Mechanisms of HIV-1 Hypersensitivity to Islatravir

项目摘要/摘要 截至2020年，3770万患者感染了人类免疫缺陷病毒1型(HIV-1)。组合 抗逆转录病毒疗法(CART)用于治疗HIV-1感染者，但这些CART的有效性 受到病毒耐药性突变的威胁，最常见的原因是患者不坚持每日服药 养生法。迫切需要具有长效潜力的有效抗病毒药物来防止耐药性突变。 伊斯拉拉韦(4‘-乙炔基-2-氟-2’-脱氧腺苷)，通常被称为EFdA，是一种高效的长效HIV- 1核苷逆转录酶(RT)易位抑制剂目前处于III期临床试验。在这些临床上 在试验中，EFdA与包括非核苷类RT抑制剂在内的其他HIV-1抗病毒药物联合使用。 有趣的是，非核苷逆转录酶抑制剂(NNRTI)的治疗包括多拉韦林、依曲韦林、利培韦林或 埃尔法韦林导致RT F227C突变，显著使HIV-1对EFdA更敏感。 然而，为什么位于遥远的非核苷类RT抑制因子口袋的突变会导致RT EFdA是一种结合在聚合酶活性部位的化合物，对EFdA敏感，目前还不清楚。研究是 需要了解RTF227C对EFdA超敏的机制以及该突变如何影响HIV-1 对各种RT抗病毒药物的敏感性。我推测NNRTI结合口袋上的F227C突变 从结构上改变聚合酶活性部位，导致对EFdA过敏。为了检验这一假设，我首先 用X射线结晶学确定RTF227C对EFdA超敏的结构基础(目标1)。第二, 我将通过一组生化电池来确定RTF227C对EFdA超敏的生化基础 化验。这些检测将测试F227C突变如何影响EFdA-三磷酸掺入，能力 RT解锁EFdA终止的引子，以及RT沿核酸移位的能力(目标2)。 最后，我将确定F227C突变如何影响HIV-1对各种EFdA和RT抑制剂的易感性 通过生化和基于细胞的分析进行组合(目标3)。我会充分利用我们实验室的 RT生物学背景和在EFdA发展中的作用，因为它提供了必要的环境， 设备，和简化的协议，以完成这些拟议的实验，并对我的培训做出贡献。 这些计划中的研究不仅将促进对RT耐药突变如何传递的理解 对EFdA过敏，但也将有助于开发与EFdA的优化组合，以治疗 HIV-1的治疗。

PROJECT SUMMARY/ ABSTRACT As of 2020, 37.7 million patients are living with human immunodeficiency virus type 1 (HIV-1). Combination antiretroviral therapies (cARTs) are used to treat HIV-1 infected patients, but the effectiveness of these cARTs is threatened by viral drug resistance mutations most frequently caused by patient non-adherence to daily dosing regimens. Potent antivirals with long-acting potential are urgently needed to prevent drug resistance mutations. Islatravir (4’-ethynyl-2-fluoro-2’-deoxyadenosine), often referred to as EFdA, is a highly potent long-acting HIV- 1 nucleoside reverse transcriptase (RT) translocation inhibitor currently in phase III clinical trials. In these clinical trials, EFdA is administered in combination with other HIV-1 antivirals including non-nucleoside RT inhibitors. Interestingly, non-nucleoside RT inhibitor (NNRTI) treatment, which include Doravirine, Etravirine, Rilpivirine, or Elsulfavirine, leads to the RT F227C mutation, which remarkably makes HIV-1 more susceptible to EFdA. However, why a mutation located at the distant non-nucleoside RT inhibitor pocket would cause RT to become hypersensitive to EFdA, a compound that binds at the polymerase active site is not understood. Studies are needed to understand the mechanism of RTF227C hypersensitivity to EFdA and how this mutation affects HIV-1 susceptibility to various RT antivirals. I hypothesize that the F227C mutation at the NNRTI binding pocket structurally alters the polymerase active site, causing hypersensitivity to EFdA. To test this hypothesis, I will first determine the structural basis of RTF227C hypersensitivity to EFdA using X-ray crystallography (Aim 1). Second, I will determine the biochemical basis of RTF227C hypersensitivity to EFdA through a battery of biochemical assays. These assays will test how the F227C mutation impacts EFdA-triphosphate incorporation, the ability of RT to unblock EFdA-terminated primers, and the ability of RT to translocate along the nucleic acid (Aim 2). Finally, I will determine how the F227C mutation affects HIV-1 susceptibility to various EFdA and RT inhibitor combinations through biochemical and cell-based assays (Aim 3). I will take full advantage of our lab’s background in RT biology and role in the development of EFdA, as it provides the necessary environment, equipment, and streamlined protocols to complete these proposed experiments and contribute to my training. These planned studies will not only advance the understanding of how RT drug resistance mutations impart hypersensitivity to EFdA but will also aid in the development of optimized combinations with EFdA for the treatment of HIV-1.