Inhibitors of Na,K-ATPase alpha4 as male contraceptives

描述（由申请人提供）：快速增长的世界人口和高意外怀孕率使避孕成为任何公共卫生计划的需要和优先事项。虽然目前有几种妇女避孕方法，但更全面的节育方法需要将避孕扩大到男性。然而，仍然没有一种安全、有效和可逆的男子避孕方法。开发男性避孕药的一个有吸引力的方法在于靶向精子中特异性表达的蛋白质，这些蛋白质是精子生育力所必需的。Na，K-ATP酶α4是一种精子特异性质膜转运蛋白，能将细胞内的Na ~+交换为细胞外的K ~+，是男性避孕的重要靶点之一。Na，K-ATP酶α4只在减数分裂后的精巢生殖细胞中表达，在精子鞭毛中大量表达。重要的是，α4具有非常独特的功能特征，对精子功能至关重要。α4的活性对于维持精子细胞内Na+水平（[Na+]i）和几个重要的精子参数，包括膜电位（Vm），细胞内Ca+2（[Ca 2 +]i）和pH值至关重要。重要的是，α4对精子运动和超活化至关重要，这是与精子获能相关的关键事件。因此，在小鼠中敲除α4导致完全的雄性不育。从生物化学的角度来看，α4对卡登诺哇巴因具有特别高的亲和力。我们已经开发了一系列具有甾体和非甾体骨架结构的合成化合物，它们通过靶向α4的哇巴因结合位点，选择性地抑制α4并影响精子运动。这些结果为α4作为控制雄性生育力的药理学靶点的适用性提供了强有力的证据。靶向Na，K-ATP酶α4的关键是抑制该蛋白质将允许特异性干扰精子功能，而不影响精子发生并提供暂时和可逆的避孕。此外，体细胞中α4的缺乏减少了其失活产生副作用的机会。然而，在α4抑制剂可以作为男性避孕药应用之前，有必要确定其疗效、药物-靶标相互作用、体外和体内特异性生物标志物、副作用和作用机制，并优化其未来的临床使用。我们将通过测定我们的抑制剂对Na，K-ATP酶α4活性、精子活力和受Na，K-ATP酶α4活性控制的精子参数（包括精子[Na+]i、[Ca 2 +]i、pH、Vm、精子超活化、活力和生育力）的有效性和选择性来对此进行测试。最后，我们将确定Na，K-ATP酶α4抑制剂在小鼠交配试验中的药代动力学参数、安全性和避孕有效性。该研究的基本原理是，一旦在体外和体内验证了强心苷作为Na，K-ATP酶α4靶点的适用性，则可将其作为可逆控制男性生育力的药物。

DESCRIPTION (provided by applicant): The rapidly growing world population and the high rate of unintended pregnancies make contraception a need and a priority for any public health program. While several contraceptive methods for women are currently available, a more comprehensive approach to birth control requires extending contraception to males. However, a safe, effective and reversible contraceptive for men is still unavailable. An attractive approach t develop male contraceptives consists in targeting proteins that are specifically expressed in sperm and are required for sperm fertility. Evidence from our laboratory has shown that Na,K-ATPase α4, a sperm specific plasma membrane transporter, which exchanges cytoplasmic Na+ for extracellular K+ is one of these attractive targets for male contraception. Na,K-ATPase α4 is only expressed in male germ cells of the testis after meiosis and abundant in the sperm flagellum. Importantly, α4 has functional characteristics that are highly unique and critical for sperm function. Activity of α4 is essential for maintaining sperm intracellular Na+ levels ([Na+]i) and several vital sperm parameters, including membrane potential (Vm), intracellular Ca+2 ([Ca2+]i) and pH. Importantly, α4 is crucial for sperm motility and hyperactivation, a key event associated with sperm capacitation. Accordingly, knockout of α4 in mice results in complete male infertility. From a biochemical standpoint, α4 has a particularly high affinity for the cardenolide ouabain. We have developed a series of synthetic compounds with steroidal and non-steroidal backbone structure, which, by targeting the ouabain binding site of α4, selectively inhibit α4 and affect sperm motility. These results provide strong evidence for the suitability of α4 as a pharmacological target for the control of male fertility. The key to targeting Na,K-ATPase α4 is that inhibition of this protein will allow to specifically interfere with sperm function, without affecting spermatogenesis and providing temporary and reversible contraception. In addition, the lack of α4 in somatic cells reduces the chances that its inactivation will produce side effects. However, before the α4 inhibitors can be moved forward into their application as male contraceptives, it is necessary that their efficacy, drug-target interaction, biomarkers for their in vitro and vivo specificity, side effects and mechanisms of action are identified and optimized for future clinical use. We will test this here by determining the efficacy and selectivity of action of our inhibitors on Na,K-ATPase α4 activity, on sperm motility and on sperm parameters that are controlled by Na,K-ATPase α4 activity, including sperm [Na+]i, [Ca2+]i, pH, Vm, sperm hyperactivation, motility and fertility. Finally, we will determine the pharmacokinetic parameters, safety and contraceptive effectiveness of Na,K-ATPase α4 inhibitors in mating trials in mice. The rationale for the research is that once the suitability of cardenolides as targets of Na,K-ATPase α4 is validated, both in vitro and in vivo, they can be advanced as agents for the reversible control of male fertility.