RNA-based PROTACs Targeting HIV-1

项目摘要/摘要 治疗艾滋病毒感染的广泛适用的治疗方法仍然难以找到，部分原因是许多病毒和细胞蛋白 在治疗策略中可能成为靶点的既不是酶也不是受体，不适合传统的 药物发现的方法。这项拟议中的研究将优化一个系统，用于开发引导分子 这些“无法下药”的靶蛋白的降解。这样的方法可以用来靶向 是艾滋病毒免疫逃避的关键，如辅助蛋白，或对 维持病毒潜伏期，这是根除病毒的关键障碍。 这种方法涉及蛋白质降解靶向嵌合体(PROTACs)、bi-Tacs和 将目标蛋白招募到E3泛素连接酶以引发其降解的功能分子，与RNA一起 适配子，无论蛋白质是否已知，都可以选择与蛋白质靶标结合的短RNA 在体内结合RNA。开发的基于RNA的PROTAC将与RNA适配子末端结合它们的靶标 蛋白质，并在PROTAC末端招募泛化目标蛋白质的细胞机械，从而 指挥着它的毁灭。与典型的药物靶向不同，RNA-PROTAC不需要适合于 蛋白质或直接抑制其在该系统中发挥作用的活性。识别具有特定蛋白质的新RNA的方法- 结合活性得到了很好的描述。高亲和力单链短RNA可在体外从大鼠 通过一种称为指数富集法的配体系统进化(SELEX)的过程构建文库，产率较低 或亚纳摩尔蛋白质配体。 在这项概念验证研究中，两种与已知RNA序列Rev和Tat特异结合的HIV蛋白质， 将被用作原型目标。首先，他们已知的RNA靶标将与PROTAC偶联 点击化学，传递到细胞，然后测试目标降解，以优化和验证方法 将靶向RNA连接到招募E3连接酶的小分子上。我们还将使用SELEX来识别小说 适配子作为靶标结合部分，进化修饰的RNA以实现最佳的配体结合。的共轭 通过点击化学方法将RNA适配子结合到PROTAC需要掺入修饰的核糖核苷酸， 当它们以不同的比例存在时，可能会影响靶标结合。提议的程序的优点是 SELEX过程将使用不同数量的修饰核糖核苷酸来优化靶向 感兴趣的蛋白质。在我们的原型目标REV和TAT的情况下，最优修改的序列 SELEX确定的RNA适配子可能与其已知的RNA靶标不同。拟议中的实验 将开发一种针对艾滋病毒的药物设计的新战略。未来的研究将针对其他“无法用药”的病毒和 细胞靶点可以使艾滋病毒感染细胞对免疫监视敏感，并有助于逆转 病毒潜伏期。

Project Summary/Abstract A broadly applicable cure for HIV infection remains elusive, in part because many of the viral and cellular proteins that could be targeted in cure strategies are neither enzymes nor receptors, poorly suited to conventional approaches to drug discovery. The proposed research will optimize a system for developing molecules that direct the degradation of these “undruggable” target proteins. Such an approach could be used to target proteins that are critical for immune evasion by HIV, such as the accessory proteins, or cellular proteins critical for the maintenance of viral latency, a critical barrier to viral eradication. This approach involves the innovative combination of PROteolysis-TArgeting Chimeras (PROTACs), bi- functional molecules that recruit target proteins to an E3 ubiquitin ligase to trigger their degradation, with RNA aptamers, short RNAs that can be selected to bind a protein target regardless of whether the protein is known to bind RNA in vivo. The developed RNA-based PROTACs will, with the RNA aptamer end, bind their target proteins, and, with the PROTAC end, recruit cellular machinery that ubiquitinates the target protein, thereby directing its destruction. Unlike typical drug targeting, the RNA-PROTAC need not fit into a particular site on the protein or directly inhibit its activity to function in this system. Methods to identify novel RNAs with specific protein- binding activity are well-described. High-affinity single-stranded short RNAs can be isolated in vitro from large libraries by a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX), yielding low or sub-nanomolar protein ligands. In this proof-of-concept study, two HIV proteins that bind specifically to known RNA sequences, Rev and Tat, will be employed as prototype targets. First, their known RNA targets will be conjugated to PROTACs using CLICK chemistry, delivered to cells and then tested for target degradation to optimize and validate the methods to link targeting RNAs to small molecules that recruit E3 ligases. We will also use SELEX to identify novel aptamers as the target-binding moiety, evolving modified RNAs for optimal ligand binding. The conjugation of the RNA aptamer to the PROTAC by CLICK chemistry requires the incorporation of modified ribonucleotides, which, when present in different ratios could affect target binding. The advantage of the proposed procedure is that the SELEX process will be performed with variable amounts of modified ribonucleotides to optimize targeting of the proteins of interest. In the case of our prototype targets, Rev and Tat, the sequences of the optimal modified RNA aptamers determined by SELEX may be different from their known RNA targets. The proposed experiments will develop a new strategy for drug design targeting HIV. Future studies will target other “undruggable” viral and cellular targets that could sensitize HIV-infected cells to immune surveillance and contribute to the reversal of viral latency.