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)是一种致癌性人类逆转录病毒，感染人数超过2000万 全世界。HTLV-1感染可导致成人T细胞淋巴瘤(ATL)和其他严重的炎症性疾病。 据估计，5%-10%的HTLV-1感染者将发展为ATL等严重疾病，这种疾病已经 4年存活率低，复发率高。HTLV-1在全球范围内具有持续的感染率，并 在某些社区中的流行率高达45%。尽管对人类健康有这种影响，但没有直接作用于 抗HTLV-1的抗病毒药物(DAA)或疫苗。HIV-1和HTLV-1来自同一个病毒家族，编码 一种同源二聚体天冬氨酸蛋白酶，对从病毒多蛋白中切割功能蛋白至关重要。活动 HIV-1和HTLV-1蛋白酶的活性对它们的病毒生命周期是必不可少的。希弗实验室拥有广泛的 病毒蛋白水解酶结晶学和抑制经验，尤其是HIV-1、丙型肝炎病毒的病毒蛋白水解酶 NS3/4A和SARS-CoV-2主要蛋白水解酶。这种专业知识使我在设计、合成和 鉴定针对HTLV-1蛋白酶的有效的、抗药性受阻的蛋白酶抑制剂。防病防病 DAA的设计是必不可少的，因为在DAA治疗过程中施加了选择性压力。一个理想而又久经考验的 开发高效和抗药性病毒蛋白水解酶抑制剂的策略是针对活性 网站通过合理设计，采用了衬底封套。HTLV-1酶的底物包膜还没有 但我们对其底物的专一性缺乏详细的了解。我假设 通过将我们设计的HIV-1蛋白酶抑制剂的策略转化为HTLV-1蛋白酶的特征 根据底物的特异性，我可以设计出针对HTLV-1蛋白酶的有效的、耐药的DAAs。 目的1：研究HTLV-1蛋白酶底物专一性的结构基础。HTLV-1蛋白酶 通过识别病毒多蛋白的单个蛋白质之间的裂解位置来裂解六种底物。这就做 通过确定共晶体来研究这种识别的分子基础，其潜在的蛋白酶特异性。 具有结合底物的蛋白酶的结构。底物所占据的保守体积将定义 HTLV-1蛋白水解酶底物包膜及信息抑制剂设计。目标二：合理设计、综合、 并对HTLV-1蛋白酶抑制剂进行表征，以优化效力。HIV-1和HTLV-1蛋白酶共享一个 活性部位氨基酸序列同源性为45%，结构相似性高。因此，我将开始抑制 通过测试我们精选的一系列内部HIV-1蛋白酶抑制剂进行设计，这些药物已经显示出低(1微米)到 对HTLV-1蛋白酶的中等效力(30 NM)。我将把实验性的抑制试验与共晶体结合起来 结构分析以确定用于缓蚀剂设计的先导化合物。我将利用底物的专一性 通过将抑制剂设计移动到模仿底物形状的化合物上，利用 底物包膜(目标1)，以及蛋白酶与底物之间的相互作用。我的目标是制作新奇的，高度的 有效的(亚纳米级)抑制剂，有望为进一步研究HTLV-1提供DAA。

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.