Small molecule degraders of HIV-1 Nef

项目摘要/摘要 小分子探针在研究蛋白质的生理功能时常常是有用的。他们还提供服务 作为原型药物来验证新的治疗靶点。在努力开发病毒小分子抑制剂的同时 酶产生了无价的机械探针和有效的药物(例如，雷替格列韦、利托那韦、齐多夫定)， 这些成功的数量超过了生化功能较差的基本病毒蛋白(S) 可以理解，并且被认为是“无法下药的”。在大多数情况下，这些蛋白质不服从于 常规的基于结构的抑制剂设计和高通量筛选。人类的Nef蛋白 免疫缺陷病毒1(HIV-1)就是一个很好的例子。作为一种多功能蛋白质，Nef被认为是 病毒感染性和致病机制的关键因素；然而，它在很大程度上与传统的 药物发现即将到来。目前的Nef抑制剂的确定和优化很大程度上是基于对 NEF与造血细胞激酶SH3结构域的相互作用，但目前尚不清楚 抑制这种相互作用就足以解决Nef的所有生物学功能。 作为功能性抑制剂的替代品，我们已经开始开发小分子，通过诱导 目的蛋白的降解。这是通过合成双功能分子(称为 “PROTAC”、“DECRONIMID”和“DECRONIDERS”)，它们含有化学上特定于感兴趣目标的配体 通过连接物连接到特定E3泛素连接酶的配体上。我们建议应用这一战略来发展 诱导HIV-1 Nef降解的小分子。为了迅速推进这项工作，我们将利用 已发表的Nef配体作为靶向配体。候选NEF降解剂的设计和合成将使用 药物化学和分子模拟改变Nef配体上的连接位置、长度和组成 以及E3泛素连接酶配基。这些候选Nef降解器将针对交互进行优化 与Nef相互作用，与E3泛素连接酶相互作用，形成Nef-dearder-E3连接酶三元复合体，以及 成功的，依赖于蛋白酶体的Nef降解。小分子被证明能诱导降解 纤维素中的NEF将在细胞培养中测试对HIV-1的抗病毒活性。 这项工作建立在我们最近成功开发丙型肝炎病毒NS3小分子降解剂的基础上- 4A水解酶。NEF是这种方法特别有吸引力的目标，因为它有可能退化- 诱导药理学相对于目前表现出传统的、 占有率驱动的药理学。我们在这项探索性工作中的目标是为使用 小分子诱导蛋白质降解靶向Nef并生成发育所需的先导化合物 可用于探测Nef生物学并在体内制作Nef抗病毒药物原型的Nef降解物。更广泛地说，我们 相信这项工作将为开发类似的降解剂提供重要的概念验证 用传统方法“无法下药”的其他病毒蛋白。

PROJECT SUMMARY / ABSTRACT Small molecules probes are often useful in studying the physiological function of proteins. They also serve as prototype drugs to validate new therapeutic targets. While efforts to develop small molecule inhibitors of viral enzymes have yielded invaluable mechanistic probes and effective drugs (e.g., raltegravir, ritonavir, zidovudine), these successes are outnumbered by the essential viral proteins whose biochemical function(s) are poorly understood and that are considered “undruggable.” In most cases, these proteins are not amenable to conventional structure-based inhibitor design and high-throughput screening. The Nef protein of human immunodeficiency virus 1 (HIV-1) is a prime example. As a multifunctional protein, Nef has been implicated as a critical factor in both viral infectivity and pathogenesis; however, it has been largely recalcitrant to conventional drug discovery approaches. Current Nef inhibitors were identified and optimized largely based on disruption of Nef's interaction with the SH3 domain of hematopoeietic cell kinase (Hck), but it remains unclear whether inhibiting this interaction is sufficient to address all of Nef's biological functions. As an alternative to functional inhibitors, we have begun developing small molecules that act by inducing degradation of the target protein. This is accomplished by synthesis of bifunctional molecules (known as “PROTACs,” “degronimids,” and “degraders”) that contain a ligand specific for the target of interest chemically conjugated via a linker to a ligand for a specific E3 ubiquitin ligase. We propose to apply this strategy to develop small molecules that induce degradation of HIV-1 Nef. To quickly advance this work, we will take advantage of published ligands of Nef as targeting ligands. Candidate Nef degraders will be designed and synthesized using medicinal chemistry and molecular modeling to vary the linkage site on the Nef ligand, the length and composition of the linker, and the E3 ubiquitin ligase ligand. These candidate Nef degraders will be optimized for interaction with Nef, interaction with the E3 ubiquitin ligase, formation of the ternary Nef-degarder-E3 ligase complex, and successful, proteasome-dependent degradation of Nef. Small molecules demonstrated to induce degradation of Nef in cellulo will be tested for antiviral activity against HIV-1 in cell culture. This work builds upon our recent success developing small molecule degraders of the hepatitis C virus NS3- 4A protease. Nef is a particularly attractive target for this approach because there is the potential for degradation- induced pharmacology to be differentiated and superior relative to current inhibitors that exhibit traditional, occupancy-driven pharmacology. Our goals in this exploratory work are to establish proof-of-concept for using small molecule-induced protein degradation to target Nef and to generate lead compounds for the development of Nef degraders that can be used to probe Nef biology and to prototype Nef antivirals in vivo. More broadly, we believe that this work will provide important proof-of-concept for the development of analogous degraders against other viral proteins that have been “undruggable” using conventional approaches.