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.
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