Targeting testis-specific ubiquitin-proteasome pathways for male contraception

针对男性避孕的睾丸特异性泛素蛋白酶体途径

基本信息

批准号：
10018522
负责人：
MARTIN M. MATZUK
金额：
$ 40万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-13 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10018522
关键词：
Applications Grants Binding Binding Proteins Bioinformatics Biology Bromodomain CRISPR/Cas technology Cell physiology Charge Chemicals Chemistry Collection Contraceptive Agents DNA Data Devices Drug Targeting Enzymes Fertility Gene Family Genes Genetic Goals Health Hormonal Human In Vitro Infertility Knockout Mice Knowledge Lead Ligase Link Male Contraceptive Agents Mediating Methods Mission Mus Mutation National Institute of Child Health and Human Development Pathway interactions Pharmaceutical Chemistry Pharmaceutical Preparations Phase Physiological Post-Translational Protein Processing Process Protac Proteins Proteolysis Research Research Support Reverse Transcriptase Polymerase Chain Reaction Ring Finger Domain Role Technology Testing Testis Ubiquitin Ubiquitin-Activating Enzymes Ubiquitin-Conjugating Enzymes Ubiquitination United States National Institutes of Health Zinc Zinc Fingers base contraceptive target human tissue in vivo inhibitor/antagonist innovation male male fertility mouse genetics multicatalytic endopeptidase complex novel pre-clinical protein degradation reproductive reproductive function reproductive tract research and development small molecule ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY Ubiquitination is a post-translational modification of proteins that is required for many cellular processes, including protein degradation by the proteasome, and requires the sequential action of three enzymes called E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin-ligase). While there is a single E1 enzyme and ~40 E2 enzymes, there are over 600 E3 enzymes in humans, with the RING (Really Interesting Novel Gene) family of E3 ubiquitin ligases being the most abundant. The RING E3 ubiquitin ligases recognize specific substrate proteins and interact with the ubiquitin-charged E2 enzymes to transfer the ubiquitin to the substrate. Recently, this pathway has been utilized to target physiologically relevant proteins for degradation using proteolysis-targeting chimeric (PROTAC) small molecules that bind to both an E3 ubiquitin ligase and a target protein. In the R61 component of this NIH grant proposal, we have chosen to validate the fertility essential functions of four evolutionarily-conserved testis-enriched ubiquitin-proteasome pathway proteins using a CRISPR/Cas9 knockout mouse strategy. Using CRISPR/Cas9, we have created mutations in all four of these genes, and preliminary data has revealed that at least one of these genes is required for male fertility. Our next step is to identify small molecules that bind these proteins using DNA-Encoded Chemistry Technology (DEC-Tec), in which each protein will be screened in vitro against our over 2.8 BILLION molecule collection. DEC-Tec is an economical process to rapidly uncover lead molecules, which will be tested directly for their inhibitory action or linked with other contraceptives to form PROTACs for targeted degradation of other spermatogenic-specific contraceptive targets. In the R33 component of this NIH grant proposal, we will use medicinal chemistry to optimize inhibitors for direct contraceptive action and create PROTACS that bind these E3 ubiquitin protein ligases and known contraceptives. Our R61/R33 proposal is an innovative, discovery- based, and integrated strategy to use mouse genetics to define the essential roles of these four testis-enriched ubiquitination-proteasome pathway proteins, to use DEC-Tec to rapidly identify small molecule binders, and to directly use these small molecules as non-hormonal contraceptives or to link them to other lead contraceptive compounds (as PROTACs) to direct the contraceptive target for degradation.

项目概要泛素化是许多细胞过程所需的蛋白质翻译后修饰，包括蛋白酶体的蛋白质降解，并且需要三种酶的连续作用 E1（泛素激活酶）、E2（泛素结合酶）和 E3（泛素连接酶）。虽然有一个单个 E1 酶和约 40 种 E2 酶，人类中有超过 600 种 E3 酶，其中 RING（真的有趣的新基因）E3 泛素连接酶家族是最丰富的。 RING E3 泛素连接酶识别特定的底物蛋白并与带泛素的 E2 酶相互作用以转移泛素到底物上。最近，该途径已被用来靶向生理相关蛋白质使用与 E3 泛素结合的蛋白水解靶向嵌合 (PROTAC) 小分子进行降解连接酶和目标蛋白。在此 NIH 拨款提案的 R61 部分中，我们选择验证四个进化保守的睾丸富集泛素蛋白酶体途径的生育基本功能使用 CRISPR/Cas9 敲除小鼠策略的蛋白质。使用 CRISPR/Cas9，我们在所有这四种基因，初步数据显示，男性至少需要其中一种基因生育能力。我们的下一步是使用 DNA 编码化学来识别结合这些蛋白质的小分子技术 (DEC-Tec)，其中每种蛋白质都将根据我们超过 28 亿个分子进行体外筛选收藏。 DEC-Tec 是一种快速发现先导分子的经济方法，并将直接进行测试因其抑制作用或与其他避孕药结合形成 PROTAC 来定向降解其他避孕药生精特异性避孕目标。在此 NIH 拨款提案的 R33 部分中，我们将使用药物化学优化直接避孕作用的抑制剂并创建结合这些抑制剂的 PROTACS E3 泛素蛋白连接酶和已知的避孕药。我们的 R61/R33 提案是一项创新的发现- 基于综合策略，利用小鼠遗传学来定义这四种睾丸富集的基本作用泛素化-蛋白酶体途径蛋白，使用 DEC-Tec 快速识别小分子结合物，并直接使用这些小分子作为非激素避孕药或将它们与其他主要避孕药联系起来化合物（如 PROTAC）引导避孕目标降解。