BACKGROUND AND PURPOSE Asthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high-throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca2+ ]i ), promote human sperm cell motility, and systematically determine the mechanism of action. EXPERIMENTAL APPROACH A validated HTS fluorometric [Ca2+ ]i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. KEY RESULTS Fluorometric assays identified trequinsin as an efficacious agonist of [Ca2+ ]i , although less potent than progesterone. Functionally, trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. Trequinsin-induced [Ca2+ ]i responses were cross-desensitised consistently by PGE1 but not progesterone. Whole-cell patch clamp electrophysiology confirmed that trequinsin activated CatSper and partly inhibited potassium channel activity. Trequinsin also increased intracellular cGMP. CONCLUSION AND IMPLICATIONS Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new agents to improve patient sperm function and fertilisation potential.

中文翻译：

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盐酸trequinsin的新药理作用可改善人类精子的运动能力和功能。

背景与目的弱精子症是男性不育症的主要原因，但由于缺乏有效的筛查平台和对合适的分子靶标的了解，阻碍了提高精子活力的药理学的发展。我们已经证明，高通量筛选（HTS）策略和已建立的体外测试可以鉴定和表征可提高精子活力的化合物。在这里，我们应用HTS从新颖的小分子文库中识别出新化合物，这些新化合物可增加细胞内钙（[Ca2 +] i），促进人类精子细胞运动并系统地确定其作用机理。实验方法采用经过验证的HTS荧光[Ca2 +] i分析方法筛选内部化合物库。选择了盐酸Trequinsin（一种PDE3抑制剂）进行详细的分子（酶标仪分析，电生理学和环核苷酸测量）和功能（活力和顶体反应）测试，这些测试来自健康的志愿者供体以及可能的患者的精子。关键结果荧光分析法确定曲霉毒素是[Ca2 +] i的有效激动剂，尽管效力不及黄体酮。从功能上讲，trequinsin显着增加了所有供体精子样本中的细胞过度活化和渗透到粘性介质中，并增加了22/25（88％）患者精子样本中的细胞过度活化。Trequinsin诱导的[Ca2 +] i反应始终被PGE1交叉脱敏，但孕酮没有。全细胞膜片钳电生理学证实，trequinsin激活CatSper并部分抑制钾通道活性。Trequinsin还增加了细胞内cGMP。结论和意义Trequinsin在人类精子中显示出新的药理学特征，可能是开发新型药物以改善患者精子功能和受精潜力的合适先导化合物。