Ultrapotent Inhibitors of Wild-type and Multi-drug Resistant HIV

项目总结 核苷/核苷酸逆转录酶抑制剂(NRTI)有效地抑制HIV，并作为骨架 抗逆转录病毒疗法。然而，需要新的治疗方法来继续控制艾滋病毒感染。4‘- 乙炔基-2-氟-2‘-脱氧腺苷(EFdA)是一种高效、稳定、独特的NRTI 对抗艾滋病毒的行动机制，已获得默克公司的许可。EFdA(也称为MK-8591或Islatavir) 已经成功地在第一阶段使用，最近在第二阶段临床试验中推出。EFdA已生成 在极低剂量(最高可达)的情况下，对患者进行测试，得出了令人信服的早期治疗和预防结果 &gt；剂量比目前的NRTI药物低10,000倍)，目标是每周口服一次，每年一次缓释 剂量，前所未有的艾滋病毒治疗方式。因此，这项工作与美国国立卫生研究院的指南直接相关 高度优先的艾滋病资金(非OD-15-137)，用于“下一代疗法…”他们表演的时间更长了。 我们已经证明：a)EFdA在体外、在细胞培养(PBMCs的EC50=50 PM)、小鼠和非 人类灵长类动物模型。虽然EFdA保留了3‘-羟基，但它阻止了HIV逆转录酶(RT)在 在体外，主要作为直接/专性的，有时由于EFdA-困难而延迟的链终止物- 终止病毒DNA转位。因此，EFdA被称为核苷酸逆转录酶易位 抑制剂(NRTTI)。然而，EFdA在HIV感染细胞(原代或细胞系)中的抑制机制仍然存在 未知。为此，Co-I Malim建立了一种创新的分析方法，使EFdA的研究成为可能 HIV感染细胞中的抗病毒机制。此外，体外传代实验已经确定了突变。 通过两种范式转换的双重机制或抵抗来传递EFdA抵抗：减少 合并/增强切除和减少合并/增强易位。EFdA模拟器上的数据 建议有效地抑制耐EFdA的艾滋病毒，尽管这种现象的机制并不是 明白了。值得注意的是，使用关键的新一代NNRTI多拉韦林(DOR)治疗会导致耐药性突变 这使EFdA具有超敏性，为EFdA/DOR联合疗法铺平了道路。我们假设 EFdA及其类似物的结构属性影响与各种RT的相互作用(野生型、药物- 抗药性，来自不同的分支，病毒)，导致临床上重要的抑制效率差异， 抗性、过敏性和生化作用机制。这一假设将通过 三个具体的目标，努力使用新的排序的组合来回答上述问题， 生化、结构和病毒学方法。这项工作将有助于优化组合疗法， 减少药物负担并具有非凡的长效潜力，应对慢性艾滋病毒的坚持挑战 治疗。

PROJECT SUMMARY Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) efficiently suppress HIV and serve as backbone of antiretroviral therapies. However, new therapeutics are needed for continued control of HIV infection. 4’- Ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) is an NRTI with exceptional potency, stability, and unique mechanism of action against HIV, which has been licensed by Merck. EFdA (also known as MK-8591 or Islatavir) has been successfully used in Phase I and recently introduced in Phase II clinical trials. EFdA has generated “compelling early results for both treatment and prevention” in patients, tested at remarkably low doses (up to >10,000-fold lower doses than current NRTI drugs) aiming at once-weekly oral and once-yearly slow-release dosing, unprecedented modalities for HIV therapies. Hence, this work is directly relevant to NIH guidelines for high priority AIDS funding (NOT-OD-15-137) for “next generation therapies…that are longer acting.” We have shown that a) EFdA has high potency in vitro, in cell culture (EC50=50 pM in PBMCs), mice and non- human primate animal models. Although EFdA retains the 3’-OH, it blocks HIV reverse transcriptase (RT) in vitro, primarily as an immediate/obligate and at times delayed chain terminator due to difficulty of EFdA- terminated viral DNA to translocate. Thus, EFdA is termed a nucleotide reverse transcriptase translocation inhibitor (NRTTI). However, the inhibition mechanism of EFdA in HIV-infected cells (primary or cell lines) remains unknown. Toward that end, Co-I Malim has established an innovative assay that enables study of the EFdA antiviral mechanism in HIV-infected cells. Additionally, in vitro passage experiments have identified mutations that impart EFdA resistance through two paradigm-shifting dual mechanisms or resistance: decreased incorporation/enhanced excision and decreased incorporation/enhanced translocation. Data on an EFdA analog suggest efficient inhibition of EFdA-resistant HIV, although the mechanism of this phenomenon is not understood. Notably, treatment with key new generation NNRTI doravirine (DOR) leads to resistance mutations that impart hypersusceptibility to EFdA, paving the way for EFdA/DOR combination therapies. We hypothesize that the structural attributes of EFdA and its analogs impact interactions with diverse RTs (wild-type, drug- resistant, from diverse clades, viruses), leading to clinically important differences in efficiency of inhibition, resistance, hypersusceptibility, and biochemical mechanism of action. This hypothesis will be tested through three specific aims, which endeavor to answer the above questions using a combination of novel sequencing, biochemical, structural, and virological approaches. This work will help optimize combination therapies that reduce drug burden and have exceptional long-acting potential, addressing adherence challenges of chronic HIV treatment.