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

使用 EFdA 靶向多种亚型的 HIV：针对临床耐药突变体的抑制机制和功效

• 批准号: 10257815
• 负责人: Maria Cilento
• 金额: $ 4.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10257815
• 关键词: Using; EFdA; target; HIV; various

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.

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 3800 万人。有几个 已报道药物敏感性和耐药性差异的艾滋病毒亚型。了解这样的 差异对于艾滋病毒的预防和治疗策略非常重要。患者坚持当前 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 目前的疗法。这项工作将有助于开发长效艾滋病毒治疗方案，预计将 提高患者的依从性。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，它可以为全球预防和治疗艾滋病毒的长效方案做出贡献 规模，特别是在低收入国家。已有多项 EFdA 抑制和耐药研究， 然而，这些研究主要使用 HIV B 亚型（HIV-B：主要发现于北美、欧洲和 日本）和 HIV-B 仅占 HIV 感染的 11%。为了在全球范围内实施 EFdA，HIV- 需要对占全球艾滋病毒感染绝大多数的非 B 亚型进行研究。其他亚型 本提案中将研究的内容包括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），这些国家的艾滋病毒感染率约占全球的 60%。因此，至关重要的是 在这些不同亚型的背景下理解 EFdA。总体假设是亚型特异性 序列差异决定了抑制机制，从而决定了病毒对 EFdA 的敏感性。这个假设 将通过两个具体目标来解决。目标 1 将确定临床耐药突变对现有药物的影响 药物对 HIV-nonB 病毒对 EFdA 的易感性和病毒适应性的影响。这将由剂量决定- 反应曲线、病毒复制、竞争和病毒传代测定。此外，EFdA的作用机制 抑制和耐药性已主要在体外仅使用 HIV B 亚型 RT 进行研究。因此，目标 2 将 确定各种 HIV 亚型中 EFdA 抑制和耐药的体外和细胞机制。 这些体外基于凝胶的生化测定将利用纯化的 RT 酶。此外，一项新技术将 用于确定 EFdA 的细胞内抑制机制，该机制在 3'- 处具有单核苷酸分辨率 逆转录酶产物的末端。这些研究将使用多种细胞系进行 使耐药性易于发展。这项工作将揭示 EFdA 在 不同亚型的细胞环境和 EFdA 功效，从而产生更好的基于 EFdA 的疗法 可以帮助全世界预防和治疗艾滋病毒。