Investigation of ATV-Based Heterobifunctional Degraders to Combat Growing HIV-1 PR Inhibitor Resistance

研究基于 ATV 的异双功能降解剂以对抗日益增长的 HIV-1 PR 抑制剂耐药性

• 批准号: 10484347
• 负责人: CHRISTINA OCHSENBAUER
• 金额: $ 22.7万
• 依托单位: UNIVERSITY OF ALABAMA IN TUSCALOOSA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-04 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10484347
• 关键词: Affinity; Antiviral Agents; Atazanavir; Autoimmune Diseases; Binding; Binding Proteins; Binding Sites; Biological; Biological Assay; Biological Availability; Biology; Cell membrane; Cells; Clinic; Clinical Trials; Complex; Computing Methodologies; Development; Dimerization; Drug resistance; Epidemic; Event; Exhibits; Failure; Foundations; Future; Goals; HIV; HIV Infections; HIV Protease; HIV Protease Inhibitors; HIV resistance; HIV-1; HIV-1 protease; Impairment; Infection; Investigation; Lead; Length; Ligands; Ligase; Mediating; Membrane; Modality; Modeling; Molecular Conformation; Multi-Drug Resistance; Mutation; Nuclear; Nuclear Envelope; Organic Synthesis; Patients; Peptide Hydrolases; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacotherapy; Predisposition; Probability; Process; Property; Protease Inhibitor; Proteins; Reagent; Regimen; Resistance; Resistance development; Series; Site; Structure; Testing; Therapeutic; Therapeutic Intervention; Ubiquitination; Variant; Viral; Virion; Work; analog; antimicrobial drug; antiretroviral therapy; assay development; base; combat; design; dimer; drug development; drug discovery; experience; high reward; high risk; in vitro activity; in vivo; inhibitor; lead optimization; membrane assembly; next generation; novel; novel strategies; novel therapeutic intervention; particle; pol Gene Products; pre-exposure prophylaxis; prevent; prospective; prototype; recruit; refractory cancer; resistant strain; screening; simulation; small molecule inhibitor; trend; ubiquitin-protein ligase; viral resistance

PROJECT SUMMARY/ABSTRACT Heterobifunctional targeted protein degraders (TPDs) offer advantages over traditional occupancy-based inhibitors including a unique catalytic mechanism of action (MOA), greater target selectivity, and a reduced probability for resistance development. TPDs are known to effectively target not only cytosolic proteins, but also nuclear and membrane-bound proteins. This therapeutic modality shows great promise for treating drug-resistant cancers and autoimmune diseases but has seen only limited application in antiviral drug discovery. HIV-1 protease (PR) is essential for proteolytic cleavage of Gag and Gag-Pol polyproteins and, thus, virion maturation and infectivity. Gag-Pol forms dynamic dimers at plasma membrane assembly/budding sites, allowing the embedded precursor PR to dimerize as a prerequisite for auto-processing. We identified precursor PR/Gag-Pol as a promising target for protease inhibitor (PI)-based TPDs. The widely used HIV-1 PI, Atazanavir (ATV), is amenable to conjugation with linkers and ubiquitin E3 ligase recruiting ligands to serve as prototype HIV-1 TPDs. Importantly, ATV inhibits activity of mature PR as well as autoprocessing of precursor PR/Gag-Pol during the assembly and budding processes. The OBJECTIVE of this study is to show proof-of-concept that HIV-1 Gag- Pol assembling on the inner leaflet of the plasma membrane can be targeted for aberrant ubiquitination, degradation, and/or inhibition, thereby impairing HIV infectivity. Importantly, due to TPDs’ established MOA, even low-affinity Gag-Pol/TPD interactions are likely to lead to impaired Gag-Pol function. Thus, we pose the HYPOTHESIS that novel ATV-based TPDs will not only augment the inhibition of ATV-sensitive HIV-1 strains compared to ATV, but also exhibit increased and broader biological activity against drug-resistant variants. The objective of AIM 1 is to design and synthesize ATV-based TPDs built on state-of-the-art computational methods and predictive physicochemical properties currently accepted for in vivo active TPDs. Our approach will feature a modular TPD design to establish the appropriate ATV attachment point, E3 ligase recruiter, and length and composition of linker required for HIV-1 Gag-Pol ubiquitination/proteasomal degradation. The goal of AIM 2 is to provide proof-of-concept that ATV-TPDs exert superior activity versus ATV against wild type and PI-resistant HIV-1 strains. We will systematically screen four series of novel ATV-TPD for activity in vitro, in single-round infectivity, and in multi-round replication assays to identify the most promising ATV-TPD candidates. The latter will be tested in complementary limited-scope mechanistic studies, including comparing ATV-TPDs with analogs bearing inactive E3-ligase ligands, to probe if antiviral activity is consistent with the proposed MOA. The IMPACT will be TPDs targeting HIV-1 Gag-Pol and precursor PR that limit infectivity and replication through a mechanism distinct from occupancy-based HIV-1 PI. This will spur the future development of efficacious regimens against PI-resistant HIV strains with reduced susceptibility for resistance development.

项目概要/摘要 异双功能靶向蛋白质降解剂 (TPD) 比传统的基于占用的蛋白质降解剂具有优势 抑制剂包括独特的催化作用机制 (MOA)、更高的靶标选择性以及更低的 产生耐药性的概率。众所周知，TPD 不仅可以有效靶向胞质蛋白，还可以有效靶向胞浆蛋白。 核和膜结合蛋白。这种治疗方式显示出治疗耐药性的巨大前景 癌症和自身免疫性疾病，但在抗病毒药物发现中的应用有限。 HIV-1 蛋白酶 (PR) 对于 Gag 和 Gag-Pol 多蛋白的蛋白水解切割以及病毒颗粒的成熟至关重要 和传染性。 Gag-Pol 在质膜组装/出芽位点形成动态二聚体，从而允许 嵌入前体 PR 进行二聚化作为自动处理的先决条件。我们确定了前体 PR/Gag-Pol 作为基于蛋白酶抑制剂 (PI) 的 TPD 的一个有前途的目标。广泛使用的 HIV-1 PI 阿扎那韦 (ATV) 是 适合与接头和泛素 E3 连接酶招募配体缀合，作为原型 HIV-1 TPD。 重要的是，ATV 抑制成熟 PR 的活性以及前体 PR/Gag-Pol 的自动加工过程。 组装和萌芽过程。本研究的目的是证明 HIV-1 Gag- 质膜内叶上的 Pol 组装可针对异常泛素化， 降解和/或抑制，从而损害 HIV 感染性。重要的是，由于 TPD 已建立 MOA，甚至 低亲和力的 Gag-Pol/TPD 相互作用可能会导致 Gag-Pol 功能受损。因此，我们提出 假设新型基于 ATV 的 TPD 不仅会增强对 ATV 敏感的 HIV-1 毒株的抑制作用 与 ATV 相比，它还表现出针对耐药变异体的更高和更广泛的生物活性。 AIM 1 的目标是设计和综合基于 ATV 的 TPD，该 TPD 建立在最先进的计算基础上 目前体内活性 TPD 所接受的方法和预测理化性质。我们的方法 将采用模块化 TPD 设计来建立适当的 ATV 连接点、E3 连接酶招募器和 HIV-1 Gag-Pol 泛素化/蛋白酶体降解所需的接头长度和组成。目标 AIM 2 的目的是提供概念证明，证明 ATV-TPD 对野生型和 ATV 具有优于 ATV 的活性 PI 耐药 HIV-1 菌株。我们将系统地筛选四个系列的新型 ATV-TPD 的体外活性、 单轮感染性和多轮复制测定以确定最有希望的 ATV-TPD 候选者。 后者将在补充性的有限范围机制研究中进行测试，包括比较 ATV-TPD 与带有无活性 E3 连接酶配体的类似物，以探测抗病毒活性是否与提议的 MOA 一致。 IMPACT 将是针对 HIV-1 Gag-Pol 和前体 PR 的 TPD，通过限制感染性和复制 一种与基于占用的 HIV-1 PI 不同的机制。这将促进未来有效的发展 针对 PI 耐药 HIV 菌株的治疗方案，降低耐药发展的易感性。