A New Paradigm for HIV Treatment: Targeted Degradation of HIV Reverse Transcriptase via the Ubiquitin-Proteasome Pathway

项目总结/摘要 根据RFA-AI-19-072，针对细胞内HIV靶标的新型治疗药物，我们建议使用 靶向蛋白质降解（TPD）对基本的HIV逆转录酶（RT）。RFA指出 “通过控制细胞内蛋白质水平来控制蛋白质功能已经发展成为一种有前途的新方法， 治疗策略这可以通过靶向降解细胞内蛋白质来实现，通过泛素- 蛋白酶体途径。”异双功能靶向蛋白降解剂（TPD）正被积极追求， 与几种癌症特异性相关的蛋白质的蛋白酶体破坏的增强剂。这些代理商提供 与传统的基于占据性的抑制剂相比的优点包括独特的催化作用机制， 靶向选择性和降低的抗性发展的可能性。令人惊讶的是，这种有前途的治疗方法 最近才通过成功的基于特拉帕韦的TPD将该模式应用于抗病毒药物的发现 对HCV蛋白酶的野生型和抗性形式有效。报告的HIV-1药物结合的分析 结构表明，必需的病毒RT应该容易适应靶向蛋白质降解。的 一种有效的临床上使用的HIV-1 RT抑制剂（RTI），阿匹韦林（RPV）结合Gag-Pol中的RT位点， 通过结合非核苷RTI（NNRTI）结合口袋变构抑制p66/p51 RT功能 （NNIBP）对p66亚基的作用。RPV易于与接头偶联和泛素E3连接酶募集 配体，以用于设计和制备原型HIV-1 RT TPD。本研究的目的是 显示了一种新抑制机制的概念验证，通过该机制HIV-1 RT可以靶向降解， 削弱HIV的传染性和复制。重要的是，由于TPD的独特作用机制，即使是低- 亲和RT/TPD相互作用将可能导致有效的靶降解。因此，我们提出假设， 基于RPV的TPD不仅可以增强RTI敏感RT对HIV-1的抑制作用， 抗RTI的RT变体。最终，这种方法可以减少耐药性的发展， 延长治疗方案的使用寿命，以对抗艾滋病毒疾病。AIM 1的目标是设计和制备RPV- 目前，基于最先进的计算方法和预测物理化学性质的TPD 接受体内活性TPD。在AIM 2中，我们将筛选两个系列的TPD的抗病毒活性， HIV-1单轮感染（在TZM-bl试验中）和复制（在初级试验中）的补充体外模型 CD 4 + T淋巴细胞）。发现限制传染性和感染性的靶向HIV-1 RT降解剂的影响 通过一种不同于基于占有的HIV-1 RTIs的机制进行复制将是识别病原体的方法 其对RTI抗性HIV毒株有效且限制RTI对病毒抗性的易感性 发展更重要的是，这项研究将证明TPD方法在治疗原发性肝癌方面具有上级优势。 艾滋病毒疾病，并将为推动针对其他艾滋病毒目标的TPD奠定基础， 预防耐药性的发展并伴随增强的临床治疗结果。

PROJECT SUMMARY/ABSTRACT In accordance with RFA-AI-19-072, Novel Therapeutics Directed to Intracellular HIV Targets, we propose using targeted protein degradation (TPD) against the essential HIV reverse transcriptase (RT). The RFA states “Controlling protein function by controlling intracellular protein levels has evolved as a promising and novel therapeutic strategy. This can be achieved by the targeted degradation of intracellular proteins thru the ubiquitin- proteasome pathway.” Heterobifunctional targeted protein degraders (TPDs) are being actively pursued as enhancers of proteasomal destruction of proteins specifically associated with several cancers. Such agents offer advantages over traditional occupancy-based inhibitors including a unique catalytic mechanism of action, greater target selectivity, and a reduced probability for resistance development. Surprisingly, this promising therapeutic modality has only recently been applied to antiviral drug discovery through a successful Telapravir-based TPD effective against the wild type and resistant forms of the HCV protease. Analysis of reported HIV-1 drug-bound structures suggests that the essential viral RT should be readily adaptable to targeted protein degradation. The potent and clinically used HIV-1 RT inhibitor (RTI), Rilpivirine (RPV) binds the RT site in Gag-Pol and also allosterically inhibits p66/p51 RT function by binding the non-nucleoside-RTI (NNRTI) Binding Pocket (NNIBP) on the p66 subunit. RPV is amenable to conjugation with linkers and ubiquitin E3 ligase recruiting ligands to serve in the design and preparation of prototype HIV-1 RT TPDs. The OBJECTIVE of this study is to show proof-of-concept of a new inhibitory mechanism by which HIV-1 RT can be targeted for degradation, impairing HIV infectivity and replication. Importantly, due to TPDs’ unique mechanism of action, even a low- affinity RT/TPD interaction will likely lead to effective target degradation. Thus, we pose the HYPOTHESIS that RPV-based TPDs will not only augment inhibition against HIV-1 with RTI-sensitive RT but will remain effective against RTI-resistant RT variants. Ultimately, this approach can reduce resistance development and potentially extend regimen lifetimes in the fight against HIV disease. The objective of AIM 1 is to design and prepare RPV- based TPDs built on state-of-the-art computational methods and predictive physicochemical properties currently accepted for in vivo active TPDs. In AIM 2, we will screen the two series of TPDs for antiviral activity in complementary in vitro models of HIV-1 single-round infection (in the TZM-bl assay), and of replication (in primary CD4+ T lymphocytes). The IMPACT of the discovery of targeted HIV-1 RT degraders that limit infectivity and replication through a mechanism distinct from occupancy-based HIV-1 RTIs will be the identification of agents that are effective against RTI-resistant HIV strains and that limit RTI susceptibility to viral resistance development. Grandly, this research will demonstrate that TPD approaches can be superior in the treatment of HIV disease and will set the stage for the advancement of TPDs against other HIV targets with improvements in the prevention of resistance development and with concomitant enhanced clinical treatment outcomes.