Structure-based Antiviral Design against HTLV-1 Protease

项目摘要 人类T细胞白血病病毒1型（HTLV-1）是一种致癌的人类逆转录病毒，影响超过2000万人 国际吧HTLV-1感染可引起成人T细胞淋巴瘤（ATL）等严重炎症性疾病。 据估计，5-10%的HTLV-1感染患者将发展成严重的病症，如ATL，其具有 4年生存率低，复发率高。HTLV-1在地球仪上具有持续的感染率， 在某些社区的患病率高达45%。尽管对人类健康有这种影响， 抗病毒药物（DAA）或针对HTLV-1的疫苗。HIV-1和HTLV-1来自相同的病毒家族， 一种对从病毒多聚蛋白中切割功能蛋白至关重要的同型二聚体乙酰基蛋白酶。活动 HIV-1和HTLV-1蛋白酶的活性对它们的病毒生命周期至关重要。希弗实验室拥有广泛的 具有病毒蛋白酶晶体学和抑制的经验，尤其是HIV-1、HCV病毒蛋白酶 NS 3/4A和SARS-CoV-2主要蛋白酶。这种专业知识使我能够设计，合成， 表征针对HTLV-1蛋白酶的有效的抵抗性阻碍蛋白酶抑制剂。防阻 DAA设计是必不可少的，因为在DAA治疗过程中施加的选择性压力。一个理想的和证明 开发高效和耐药性预防病毒蛋白酶抑制剂的策略是靶向活性 网站通过合理的设计使用基板信封。HTLV-1蛋白酶的底物包膜尚未 已经被表征，我们缺乏对蛋白酶底物特异性的详细了解。我假设 通过翻译我们设计的HIV-1蛋白酶抑制剂的策略，即表征HTLV-1蛋白酶的 底物特异性，我可以设计有效的和耐药性预防的DAA的HTLV-1蛋白酶。 目的1：研究HTLV-1蛋白酶底物特异性的结构基础。HTLV-1蛋白酶 通过识别病毒多蛋白的单个蛋白质之间的切割位点来切割六种底物。我会 通过测定共晶体来研究这种识别的分子基础， 蛋白酶与结合底物的结构。基质占据的守恒体积将定义 该底物包被并通知针对HTLV-1蛋白酶的抑制剂设计。目标二：合理设计、合成， 并表征HTLV-1蛋白酶的抑制剂以优化效力。HIV-1和HTLV-1蛋白酶共享一个 活性位点氨基酸序列同源性为45%，结构相似性高。因此，我将开始抑制剂 通过测试我们的内部HIV-1蛋白酶抑制剂的选择来设计，这些抑制剂已经显示出低（1 µM）至 对HTLV-1蛋白酶的中等（30 nM）效力。我将联合收割机实验性抑制试验与共晶 结构分析以鉴定用于抑制剂设计的先导化合物。我将利用底物特异性 通过将抑制剂设计转向模拟底物形状的化合物，利用底物的性质， 底物包被（Aim 1），以及蛋白酶与底物之间的相互作用。我的目标是创作小说， 有效的（亚nM）抑制剂，将是有前途的DAA进一步研究对HTLV-1。

Project Summary Human T-cell leukemia virus type-1 (HTLV-1) is an oncogenic human retrovirus affecting over 20 million people worldwide. HTLV-1 infection can cause adult T-cell lymphoma (ATL) and other serious inflammatory diseases. Estimates report that 5-10% of HTLV-1 infected patients will develop a serious condition such as ATL, which has poor 4-year survival and high relapse rates. HTLV-1 has persistent infection rates across the globe and reaches up to 45% prevalence in certain communities. Despite this impact on human health, there are no direct-acting antivirals (DAAs) or vaccines against HTLV-1. HIV-1 and HTLV-1 are from the same viral family and encode for a homodimeric aspartyl protease crucial for cleavage of functional proteins from viral polyproteins. The activity of HIV-1 and HTLV-1 protease is essential to their viral life cycles. The Schiffer laboratory has extensive experience with viral protease crystallography and inhibition, especially with viral proteases for HIV-1, HCV NS3/4A, and SARS-CoV-2 main protease. This expertise uniquely positions me to design, synthesize, and characterize potent, resistance-thwarting protease inhibitors against HTLV-1 protease. Resistance-preventing DAA design is essential because of the selective pressure applied during DAA treatment. An ideal and proven strategy for developing a highly potent and resistance-preventing viral protease inhibitor is to target the active site through rational design using the substrate envelope. The substrate envelope for HTLV-1 protease has not been characterized and we lack a detailed understanding of the protease substrate specificity. I hypothesize that by translating strategies from our design of HIV-1 protease inhibitors, namely characterizing HTLV-1 protease’s substrate specificity, I can design potent and resistance-preventing DAAs for HTLV-1 protease. Aim 1: Characterize the structural basis for HTLV-1 protease substrate specificity. HTLV-1 protease cleaves six substrates by recognizing cleavage sites between individual proteins of the viral polyprotein. I will investigate the molecular basis of this recognition underlying protease specificity by determining cocrystal structures of the protease with bound substrates. The conserved volume inhabited by the substrates will define the substrate envelope and inform inhibitor design for HTLV-1 protease. Aim 2: Rationally design, synthesize, and characterize inhibitors of HTLV-1 protease to optimize potency. HIV-1 and HTLV-1 proteases share an active site amino acid sequence identity of 45% and high structural similarity. Therefore, I will begin inhibitor design by testing a selection of our in-house HIV-1 protease inhibitors, which have already shown low (1 µM) to moderate (30 nM) potency against HTLV-1 protease. I will combine experimental inhibition assays with cocrystal structure analysis to identify lead compounds for inhibitor design. I will leverage substrate specificity of the protease by moving inhibitor design towards compounds that mimic the shape of substrates, leveraging the substrate envelope (Aim 1), and the interactions between protease and substrate. I aim to produce novel, highly potent (sub-nM) inhibitors that will be promising DAAs for further investigation against HTLV-1.