Small molecule degraders of HIV-1 Nef

项目总结/摘要 小分子探针在研究蛋白质的生理功能中经常是有用的。他们还提供 作为原型药物来验证新的治疗靶点。虽然开发病毒的小分子抑制剂的努力 酶已经产生了无价的机理探针和有效的药物（例如，雷特格韦、利托那韦、齐多夫定）， 这些成功的数量被其生化功能差的基本病毒蛋白质所超过 被认为是“不可接受的”。在大多数情况下，这些蛋白质不适合 传统的基于结构的抑制剂设计和高通量筛选。人Nef蛋白 免疫缺陷病毒1（HIV-1）就是一个最好的例子。作为一种多功能蛋白，Nef被认为是 病毒感染性和发病机制的关键因素;然而，它在很大程度上已被排除在常规 药物发现方法。目前的Nef抑制剂主要是基于破坏 Nef与造血细胞激酶（Hck）的SH 3结构域相互作用，但目前尚不清楚Nef是否与Hck的SH 3结构域相互作用。 抑制这种相互作用足以解决Nef的所有生物功能。 作为功能性抑制剂的替代品，我们已经开始开发小分子， 降解目标蛋白。这是通过合成双功能分子（称为 “PROTAC”、“去甲亚胺”和“降解剂”），其含有对感兴趣的靶标具有化学特异性的配体 通过接头与特异性E3泛素连接酶的配体缀合。我们建议将这一战略应用于发展 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 公开的Nef配体作为靶向配体。候选Nef降解剂将设计和合成使用 药物化学和分子建模来改变Nef配体上的连接位点、长度和组成， 和E3泛素连接酶配体。这些候选Nef降解剂将针对相互作用进行优化 与Nef，与E3泛素连接酶相互作用，形成三元Nef-degarder-E3连接酶复合物，和 Nef的成功的蛋白酶体依赖性降解。小分子被证明可以诱导 将在细胞培养物中测试Nef在细胞中对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NS 3的小分子降解剂的基础上， 4A蛋白酶。Nef是这种方法的一个特别有吸引力的目标，因为它有降解的潜力- 诱导药理学与目前的抑制剂相比是不同的和上级的， 职业驱动的药理学我们在这项探索性工作中的目标是建立概念验证， 小分子诱导的蛋白质降解靶向Nef并产生用于开发的先导化合物 Nef降解剂，可用于探测Nef生物学和原型体内Nef抗病毒药物。更广泛地说，我们 我相信，这项工作将提供重要的概念验证，为发展类似的降质剂， 其他病毒蛋白质使用常规方法是“不可用药的”。

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.