RNA-based PROTACs Targeting HIV-1

基于 RNA 的针对 HIV-1 的 PROTAC

• 批准号: 10674998
• 负责人: Mary Kathleen Lewinski
• 金额: $ 7.18万
• 依托单位: VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10674998
• 关键词: Affect; Affinity; Binding; Binding Proteins; Biotin; Cells; Chemistry; Development; Drug Design; Drug Targeting; Enzymes; Evolution; Future; Genetic Transcription; Guide RNA; HIV; HIV Infections; HIV-1; Immune Evasion; Immunologic Surveillance; In Vitro; Libraries; Ligand Binding; Ligands; Link; Lipids; Maintenance; Methods; Modeling; Modification; Natural regeneration; Nucleic Acids; Pathogenesis; Procedures; Process; Protac; Proteins; Protocols documentation; RNA; RNA Binding; RNA Sequences; RNA chemical synthesis; Reaction; Recombinants; Research; Response Elements; Ribonucleotides; Site; Streptavidin; System; Systems Development; Testing; Transactivation; Ubiquitination; Viral; Viral Proteins; Virus Diseases; Virus Latency; Virus Replication; aptamer; cellular targeting; design; drug discovery; experimental study; genetic regulatory protein; in vivo; innovation; interest; nanomolar; novel; particle; pomalidomide; prototype; receptor; recruit; response; rev Protein; screening; small molecule; tat Protein; ubiquitin-protein ligase

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.

项目总结/摘要 一种广泛适用的治疗艾滋病毒感染的方法仍然难以捉摸，部分原因是许多病毒和细胞蛋白质 在治疗策略中可以靶向的既不是酶也不是受体，不适合常规治疗。 药物发现的方法。这项拟议中的研究将优化一个系统，用于开发直接 这些“不可用药的”靶蛋白的降解。这种方法可以用于靶向蛋白质， 对于HIV的免疫逃避至关重要，例如辅助蛋白，或对于免疫逃避至关重要的细胞蛋白。 维持病毒潜伏期，这是病毒根除的关键障碍。 该方法涉及PROTAC（PROteolysis-Targeting Chimeras）、双- 功能性分子，其将靶蛋白募集至E3泛素连接酶以触发其降解， 适体，可以选择结合蛋白质靶标的短RNA，无论蛋白质是否已知 在体内结合RNA。所开发的基于RNA的PROTAC将与RNA适体末端结合其靶标 蛋白质，并与PROTAC结束，招募细胞机制，泛素化的目标蛋白，从而 指挥它的毁灭。与典型的药物靶向不同，RNA-PROTAC不需要适合靶向药物上的特定位点。 蛋白质或直接抑制其活性以在该系统中发挥作用。鉴定具有特异性蛋白质的新RNA的方法- 结合活性已被充分描述。高亲和力的单链短RNA可以在体外从大RNA中分离出来。 通过称为指数富集配体系统进化（SELEX）的过程， 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序列特异性结合的HIV蛋白Rev和达特， 将被用作原型目标。首先，它们已知的RNA靶标将与PROTAC缀合， 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连接到募集E3连接酶的小分子上。我们还将使用SELEX来识别新的 适体作为靶结合部分，进化修饰的RNA以实现最佳配体结合。的缀合 通过CLICK化学作用的PROTAC的RNA适体需要掺入修饰的核糖核苷酸， 当以不同比例存在时，其可影响靶结合。建议程序的优点是 SELEX过程将使用可变量的修饰核糖核苷酸进行，以优化靶向 我们感兴趣的蛋白质在我们的原型目标Rev和达特的情况下，最佳修改的序列 通过SELEX确定的RNA适体可能不同于其已知的RNA靶标。拟议的实验 将为针对艾滋病毒的药物设计制定新的策略。未来的研究将针对其他“不可治疗”的病毒和 细胞靶点，可以使HIV感染细胞对免疫监视敏感，并有助于逆转 病毒潜伏期