Small Molecule Inhibitors of DMRT1-Regulated Target Genes as Male Contraceptives

DMRT1 调控靶基因的小分子抑制剂作为男性避孕药

• 批准号: 7789623
• 负责人: LESLIE L. HECKERT
• 金额: $ 28.51万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7789623
• 关键词: Adult; Animals; Antibodies; Binding; Binding Sites; Biological; Biological Assay; Cell Survival; Cells; Computer Simulation; Contraceptive Agents; Cultured Cells; Development; Drug Delivery Systems; Electrophoretic Mobility Shift Assay; Elements; Fertility; Fluorescence; Fluorescence Polarization; Future; Gene Targeting; Genes; Germ Cells; Goals; Gonadal structure; Homidium Bromide; In Vitro; Kansas; Laboratories; Lead; Male Contraceptive Agents; Molecular; Molecular Profiling; Monitor; Physiological; Production; Proteins; Rattus; Research; Response Elements; Spermatogenesis; Testing; Testis; Transfection; Universities; base; chromatin immunoprecipitation; contraceptive target; design; drug development; drug discovery; high throughput screening; in vivo; inhibitor/antagonist; interest; male; novel; sertoli cell; small molecule; transcription factor

The evolutionary conserved protein Dmrtl (Doublesex and Mab-3 related transcription factor-1) is a testisspecific transcription factor that is essential for gamete maturation and fertility. Its expression is restricted to the gonad and, postnatally, it is male-specific, where it found only in a subset of premeiotic germ cells and Sertoli cells in the testis. Its requirement for gamete maturation and its testis-specific expression make Dmrtl an excellent candidate target for the development of new male contraceptives, as compounds that disrupt its activity, or activity of its downstream target genes, will likely arrest spermatogenesis and block fertility. The project goals are to identify lead contraceptive compounds that function by disrupting Dmrtl activity and new contraceptive targets by revealing genes activated by Dmrtl. Aim 1 will develop an assay for high throughput screening (HTS) to identify novel small molecules that disrupt interactions between Dmrtl and its DMA binding element. Following production of purified Dmrtl, more than 120,000 compounds, available through the Drug Discovery, Design & Synthesis core (Core B) HTS facility, will be screened using a fluorescence-based assay adapted to a 384-well format. Aim 2 will evaluate lead compounds identified in Aim 1 to determine their biological effects in cell-based and whole animal studies. Cell viability, transient transfection analysis, and chromatin immunoprecipitation will be used to test compound activity in culture cells and compounds of continued interest will be administered to male rats and their toxicological and fertility effects evaluated. In aim 3, chromatin immunoprecipitation will be used to isolate and clone Dmrtl target genes. Identified clones will be examined for the presence of Dmrtl binding sites, functionally tested for Dmrtl response elements, and their genes reexamined for Dmrtl binding in vivo. Verified targets will be characterized to establish their expression profile, gauge their function, and determine their potential as drug targets for future contraceptive development. It is anticipated that the proposed studies will identify new compounds for use as male contraceptives as well as new targets for future contraceptive drug development.

进化保守蛋白 Dmrtl（Doublesex 和 Mab-3 相关转录因子-1）是一种睾丸特异性蛋白 对配子成熟和生育力至关重要的转录因子。其表达仅限于 性腺，出生后，它是男性特异性的，仅在减数分裂前生殖细胞的子集中发现 睾丸中的支持细胞。它对配子成熟的要求及其睾丸特异性表达使得 Dmrtl 是开发新型男性避孕药的绝佳候选靶点，因为它是一种化合物 破坏其活性或其下游靶基因的活性，可能会阻止精子发生并阻碍 生育能力。该项目的目标是确定通过破坏 Dmrtl 发挥作用的先导避孕化合物 通过揭示 Dmrtl 激活的基因来了解活性和新的避孕目标。目标 1 将开发一种检测方法 用于高通量筛选 (HTS)，以识别破坏之间相互作用的新型小分子 Dmrtl 及其 DMA 绑定元素。生产出纯化的 Dmrtl 后，超过 120,000 种化合物， 可通过药物发现、设计和合成核心（核心 B）HTS 设施获得，将使用 适合 384 孔格式的基于荧光的测定。目标 2 将评估中确定的先导化合物 目标 1 确定它们在细胞研究和整体动物研究中的生物效应。细胞活力，瞬时 转染分析和染色质免疫沉淀将用于测试培养物中的化合物活性 持续感兴趣的细胞和化合物将被给予雄性大鼠，并观察它们的毒理学和 评估生育效果。在目标 3 中，染色质免疫沉淀将用于分离和克隆 Dmrtl 目标基因。将检查已识别的克隆是否存在 Dmrtl 结合位点，并进行功能测试 用于 Dmrtl 反应元件，并重新检查它们的基因在体内的 Dmrtl 结合。已验证的目标将是 确定其表达谱、评估其功能并确定其作为药物的潜力 未来避孕药具发展的目标。预计拟议的研究将确定新的 用作男性避孕药的化合物以及未来避孕药的新靶点 发展。