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

项目摘要 艾滋病毒是一项重大的公共卫生挑战，目前影响着全世界3 800万人。有几 已报告在药物敏感性和耐药性方面存在差异的HIV亚型。理解这些 这些差异对于艾滋病毒的预防和治疗战略很重要。患者对当前药物的依从性 抗病毒药物是一个主要问题，因为不依从可能导致病毒耐药性突变，从而导致治疗失败。 目前的治疗。这项工作将有助于开发长效艾滋病毒治疗方案， 提高患者依从性。该提案使用了一种新型核苷逆转录酶易位抑制剂 （NRTTI），称为4'-乙炔基-2-氟-2'-脱氧腺苷（EFdA、MK-8591或Islatravir）。如果实施EFdA 作为一种治疗方法，它可以有助于在全球范围内预防和治疗艾滋病毒的长效方案。 特别是在低收入国家。已经进行了几项EFdA抑制和抗性研究， 然而，这些研究主要使用HIV的B亚型（HIV-B：主要发现于北美、欧洲， 日本）和HIV-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。其他亚型， 将在本提案中研究的项目包括CRF_AE和CRF_AG（西非和东南亚）以及C（撒哈拉以南非洲 非洲、印度、巴西），占全球艾滋病毒流行率的60%左右。因此，关键是 在这些不同亚型的背景下理解EFdA。总体假设是， 序列差异决定了抑制机制，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。目的1将确定临床耐药突变对现有耐药基因的影响。 药物对HIV-非B病毒对EFdA的易感性和病毒适应性的影响。这将取决于剂量- 响应曲线、病毒复制、竞争和病毒传代测定。此外，EFdA的作用机制 抑制和耐药性已在体外仅使用HIV亚型B RT进行了初步研究。因此，Aim 2将 确定各种HIV亚型中EFdA抑制和耐药性的体外和细胞内机制。 这些基于凝胶的体外生物化学测定将利用纯化的RT酶。此外，一种新技术将 用于确定EFdA的细胞内抑制机制，EFdA在3'- 逆转录酶产物的末端。这些研究将使用多种细胞系进行， 容易产生抗药性。本工作将揭示EFdA在细胞凋亡中的抑制机制。 细胞环境和EFdA在各种亚型中的疗效，从而产生更好的基于EFdA的疗法， 可以帮助全球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.