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。虽然目前有几种针对女性的避孕方法，但更全面的节育方法需要将避孕扩大到男性。然而，安全、有效和可逆的男性避孕药仍然无法获得。开发男性避孕药的一个有吸引力的方法是靶向在精子中特异表达的蛋白质，这些蛋白质是精子生育所必需的。本实验室的研究表明，精子特异性质膜转运蛋白Na，K-ATPaseα4是一种将胞内Na+交换为胞外K+的重要靶标。Na，K-ATPaseα4仅在减数分裂后的雄性生殖细胞中表达，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)和一些重要的精子参数是必不可少的，这些参数包括膜电位(Vm)、细胞内钙离子([Ca+]i)和pH。重要的是，α4对精子的运动和过度激活是至关重要的，这是与精子获能有关的关键事件。因此，在小鼠中敲除α4会导致完全的男性不育。从生化的角度来看，α4对哇巴因具有特别高的亲和力。我们开发了一系列具有甾体和非甾体骨架结构的合成化合物，这些化合物通过靶向α4的哇巴因结合部位，选择性地抑制α4并影响精子的活力。这些结果为α-4作为控制男性生育能力的药理靶点提供了强有力的证据。靶向Na，K-α酶4的关键是抑制该蛋白将允许特异性地干扰精子功能，而不影响精子发生，并提供临时和可逆的避孕措施。此外，体细胞中缺乏α4，降低了其失活会产生副作用的可能性。然而，在α-4抑制剂作为男性避孕药应用之前，有必要确定和优化它们的有效性、药物-靶点相互作用、体外和体内特异性的生物标志物、副作用和作用机制，以便将来临床使用。我们将在这里通过确定我们的抑制剂对Na，K-ATPaseα4活性、精子活力以及受Na，K-ATPaseα4活性控制的精子参数的有效性和选择性来测试这一点，这些参数包括精子[Na+]i、[Ca+]i、pH、Vm、精子过度激活、活动率和生育力。最后，我们将在小鼠交配试验中测定Na，K-ATP酶α4抑制剂的药代动力学参数、安全性和避孕效果。这项研究的基本原理是，一旦在体外和体内验证了Cardenoldes作为Na，K-ATPaseα4靶标的适宜性，它们就可以作为可逆控制男性生育能力的试剂而进一步发展。

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.