Using EFdA to target HIV from various subtypes:Inhibition mechanism and efficacy against clinical resistant mutants

项目摘要 艾滋病毒是一个重大的公共卫生挑战，目前影响着全世界3800万人。有几个 报告在药物敏感性和耐药性方面存在差异的艾滋病毒亚型。了解以下内容 差异对于指导艾滋病毒的预防和治疗战略很重要。患者对当前 抗病毒药物是一个主要问题，因为不坚持会导致病毒抗药性突变，从而导致失败 目前的治疗方法。这项工作将有助于开发长效艾滋病毒方案，预计将 提高患者的依从性。该方案使用了一种新的核苷逆转录酶转位抑制剂 (NRTTI)，称为4‘-乙炔基-2-氟-2’-脱氧腺苷(EFdA、MK-8591或Islatravir)。如果实施了EFdA 作为一种治疗方法，它可能有助于全球艾滋病毒的预防和治疗的长效方案 规模，特别是在低收入国家。已经有几项EFdA抑制和耐药性研究， 然而，这些研究主要使用B亚型艾滋病毒(艾滋病毒-B：主要发现于北美、欧洲和 日本)，而艾滋病毒-B仅占艾滋病毒感染的11%。为了在全球范围内实施EFdA，艾滋病毒- 占全球艾滋病毒感染绝大多数的非B亚型需要研究。其他子类型 将在本提案中研究的包括CRF_AE和CRF_AG(西非和东南亚)和C(撒哈拉以南地区 非洲、印度、巴西)，约占全球艾滋病毒流行率的60%。因此，至关重要的是 在这些不同的子类型的上下文中理解EFdA。最重要的假设是特定于子类型 序列差异决定了抑制机制(S)，从而决定了病毒对EFdA的易感性。这一假设 将在两个具体目标中加以解决。目标1将确定临床耐药突变对现有的 关于艾滋病毒-非B类病毒对EFdA易感性和病毒适应性的药物。这将由剂量决定- 反应曲线、病毒复制、竞争和病毒传代试验。此外，EFdA的作用机制 仅使用HIV B亚型RT在体外初步研究了抑制和耐药性。因此，目标2将 确定不同亚型HIV对EFdA抑制和耐药的体外和细胞内机制(S)。 这些基于凝胶的体外生化分析将使用纯化的RT酶。此外，一种新的技术将 用于确定具有3‘-单核苷酸分辨率的EFdA在细胞内的抑制机制 逆转录酶产物的终端。这些研究将对各种细胞系进行，这些细胞系 使抗药性得以迅速发展。这项工作将揭示EFdA在体内的抑制作用机制。 细胞环境和不同亚型的EFdA疗效，导致更好的基于EFdA的治疗 有助于在全球范围内预防和治疗艾滋病毒。

PROJECT ABSTRACT HIV is a major public health challenge that currently affects 38 million people worldwide. There are several subtypes of HIV that have reported differences in drug susceptibility and resistance profiles. Understanding such differences are important to inform prevention and treatment strategies of HIV. Patient adherence to current antivirals is a major concern, as non-adherence can lead to viral resistance mutations, which result in failure of current therapies. This work will contribute to the development of long-acting HIV regimens that are expected to improve patient adherence. This proposal uses a novel nucleoside reverse transcriptase translocation inhibitor (NRTTI), known as 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, MK-8591, or Islatravir). If EFdA is implemented as a therapeutic, it could contribute to long-acting regimens for the prevention and treatment of HIV on a global scale, especially in low-income countries. There have been several EFdA inhibition and resistance studies, however, these studies primarily use subtype B of HIV (HIV-B: primarily found in North America, Europe, and Japan) and HIV-B only accounts for 11% of HIV infections. In order to implement EFdA on a global scale, HIV- nonB subtypes that account for the vast majority of global HIV infections need to be studied. Other subtypes that will be studied in this proposal include CRF_AE and CRF_AG (West Africa and SE Asia), and C (sub-Saharan Africa, India, Brazil), which account for about 60% of the global prevalence of HIV. Therefore, it is critical to understand EFdA in the context of these various subtypes. The overarching hypothesis is that subtype-specific sequence differences dictate the inhibition mechanism(s) and thus virus susceptibility to EFdA. This hypothesis will be addressed in two specific aims. Aim 1 will determine the effect of clinical resistance mutations to existing drugs on the susceptibility of HIV-nonB viruses to EFdA and on viral fitness. This will be determined by dose- response curves, viral replication, competition, and viral passaging assays. In addition, the mechanism of EFdA inhibition and resistance has been primarily studied in vitro using only HIV subtype B RT. Therefore, Aim 2 will determine the in vitro and in cellulo mechanism(s) of EFdA inhibition and resistance in various HIV subtypes. These in vitro gel-based biochemical assays will utilize purified RT enzymes. In addition, a novel technique will be used to determine the in cellulo inhibition mechanism of EFdA that has single-nucleotide resolution at the 3’- termini of the reverse transcriptase products. These studies will be conducted with a variety of cell lines that enable facile development of drug resistance. This work will reveal the inhibition mechanism of EFdA in the cellular environment and EFdA efficacy across various subtypes, leading to better EFdA-based therapies that can help prevent and treat HIV worldwide.