Targeting the piRNA pathway and meiotic recombination for male contraception

 描述（由申请人提供）：男性生殖受许多基本和特定过程的调节。高度进化保守的皮尔纳途径对于小鼠中的雄性生育力是必需的。MOV 10 L1是一种推定的RNA解旋酶，是所有piRNA生物合成所必需的，因此是该途径的主要调节因子。M0 V10 L1与皮尔纳结合蛋白（包括PIWIL 1和PIWIL 2）相互作用，并且这些相互作用对于皮尔纳生物发生以及因此对于雄性生育力是关键的。减数分裂重组负责同源染色体之间遗传物质的交换和减数分裂过程中染色体的忠实分离。MEIOB是一种单链DNA结合蛋白，是一种新的减数分裂特异性因子，对减数分裂重组至关重要。MEIOB与另一个减数分裂特异性因子SPATA 22相互作用，我们发现这种相互作用对它们的相互稳定性至关重要。值得注意的是，所有这些蛋白质（MOV 10 L1、PIWIL 1、PIWIL 2、MEIOB和SPATA 22）都是生殖细胞特异性的，并且这些基因中的任何一个的敲除突变小鼠都是存活的，但由于减数分裂或精子生成的停滞而不育。由于这些突变小鼠在其他方面是健康的，它们的不育表现为“纯不育”表型。基于我们的遗传和生物化学研究，我们假设皮尔纳途径和减数分裂重组中的蛋白质组分是男性避孕的极好靶点，副作用最小。在这里，我们特别建议验证MOV 10 L1/Piwi和MEIOB/SPATA 22蛋白质-蛋白质相互作用作为新的男性避孕靶点，并筛选这些相互作用的小分子抑制剂。我们将系统地绘制MOV 10 L1/PIWIL 1/2（Aim 1）和MEIOB/SPATA 22（Aim 2）中的结合结构域，并开发强大的双分子荧光互补（BiFC）测定来监测MOV 10 L1-Piwi和MEIOB-SPATA 22相互作用。在目标3中，我们将对MOV 10 L1-Piwi和MEIOB-SPATA 22相互作用的抑制剂的小分子化合物文库进行基于细胞的高通量BiFC筛选。以遗传学为基础，我们创新的基于细胞的筛选将确定抑制这些新型男性避孕靶点的先导化合物，用于未来开发口服活性非激素避孕药。

 DESCRIPTION (provided by applicant): Male reproduction is regulated by a number of essential and specific processes. The highly evolutionarily conserved piRNA pathway is essential for male fertility in mice. MOV10L1, a putative RNA helicase, is required for biogenesis of all piRNAs and thus is a master regulator of this pathway. MOV10L1 interacts with piRNA- binding proteins, including PIWIL1 and PIWIL2, and these interactions are critical for piRNA biogenesis, and thus male fertility. Meiotic recombination is responsible for exchange of genetic materials between homologous chromosomes and faithful chromosome segregation during meiosis. MEIOB, a single-stranded DNA-binding protein, is a novel meiosis-specific factor essential for meiotic recombination. MEIOB interacts with SPATA22, another meiosis-specific factor, and we find that this interaction is critical for their mutual stability. Notably, all thes proteins (MOV10L1, PIWIL1, PIWIL2, MEIOB, and SPATA22) are germ cell-specific and knockout mutant mice for any of these genes are viable but sterile due to arrest in meiosis or spermiogenesis. As these mutant mice are otherwise healthy, their infertility demonstrates a "pure sterile" phenotype. Based on our genetic and biochemical studies, we hypothesize that the protein components in the piRNA pathway and meiotic recombination are excellent targets for male contraception with minimal side effects. Here, we specifically propose to validate MOV10L1/Piwi and MEIOB/SPATA22 protein-protein interactions as novel male contraceptive targets and to screen for small molecule inhibitors of these interactions. We will systematically map the binding domains in MOV10L1/PIWIL1/2 (Aim 1) and in MEIOB/SPATA22 (Aim 2), and develop powerful bimolecular fluorescence complementation (BiFC) assays to monitor MOV10L1-Piwi and MEIOB-SPATA22 interactions. In Aim 3, we will perform high throughput cell-based BiFC screens of libraries of small molecule compounds for inhibitors of MOV10L1-Piwi and MEIOB-SPATA22 interactions. With genetics as the foundation, our innovative cell-based screens will identify lead compounds that inhibit these novel male contraceptive targets for future development of an orally active non-hormonal contraceptive.