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

项目摘要/摘要 异双功能靶向蛋白质降解器(TPD)比传统的基于占有率的降解器具有优势 缓蚀剂包括独特的催化作用机理(MOA)、更高的靶向选择性和更低的 出现抗药性的可能性。众所周知，TPD不仅可以有效地靶向胞浆蛋白，而且还可以 核蛋白和膜结合蛋白。这种治疗方式在治疗耐药性方面显示出很大的前景。 癌症和自身免疫性疾病，但在抗病毒药物开发中的应用有限。HIV-1 蛋白水解酶(PR)对于GAG和GAG-POL多蛋白的蛋白水解性切割以及病毒粒子的成熟是必不可少的 和传染性。GAG-POL在质膜组装/萌发部位形成动态二聚体，允许 内嵌前驱体PR以二聚为前提进行自动加工。我们鉴定了前体PR/GAG-Pol 作为基于蛋白水解酶抑制剂(PI)的TPDs的一个有前途的靶点。广泛使用的HIV-1 PI，阿扎那韦(ATV)，是 可与接头和泛素E3连接酶结合，招募配体作为HIV-1 TPD的原型。 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 组装和萌芽过程。这项研究的目的是证明HIV-1Gag-1是 组装在质膜内叶上的POL可以被靶向于异常泛素化， 降解和/或抑制，从而损害艾滋病毒的传染性。重要的是，由于TPD建立了MOA，甚至 低亲和力的GAG-Pol/TPD相互作用可能导致GAG-Pol功能受损。因此，我们提出了 假设基于ATV的新型TPD不仅将增强对ATV敏感的HIV-1毒株的抑制 与ATV相比，还表现出更强和更广泛的抗耐药变异体的生物活性。 AIM 1的目标是设计和合成基于ATV的TPD，建立在最先进的计算基础上 目前公认的活体活性TPD的方法和预测的物理化学性质。我们的方法 将采用模块化的TPD设计，以建立适当的ATV连接点，E3连接招聘人员，以及 HIV-1 Gag-Pol泛素化/蛋白酶体降解所需的连接子的长度和组成。目标是 目标2的目的是提供概念证明，即ATV-TPD对野生型和 耐PI的HIV-1毒株。我们将系统地筛选四个系列的新型ATV-TPD在体外、在 单轮传染性，并在多轮复制试验中确定最有希望的ATV-TPD候选病毒。 后者将在互补性有限范围的机制研究中进行测试，包括比较ATV-TPD 带有非活性E3-连接酶配体的类似物，以探测抗病毒活性是否与建议的MOA一致。 其影响将是针对HIV-1 Gag-Pol和前体PR的TPD，它们通过 一种不同于基于占有率的HIV-1 PI的机制。这将推动威力科技的未来发展。 针对耐PI的HIV毒株的治疗方案，降低了对耐药性发展的敏感性。

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.