Novel Sperm Glycolytic Enzymes as Contraceptive Targets

描述(申请人提供)：糖酵解是哺乳动物精子功能和受精所必需的。几种精子糖酵解酶具有独特的性质，这一途径中的三种同工酶是只在生精细胞中表达的基因的产物。其中两种生殖细胞特异性同工酶，甘油醛3-磷酸脱氢酶-S和磷酸甘油酸激酶-2，位于消耗和产生三磷酸腺苷的两个阶段之间的关键转换点。选择性地抑制这两种同工酶中的任何一种都可以消除糖酵解产生的ATP，而不影响其他组织的能量产生，表明GAPDS和PGK2可能是很好的避孕靶点。我们最近的研究表明，Gapds-/-雄性小鼠是不育的，产生的精子没有前进的活力，ATP水平是正常水平的10%。这项提议将确定精子活力和男性生育是否同样需要PGK2，并将研究这种同工酶的酶和结构特征，目的是确定选择性抑制物。其具体目的是：1)确定PGK2是否对精子活力和男性生育能力是必需的。在小鼠体内，Pgk2基因将通过同源重组而被破坏。将评估Pgk2-/-动物的生育能力、精子发生和精子功能，包括精子活力、形态和ATP水平的分析。2)鉴定PGK2独特的结构特征，设计该同工酶的选择性抑制剂。分子模拟研究将被用来确定区别PGK2和PGK1的特征，PGK1是存在于体细胞组织中的同工酶。潜在的活性部位抑制剂将通过虚拟筛选来确定，最有希望的候选者将与重组的PGK2和PGK1进行测试，以确定PGK2的选择性抑制剂。3)确定PGK2在精子中的定位机制。像其他糖酵解酶一样，PGK2与精子的结构成分结合在一起。为了更好地了解蛋白质之间的相互作用，这些蛋白质对于高水平的糖酵解ATP产生以支持精子的活力是必不可少的，我们将确定PGK2是否定位于精子鞭毛的主要部分，识别与PGK2相互作用的蛋白质，并确定PGK2是否具有负责精子定位的特定靶向序列。这项提议将确定PGK2的新特征，并确定这种精子同工酶是否是开发高度特异的男性避孕药的目标。

DESCRIPTION (provided by applicant): Glycolysis is required for mammalian sperm function and fertilization. Several sperm glycolytic enzymes have distinctive properties, and three isozymes in this pathway are products of genes that are expressed only in spermatogenic cells. Two of these germ cell-specific isozymes, glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) and phophoglycerate kinase-2 (PGK2), are located at a key transition point in the pathway between the two phases that consume and produce ATP. Selective inhibition of either of these isozymes would eliminate ATP production by glycolysis without affecting energy production in other tissues, indicating that GAPDS and PGK2 may be excellent contraceptive targets. Our recent studies indicate that Gapds -/- male mice are infertile, producing sperm with no progressive motility and ATP levels that are 10% of normal levels. This proposal will determine if PGK2 is similarly required for sperm motility and male fertility, and will examine the enzymatic and structural characteristics of this isozyme with the goal of identifying selective inhibitors. The specific aims are to: 1) Determine if PGK2 is required for sperm motility and male fertility. The Pgk2 gene will be disrupted in mice by homologous recombination. Fertility, spermatogenesis and sperm function will be assessed in the Pgk2 -/- animals, including assays of sperm motility, morphology and ATP levels. 2) Identify unique structural features of PGK2 and design selective inhibitors of this isozyme. Molecular modeling studies will be used to determine features that distinguish PGK2 from PGK1, the isozyme present in somatic tissues. Potential active site inhibitors will be identified by virtual screening, and the most promising candidates will be tested against recombinant PGK2 and PGK1 to identify selective inhibitors of PGK2. 3) Determine the mechanisms for PGK2 localization in sperm. Like other glycolytic enzymes, PGK2 is bound to structural components of sperm. To provide a better understanding of protein interactions that are essential for high levels of glycolytic ATP production to support sperm motility, we will determine if PGK2 is localized in the principal piece of the sperm flagellum, identify proteins that interact with PGK2, and determine if PGK2 has a specific targeting sequence that is responsible for sperm localization. This proposal will identify novel features of PGK2 and determine if this sperm isozyme is a target for developing highly specific male contraceptives.