Development of PD-L1 Proteolysis-Targeting Chimeras (PROTACs): An Innovative Cancer Drug Discovery Route

PD-L1 蛋白水解靶向嵌合体 (PROTAC) 的开发：创新的癌症药物发现路线

• 批准号: 10605616
• 负责人: Demonta Coleman
• 金额: $ 3.81万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605616
• 关键词: Affinity; Antineoplastic Agents; Bacteriophages; Binding; Biological Assay; Biotinylation; CD44 gene; Cancer cell line; Cell Line; Cells; Clinical Trials; Consensus; Consensus Sequence; Cyclic Peptides; Cyclization; Dependence; Detection; Development; Dimensions; Docking; Drug Design; Enzymes; Epitopes; Escherichia coli; Event; Extracellular Domain; Flow Cytometry; High Pressure Liquid Chromatography; Immobilization; Immune Evasion; Immune Targeting; Immune checkpoint inhibitor; Immune response; In Vitro; Incubated; Kinetics; Large Intestine Carcinoma; Length; Libraries; Ligand Binding; Ligands; Literature; Lysosomes; MDA MB 231; Malignant Neoplasms; Medicine; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Non-Small-Cell Lung Carcinoma; Oncology; Organic Synthesis; PD-1/PD-L1; PDL1 pathway; Pathway interactions; Peptide Synthesis; Peptides; Phage Display; Phase; Polyethylene Glycols; Polyubiquitination; Positioning Attribute; Proliferating; Protac; Proteins; Proteomics; Recombinants; Reporting; Research; Research Personnel; Residual state; Resolution; Route; SARS-CoV-2 spike protein; Solid; Streptavidin; Structure; Structure-Activity Relationship; Surface; System; T-Cell Activation; T-Lymphocyte; Testing; Therapeutic; Time; Ubiquitin; Western Blotting; Work; cancer therapy; cancer type; cell growth regulation; design; drug development; drug discovery; extracellular; frontier; immunoregulation; in vivo; innovation; insight; member; multicatalytic endopeptidase complex; neoplastic cell; novel; novel therapeutics; overexpression; pharmacologic; programmed cell death ligand 1; programmed cell death protein 1; protein aminoacid sequence; protein degradation; receptor; recruit; small molecule; triple-negative invasive breast carcinoma; tumor; ubiquitin-protein ligase

Project Summary The current cancer treatment bottleneck showcases the need for more innovative approaches in drug discovery efforts. As of recent, immune-checkpoint inhibitors (ICHs) have shown promise in cancer therapeutics, and work by manipulating immunoregulatory pathways involved in tumor detection and elimination. Many cancer types overexpress the PD-L1 receptor to inhibit T cell activation and subsequently evade the immune response. Though proven effective, the PD-1/PD-L1 pathway targeting ICHs are pharmacologically limited by occupancy- based inhibition. A new drug paradigm has emerged through the development of proteolysis targeting chimeras (PROTACs) that are not limited by such a mechanism. PROTACs are heterobifunctional molecules consisting of an E3 ubiquitin (Ub) ligase ligand, a target protein warhead, and a linker connecting the two and work by bringing an E3 Ub ligase and the target protein together through bivalent binding. The formation of a ternary structure through this bivalent interaction drives the polyubiquitination and subsequent degradation of the target via the ubiquitin proteasome system (UPS). Although this drug design has shown great medicinal promise through research findings and clinical trials, there is seldom literature on the use of transmembrane E3 Ub ligase recruiting PROTACs to degrade cancer-promoting membrane proteins such as PD-L1. The proposed project is aimed at investigating the applicability of such PD-L1 PROTACs by recruitment of the transmembrane E3 Ub ligase ZNRF3. In doing so, three specific aims will be investigated: Aim 1. Identifying cyclic peptide binders of the ZNRF3 extracellular domain. Previous research in the Liu lab utilized a cyanobenzothiazole based linker that allows for cyclization of peptides on a phage surface with applications in phage selections against CD44 and SARS-CoV-2 spike protein epitopes. Recombinant bacterial expression of biotinylated ZNRF3 ECD has been developed, and the binding of selected cyclic peptides will be validated through BLI and flow cytometry. Aim 2. Synthesis and in-vitro characterization of PD-L1 PROTACs. Reported binders of PD-L1 will be incorporated in the PROTAC design via polyethylene glycol (PEG) and polyether linkages of varying lengths. The optimal positioning of linkers will be determined through structure-activity relationships (SAR) analysis, binding assays, co-cocrystal structures, and molecular docking studies of ligand-protein interactions of the ZNRF3 ECD and PD-L1. Aim 3. In-vivo characterization of PD-L1 PROTACs. Degradation potency will be evaluated with western-blotting of residual PD-L1 and ZNRF3 dependency on the induced degradation of PD-L1 and mode of action (proteosome vs lysosome) will be assessed. In addition, whole-cell proteomics studies will be conducted to investigate any consequences on normal cellular activities due to PROTAC activity. By accomplishing these aims, new and important conclusions can be made about the efficacy and mode of action of our designed PROTACs that we hope will have a lasting impact on the cancer therapeutics bottleneck and general drug discovery efforts.

项目摘要 目前的癌症治疗瓶颈表明，在药物发现方面需要更多的创新方法 努力最近，免疫检查点抑制剂（ICHs）在癌症治疗中显示出前景， 通过操纵参与肿瘤检测和消除的免疫调节途径。许多癌症类型 过表达PD-L1受体以抑制T细胞活化并随后逃避免疫应答。 尽管已被证明是有效的，但靶向ICH的PD-1/PD-L1通路受到占位性的限制。 基于抑制。通过蛋白水解靶向嵌合体的发展，出现了一种新的药物范式 （PROTAC）不受这种机制的限制。PROTAC是由以下组成的异双功能分子： E3泛素（Ub）连接酶配体、靶蛋白弹头和连接两者的接头， E3 Ub连接酶和靶蛋白通过二价结合在一起。三元结构的形成 通过这种二价相互作用，驱动多聚泛素化和随后的靶标降解， 泛素蛋白酶体系统（UPS）。虽然这种药物设计已经显示出巨大的药用前景， 研究发现和临床试验，很少有文献使用跨膜E3 Ub连接酶 招募PROTAC降解促癌膜蛋白，如PD-L1。拟建项目 目的是通过募集跨膜蛋白来研究这种PD-L1 PROTAC的适用性。 E3 Ub连接酶ZNRF 3。在这样做时，将研究三个具体目标：目标1。识别环肽 ZNRF 3胞外结构域的结合剂。Liu实验室以前的研究使用了基于氰基苯并噻唑的 允许在噬菌体表面上的肽环化的接头， CD 44和SARS-CoV-2刺突蛋白表位。生物素化ZNRF 3 ECD的重组细菌表达 已开发，并将通过BLI和流式细胞术验证所选环肽的结合。 目标二。PD-L1 PROTAC的合成和体外表征。报告的PD-L1结合物将 通过不同长度的聚乙二醇（PEG）和聚醚键将其并入PROTAC设计中。 接头的最佳定位将通过结构-活性关系（SAR）分析来确定， 结合分析，共共晶结构，以及配体-蛋白质相互作用的分子对接研究， ZNRF 3 ECD和PD-L1。目标3。PD-L1 PROTAC的体内表征。降解效价为 用蛋白质印迹法评估残留PD-L1和ZNRF 3对PD-L1诱导降解的依赖性 并评估作用模式（蛋白体vs溶酶体）。此外，全细胞蛋白质组学研究将 研究PROTAC活性对正常细胞活性的任何影响。通过 实现这些目标后，就可以得出关于功效和作用方式的新的重要结论 我们设计的PROTAC，我们希望将对癌症治疗瓶颈产生持久的影响， 一般药物发现的努力。