Chemical Biology-Based Tools to Uncover the Function of PARP16 in cancer

基于化学生物学的工具揭示 PARP16 在癌症中的功能

• 批准号: 10750279
• 负责人: Daniel Bejan
• 金额: $ 4.77万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-17 至 2025-07-16
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750279
• 关键词: ADP ribosylation; Acrylamides; Active Sites; Adenosine Diphosphate Ribose; Alkynes; Automobile Driving; Binding; Binding Proteins; Biological Assay; Biology; Cell Death; Cell Survival; Cells; Chemicals; Chemistry; Chimeric Proteins; Comparative Study; Cysteine; Diazomethane; Disease; Drug Targeting; Endoplasmic Reticulum; Environment; Enzymes; Exhibits; FDA approved; Family; Genetic; Goals; Growth; Human; Hydrogen Peroxide; Knock-out; Label; Lead; Length; Light; Link; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Maps; Mass Spectrum Analysis; Mediating; Methods; Nicotinamide adenine dinucleotide; Oncology; Phenotype; Phosphotransferases; Physiology; Poly(ADP-ribose) Polymerase Inhibitor; Post-Translational Protein Processing; Process; Protac; Protein Biosynthesis; Proteins; Proteome; Proteomics; Research Personnel; Role; Signal Transduction; Stress; Technology; Testing; Translations; Ubiquitin; Ultraviolet Rays; Western Blotting; cancer cell; cancer subtypes; cross reactivity; crosslink; drug candidate; inhibitor; insight; interest; knock-down; lung cancer cell; member; nanomolar; new therapeutic target; novel; paralogous gene; pomalidomide; pre-clinical; preservation; protein degradation; protein protein interaction; proteostasis; proteotoxicity; small cell lung carcinoma; small molecule; tandem mass spectrometry; tool; tumor progression; ubiquitin-protein ligase; ultraviolet irradiation

PROJECT SUMMARY/ABSTRACT PARP16, a member of the PARP family of enzymes responsible for carrying out the post-translational modification known as ADP-ribosylating, is emerging as a novel therapeutic target in two cancer subtypes. In ovarian cancer, PARP16 was shown to negatively regulate protein translation in order to maintain proteostasis. Genetic deletion (i.e., knockout or knockdown) of PARP16 resulted in an increase in global protein translation, forcing ovarian cancer cells to enter a state of proteotoxic stress that ultimately leads to cancer cell death. In small cell lung cancer (SCLC), PARP16 was identified as an off-target to the recently FDA-approved PARP1 inhibitor, talazoparib, suggesting that the efficacy of talazoparib in SCLC may be due to dual targeting of PARP1 and PARP16. However, knockdown of PARP16 alone also decreased SCLC viability. Both of these cancer studies point to PARP16 as actionable oncology target, however, the role of PARP16 catalytic activity in cancer has not been fully characterized. Our group has recently developed the first cysteine-targeted covalent PARP inhibitor called DB008, that displays excellent proteome-wide selectivity for PARP16 in the covalent binding mode. While DB008 inhibits PARP16 catalytic activity with nanomolar potency, neither of the aforementioned PARP16 knockout/knockdown phenotypes were observed with DB008 treatment in ovarian cancer and SCLC, suggesting that the non-catalytic activity (i.e., protein-protein interactions) of PARP16 may regulate protein homeostasis and cancer growth as opposed to PARP16 enzymatic activity. To test this hypothesis, I aim to develop two novel chemical probes, based on DB008, to evaluate the non-catalytic functions of PARP16 in cancer. In Aim 1, I will synthesize a PARP16 proteolysis targeting chimera (PROTAC) that will chemically knockdown PARP16 in ovarian cancer and SCLC. The PARP16 PROTAC will evaluate whether depletion of PARP16 and its protein-protein interactions reduces cancer cell viability as observed with genetic knockdown methods. Completion of this aim will validate PARP16 as a new cancer target and provide a lead preclinical drug candidate. In Aim 2, I introduce a novel proximity labeling strategy for identifying interactors of endogenous PARP16 in cancer. This is done by converting DB008 into a caged photo-crosslinkable probe that uses UV light to uncage and release a reactive crosslinking species that will covalently tag interacting proteins, which can then be enriched using click chemistry and identified by mass spectrometry. Completion of this aim will provide understanding for how PARP16 regulates translation and cell viability in cancer. The technology described in Aim 2 presents a new use case for covalent inhibitors that is generalizable to other enzymes families beyond PARPs. In summary, this proposal will generate invaluable chemical biology tools for uncovering the mechanism of action of PARP16 in cancer while also providing a potential lead drug candidate for combating PARP16-mediated diseases.

项目总结/摘要 PARP 16是PARP家族的一员，负责进行翻译后修饰。 被称为ADP-核糖基化的修饰正在成为两种癌症亚型的新治疗靶点。在 在卵巢癌中，PARP 16显示负调节蛋白质翻译以维持蛋白质稳态。 基因缺失（即，敲除或敲低）导致整体蛋白质翻译的增加， 迫使卵巢癌细胞进入蛋白毒性应激状态，最终导致癌细胞死亡。在 小细胞肺癌（SCLC），PARP 16被确定为最近FDA批准的PARP 1的脱靶点 抑制剂talazoparib，这表明talazoparib在SCLC中的疗效可能是由于双重靶向 PARP 1和PARP 16。然而，单独敲除PARP 16也降低SCLC活力。这两 癌症研究指出PARP 16是可操作的肿瘤学靶点，然而，PARP 16催化活性的作用 在癌症中的作用还没有完全确定。我们的团队最近开发了第一个半胱氨酸靶向的 共价PARP抑制剂称为DB 008，它在细胞中对PARP 16表现出优异的蛋白质组范围的选择性。 共价结合模式。虽然DB 008以纳摩尔效力抑制PARP 16催化活性，但两种抑制剂都没有抑制PARP 16催化活性。 用DB 008处理在卵巢癌中观察到上述PARP 16敲除/敲低表型。 癌症和SCLC，这表明非催化活性（即，蛋白质相互作用） PARP 16可以调节蛋白质稳态和癌症生长，而不是PARP 16酶促 活动为了验证这一假设，我的目标是开发两种新的化学探针，基于DB 008，以评估 PARP 16在癌症中的非催化功能。在目标1中，我将合成一个PARP 16蛋白水解靶向 嵌合体（PROTAC），其将化学敲低卵巢癌和SCLC中的PARP 16。PARP16 PROTAC将评估PARP 16的缺失及其蛋白质-蛋白质相互作用是否会减少癌细胞 用基因敲除方法观察到的生存力。这一目标的完成将验证PARP 16作为一个新的 癌症靶点，并提供一个领先的临床前候选药物。在目标2中，我介绍了一种新的邻近标记 鉴定癌症中内源性PARP 16相互作用物的策略。这是通过将DB 008转换为 一种笼状光交联探针，其使用UV光来解开并释放反应性交联物质， 将共价标记相互作用的蛋白质，然后可以使用点击化学富集并通过质量鉴定 光谱法这一目标的完成将有助于理解PARP 16如何调节翻译和细胞增殖。 在癌症中的生存能力。目标2中描述的技术提出了共价抑制剂的新用例， 可推广到PARP以外的其他酶家族。总而言之，这项提议将产生宝贵的 化学生物学工具，用于揭示PARP 16在癌症中的作用机制， 潜在的主要候选药物，用于对抗PARP 16介导的疾病。