Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem.
Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem.
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DOI：
10.1093/humrep/dex055
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发表时间：
2017-05-01
期刊：
影响因子：
--
通讯作者：
Hope AG
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文献类型：
--
作者：
Martins da Silva SJ;Brown SG;Sutton K;King LV;Ruso H;Gray DW;Wyatt PG;Kelly MC;Barratt CLR;Hope AG
可以利用药物发现方法将研究转化为男性不育症的新疗法吗？高通量筛选（HTS）是一种可行的方法，急需的药物发现男性因素不育。对于不育男性的新治疗选择，既有巨大的需求，也有真正的临床需求。然而，由于人类精子的细胞、物理和功能特性的独特挑战以及缺乏适当的测定平台，药物发现所需的时间、精力和资源目前过高。精子来自2012年1月至2016年11月期间在医院辅助生殖技术诊所接受IVF/ICSI的健康志愿者研究供体和生育力低下患者。开发了一种HTS测定法，并使用细胞内钙（[Ca 2 +]i）作为人精子运动力的替代物进行了验证。使用Flexstation酶标仪（384孔平台）检测钙荧光，并与孕酮诱发的反应进行比较，孕酮是一种已知可改变人类精子中许多生物学相关行为的化合物。对邓迪大学药物发现单位组装的离子通道聚焦文库进行单点药物筛选（10 μM）后鉴定的命中化合物进行重新筛选，以使用标准10点半对数浓度曲线确保效价，并使用液相色谱法和质谱法检测纯度和完整性。命中化合物进行分组的构效关系和五个代表性的化合物，然后进一步研究对精子的直接影响，使用计算机辅助精子评估，精子穿透试验和全细胞膜片钳。在筛选的3242种离子通道库配体中，384种化合物（11.8%）引起钙荧光的统计学显著增加，高于基线的中位绝对偏差大于3倍。对在初步筛选中引起荧光增加≥50%的74种化合物进行了重新筛选和进一步评价，得到48种产生[Ca 2 +]i浓度依赖性增加的命中化合物。精子穿透研究证实，在体外暴露于两种命中化合物（A和B）导致健康志愿者供体精子的功能运动性显著改善（A：1cm穿透指数2.54，2cm穿透指数2.49; P < 0.005; B：1cm穿透指数2.1，2cm穿透指数2.6; P < 0.005），但至关重要的是，在接受生育治疗的患者样本中也是如此（A：1厘米渗透指数2.4; P = 0.009，2厘米渗透指数3.6; P = 0.02和B：1厘米渗透指数2.2; P = 0.0004，2厘米渗透指数3.6; P = 0.002）。这主要是由于直接或间接的CatSper通道的作用，支持的证据，从电生理学研究的个人精子。[Ca ~（2+）]i的增加和流动是调节精子运动和功能（包括顶体反应）的基础。在这项研究中，钙信号作为精子活力的替代物被认为是一个潜在的限制。我们的结论是，HTS可以稳健，有效地识别新的化合物，增加[Ca 2 +]i在人类精子和功能性修改运动，并提出其作为基石，建立和改造急需的药物发现男性不育症。大部分数据是由TENOVUS Scotland和首席科学家办公室NRS奖学金提供的资金获得的。NHS Tayside、MRC项目赠款（MR/K 013343/1、MR/012492/1）和阿伯泰大学提供了额外资金。作者声明不存在利益冲突。N/A.
Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform. Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016. A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 μM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping. Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P < 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P < 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm. Increase and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study. We conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility. The majority of the data were obtained using funding from TENOVUS Scotland and Chief Scientist Office NRS Fellowship. Additional funding was provided by NHS Tayside, MRC project grants (MR/K013343/1, MR/012492/1) and University of Abertay. The authors declare that there is no conflict of interest. N/A.
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影响因子：
6.7
作者：
Amory, John K.
通讯作者：
Amory, John K.
影响因子：
1.9
作者：
Ho, Katharine;Wolff, Collin A.;Suarez, Susan S.
通讯作者：
Suarez, Susan S.
DOI：
10.1073/pnas.2536658100
发表时间：
2003-12-09
影响因子：
11.1
作者：
Carlson, AE;Westenbroek, RE;Babcock, DF
通讯作者：
Babcock, DF
DOI：
10.1053/beem.2000.0083
发表时间：
2000-09-01
影响因子：
7.4
作者：
Grootegoed, JA;Siep, M;Baarends, WM
通讯作者：
Baarends, WM
影响因子：
6.1
作者：
Kupka, M. S.;Ferraretti, A. P.;Goossens, V.
通讯作者：
Goossens, V.