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

进化保守蛋白Dmrtl（Doublemex and Mab-3 related transcription factor-1）是睾丸特异性转录因子， 配子成熟和生育力所必需的转录因子。其表达仅限于 生殖腺，出生后，它是男性特异性的，它只存在于减数分裂前生殖细胞的一个子集， 睾丸支持细胞。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极好候选靶点，作为 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断精子形成。 生育该项目的目标是确定通过破坏Dmrtl发挥作用的主要避孕化合物 活性和新的避孕目标，揭示基因激活的Dmrtl。Aim 1将开发一种检测方法， 用于高通量筛选（HTS），以鉴定破坏相互作用的新型小分子。 Dmrtl及其DMA绑定元素。在生产纯化的Dmrtl之后，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 基于荧光的测定法适用于384孔格式。目标2将评估在 目的1在细胞和整体动物研究中确定它们的生物学效应。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 将持续感兴趣的细胞和化合物给予雄性大鼠， 评价生育效果。目的3：利用染色质免疫沉淀技术分离克隆Dmrtl基因 靶基因将检查鉴定的克隆是否存在Dmrtl结合位点，功能测试 的Dmrtl反应元件，并在体内重新检查其基因的Dmrtl结合。验证目标将是 表征以建立它们的表达谱，测量它们的功能，并确定它们作为药物的潜力， 未来避孕发展的目标。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

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.