Small molecule inhibitors of HIV-1 Vif

描述（由申请人提供）：该项目的总体目标是鉴定HIV Vif的小分子抑制剂，抑制其与细胞辅助因子APOBEC3G （A3G）和其他APOBEC3蛋白的结合。Vif是HIV复制所必需的，因此是一个潜在的治疗靶点。A3G和相关胞苷脱氨酶APOBEC3F （A3F）通过诱导病毒DNA中G到A的高突变来抑制HIV复制，此外还有脱氨不依赖的机制。Vif通过结合A3G和A3F这些细胞蛋白并靶向它们进行蛋白酶体降解，从而克服了A3G和A3F固有的抗病毒活性。目前，还没有针对Vif与宿主辅因子相互作用的抗病毒药物。在初步研究中，我们开发了384孔格式的高通量筛选（HTS）来识别Vif-A3G结合抑制剂。我们通过对2640种生物活性化合物的自动筛选验证了HTS，这表明该筛选具有特异性和鲁棒性，并鉴定出15种化合物，这些化合物正在进行基于细胞的测定，以评估其抑制Vif功能和病毒复制的能力。Aim 1将使用该HTS在哈佛ICCB完成100,000种化合物的筛选，以确定Vif-A3G结合的小分子抑制剂，并在二级筛选中测试最佳命中，以确定那些特异性抑制Vif-A3G结合、Vif介导的A3G降解和HIV复制的化合物。Aim 2将选择在体外传代过程中对Aim 1中鉴定的最佳靶点产生耐药性的耐药HIV毒株，并确定耐药突变是否局限于vif基因。Aim 3将在人源化小鼠模型中进行抗病毒功效研究，以确定先导化合物是否能抑制体内HIV复制。这些研究有望鉴定出抑制Vif与A3G和其他APOBEC3蛋白结合的小分子Vif抑制剂，并可能鉴定出具有进一步开发新的抗病毒治疗潜力的新型先导化合物。

DESCRIPTION (provided by applicant): The overall goal of this project is to identify small molecule inhibitors of HIV Vif that inhibit binding to its cellular cofactor APOBEC3G (A3G) and other APOBEC3 proteins. Vif is required for HIV replication, and is therefore a potential therapeutic target. A3G and the related cytidine deaminase APOBEC3F (A3F) inhibit HIV replication by inducing G to A hypermutation in viral DNA, in addition to deamination-independent mechanisms. Vif overcomes the innate antiviral activity of A3G and A3F by binding these cellular proteins and targeting them for proteasomal degradation. Currently, there are no antivirals that target Vif or its interactions with host cofactors. In preliminary studies, we developed a high-throughput screen (HTS) in 384-well format to identify inhibitors of Vif-A3G binding. We validated the HTS by automated screening of 2,640 bioactive compounds, which demonstrated that the screen is specific and robust and identified 15 compounds that are being evaluated for their ability to inhibit Vif function and viral replication in cell-based assays. Aim 1 will use this HTS to complete screening of 100,000 compounds at the Harvard ICCB to identify small molecule inhibitors of Vif-A3G binding and test the best hits in secondary screens to identify those that specifically inhibit Vif-A3G binding, Vif- mediated degradation of A3G, and HIV replication. Aim 2 will select for drug-resistant HIV strains that develop resistance to the best hits identified in Aim 1 during passage ex vivo and determine if resistance mutations localize to the vif gene. Aim 3 will perform antiviral efficacy studies in a humanized mouse model to determine whether lead compounds inhibit HIV replication in vivo. These studies are expected to identify small molecule Vif inhibitors that inhibit Vif binding to A3G and other APOBEC3 proteins, and may identify novel lead compounds with potential for further development as new anti-viral therapies. PUBLIC HEALTH RELEVANCE: This project will use a new high throughput screening assay to identify inhibitors of the HIV Vif protein that inhibit Vif binding to its host cell targets APOBEC3G and APOBEC3F. These studies may identify small molecules that inhibit HIV Vif, including lead compounds that could be further developed as new anti-viral therapies