High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility.
High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility.
复制标题
DOI：
10.1371/journal.pone.0143243
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Reese J
中科院分区：
文献类型：
--
作者：
Herington JL;Swale DR;Brown N;Shelton EL;Choi H;Williams CH;Hong CC;Paria BC;Denton JS;Reese J
子宫肌层（UT-myo）是早产、引产和产后出血的治疗靶点。催产素刺激UT-myo细胞内Ca 2+释放是控制子宫肌层收缩的最终途径。本研究的目的是开发一种双添加试验，用于高通量筛选小分子化合物，其可以调节UT-myo细胞中的Ca 2+动员，从而调节子宫肌层收缩。将384孔板中的原代鼠UT-myo细胞加载Ca 2+敏感性荧光探针，然后筛选Ca 2+动员的诱导剂和催产素诱导的Ca 2+动员的抑制剂。该检测方法显示出稳健的筛选统计学（Z ′ = 0.73）和DMSO耐受性，并针对来自Spectrum、NIH Clinical I和II系列注释良好的化合物的2，727种小分子进行了高通量筛选。筛选结果显示，激动剂化合物的命中率为1.80%，拮抗剂化合物的命中率为1.39%。命中化合物的浓度依赖性反应表明，21种命中拮抗剂化合物的EC 50小于10μM，而只有7种命中激动剂化合物。随后的研究集中在命中拮抗剂化合物。基于抑制百分比和功能注释分析，我们选择了4个确认的命中拮抗剂化合物（苯溴马隆，双嘧达莫，氢溴酸非诺特罗和尼索地平）进行进一步分析。使用离体等长收缩试验，每种化合物在不同的效力（IC 50）下显著抑制子宫收缩。总的来说，这些结果首次证明，高通量的子宫肌层钙离子动员的小分子筛选是发现子宫收缩力调节剂的理想的主要方法。
The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility.