Novel Sperm Glycolytic Enzymes as Contraceptive Targets

描述（由申请方提供）：哺乳动物精子功能和受精需要糖酵解。几种精子糖酵解酶具有独特的特性，其中三种同工酶是仅在生精细胞中表达的基因产物。这些生殖细胞特异性同工酶中的两种，甘油醛3-磷酸脱氢酶-S（GAPDS）和磷酸甘油酸激酶-2（PGK 2），位于消耗和产生ATP的两个阶段之间的途径中的关键过渡点。选择性抑制这些同工酶中的任何一种都会通过糖酵解消除ATP的产生，而不会影响其他组织的能量产生，这表明GAPDS和PGK 2可能是极好的避孕靶点。我们最近的研究表明，Gapds -/-雄性小鼠是不育的，产生的精子没有前向运动能力，ATP水平是正常水平的10%。该提案将确定PGK 2是否同样需要精子活力和男性生育力，并将检查这种同工酶的酶和结构特征，以确定选择性抑制剂的目标。具体目标是：1）确定PGK 2是否是精子活力和男性生育力所必需的。Pgk 2基因将在小鼠中通过同源重组被破坏。将在Pgk 2-/-动物中评估生育力、精子发生和精子功能，包括精子活力、形态和ATP水平测定。2)识别PGK 2的独特结构特征并设计该同工酶的选择性抑制剂。分子模拟研究将用于确定区分PGK 2和PGK 1（体细胞组织中存在的同工酶）的特征。将通过虚拟筛选鉴定潜在的活性位点抑制剂，并将针对重组PGK 2和PGK 1测试最有希望的候选物，以鉴定PGK 2的选择性抑制剂。3)确定PGK 2在精子中定位的机制。与其他糖酵解酶一样，PGK 2与精子的结构成分结合。为了更好地了解蛋白质相互作用，这是必不可少的高水平的糖酵解ATP的生产，以支持精子活力，我们将确定PGK 2是本地化的精子鞭毛的主要部分，确定与PGK 2相互作用的蛋白质，并确定PGK 2是否有一个特定的靶向序列，负责精子定位。该提案将确定PGK 2的新功能，并确定这种精子同工酶是否是开发高度特异性男性避孕药的目标。

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.