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求和优先事项。虽然目前有几种妇女避孕方法，但更全面的避孕方法需要将避孕措施扩大到男性。然而，目前仍然没有一种安全、有效和可逆的男性避孕方法。开发男性避孕药的一个有吸引力的方法是针对精子中特定表达的蛋白质，这些蛋白质是精子生育所必需的。我们实验室的证据表明，Na，K- atp酶α4，一种精子特异性的质膜转运蛋白，将细胞质内的Na+交换为细胞外的K+，是男性避孕的一个有吸引力的靶点。Na， k - atp酶α4仅在减数分裂后的睾丸雄性生殖细胞中表达，在精子鞭毛中大量存在。重要的是，α4具有对精子功能非常独特和关键的功能特征。α4的活性对于维持精子胞内Na+水平（[Na+]i）和几个重要的精子参数（包括膜电位（Vm）、胞内Ca+2 （[Ca2+]i）和ph）至关重要。重要的是，α4对于精子运动和超激活至关重要，这是与精子获能相关的关键事件。因此，敲除小鼠α4会导致完全雄性不育。从生物化学的角度来看，α4对核桃内酯具有特别高的亲和力。我们已经合成了一系列具有甾体和非甾体骨架结构的化合物，它们通过靶向α4的瓦巴因结合位点，选择性地抑制α4，影响精子活力。这些结果为α4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 - atp酶α4的关键是抑制该蛋白可以特异性地干扰精子功能，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的缺失减少了α4失活产生副作用的可能性。然而，在α4抑制剂作为男性避孕药应用之前，有必要确定其疗效、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，并为未来的临床应用进行优化。我们将在这里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、[Ca2+]i、pH、Vm、精子过度激活、活力和生育能力）的功效和选择性来测试这一点。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。本研究的基本原理是，一旦在体外和体内验证了香梨内酯作为Na， k - atp酶α4靶点的适用性，它们就可以作为可逆控制男性生育能力的药物。

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.