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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要当前的癌症治疗瓶颈展示了对药物发现中更具创新方法的需求努力。截至最近，免疫检查点抑制剂（ICHS）在癌症治疗方面表现出了希望，并且有效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反应。尽管经过证明有效，但针对ICHS的PD-1/PD-L1途径在药理学上受到占有限制基于抑制。通过开发靶向嵌合体的蛋白水解，出现了一种新的药物范式（Protac）不受这种机制的限制。 protac是由杂功能分子组成的 E3泛素（UB）连接酶配体，靶蛋白弹头以及连接两者并通过带来工作的连接器 E3 UB连接酶和靶蛋白通过二价结合一起。三元结构的形成通过这种二价相互作用，驱动目标通过泛素蛋白酶体系统（UPS）。尽管这种药物设计通过研究发现和临床试验，很少有关于跨膜E3 UB连接酶使用的文献招募Protac以降解促进癌症的膜蛋白，例如PD-L1。拟议的项目旨在通过募集跨膜来研究此类PD-L1 Protac的适用性 E3 UB连接酶ZnRF3。这样一来，将研究三个具体目标：目标1。识别环状肽 ZnRF3细胞外域的粘合剂。刘实验室的先前研究利用了基于蓝苯二唑的接头允许在噬菌体上环化肽，并在噬菌体选择中应用 CD44和SARS-COV-2尖峰蛋白表位。生物素化Znrf3 ECD的重组细菌表达已经开发出来，选定的环状肽的结合将通过BLI和流式细胞仪验证。 AIM 2。PD-L1 Protac的合成和体外表征。报告的PD-L1的粘合剂将是通过聚乙烯乙二醇（PEG）和不同长度的聚醚链接融合在Protac设计中。接头的最佳定位将通过结构 - 活性关系（SAR）分析确定，结合测定，共晶体结构和分子对接研究 ZnRF3 ECD和PD-L1。 AIM 3。PD-L1 Protac的体内表征。降解效力将是用残留的PD-L1和ZnRF3依赖性对PD-L1降解的依赖性的西部印象进行评估将评估作用方式（蛋白质体与溶酶体）。此外，全细胞蛋白质组学研究将进行以调查由于Protac活性引起的正常细胞活性的任何后果。经过实现这些目标，可以就功效和行动方式得出新的重要结论我们希望我们设计的protac对癌症治疗剂的瓶颈和一般药物发现工作。