Targeting the piRNA pathway and meiotic recombination for male contraception

 描述(由申请人提供)：男性生殖受到一些基本和特定的过程的调节。进化上高度保守的piRNA途径对小鼠的雄性生育是必不可少的。MOV10L1是一种可能的RNA解旋酶，在所有piRNAs的生物发生中都是必需的，因此是这一途径的主要调节因子。MOV10L1与包括PIWIL1和PIWIL2在内的piRNA结合蛋白相互作用，这些相互作用对piRNA的生物发生和雄性生育至关重要。减数分裂重组负责同源染色体之间的遗传物质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因子，是减数分裂重组所必需的。MEIOB与另一种减数分裂特异因子SPATA22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(MOV10L1、PIWIL1、PIWIL2、MEIOB和SPATA22)都是生殖细胞特异性和敲除突变小鼠，因为这些基因中的任何一个都是活的，但由于减数分裂或精子发生停滞而不育。由于这些突变小鼠在其他方面都是健康的，它们的不孕不育表现出“纯不育”的表型。基于我们的遗传和生化研究，我们假设piRNA途径和减数分裂重组中的蛋白质成分是男性避孕的极佳靶标，副作用最小。在这里，我们特别建议验证MOV10L1/Piwi和MEIOB/SPATA22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。我们将系统地定位MOV10L1/PIWIL1/2(Aim 1)和MEIOB/SPATA22(Aim 2)中的结合结构域，并建立强大的双分子荧光互补(BIFC)方法来监测MOV10L1-PIwi和MEIOB-SPATA22的相互作用。在目标3中，我们将进行基于细胞的高通量BIFC筛选小分子化合物库，寻找MOV10L1-Piwi和MEIOB-SPATA22相互作用的抑制剂。以遗传学为基础，我们创新的基于细胞的筛查将识别抑制这些新型男性避孕药目标的先导化合物，以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.