developed to determine their ability to inhibit HIV-1 replication and to determine the molecular targets of antiviral activity. In conjunction with these studies, we are developing in vivo assays to define the intracellular mechanisms of action of HIV-1 IN inhibitors and are developing quantitative methods to determine the effects of potential HIV-1 IN inhibitors of 2-LTR circle formation. Finally, we are developing novel in vivo assays to identify new lead compounds that inhibit HIV-1 IN.