High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility

Abstract

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.

Increased rate of acute caesarean sections in women with epilepsy: results from the Oppland Perinatal Database in Norway

“…In‐depth examination revealed multiple obstetric reasons for acute CS in WWE. As most of the AEDs used in this study are sodium or calcium channel blockers, one factor of possible importance might be that uterine excitability is modulated by sodium channels and calcium‐channel blockers are used as tocolytic drugs . Reasons for increased acute CS risk in WWE are probably multifactorial and use of AEDs appears to be the most important epilepsy‐related risk factor.…”

Study of Magnesium Formulations on Intestinal Cells to Influence Myometrium Cell Relaxation

“…Isometric contraction measurement was performed as reported in the literature [43]. Briefly, uterine myometrial samples, obtained from wild-type mice on day 19 of pregnancy, were prepared by cutting the uterus along the mesometrial (vascularized) border, followed by removal of fetal, placental, and connective tissue/membranes obtaining uterine strips of 4 mm × 12 mm.…”

Genetically Encoded Calcium Indicators as Probes to Assess the Role of Calcium Channels in Disease and for High-Throughput Drug Discovery

“…Only a few published studies have used GECIs in the context of high-throughput screening in drug discovery; however, there has been wide use of small molecule Ca 2 + indicators in a variety of G-protein-coupled receptors and ion channel-based biomolecular screens (Behrendt, Germann, Gillen, Hatt, & Jostock, 2004;Herington et al, 2015;Wang et al, 2015). This is a strong indicator of the utility of GECIs in biomolecular screening, given the recent advances in GECIs as discussed throughout this review.…”

“…This assay was not without challenges; nematodes were imaged in microwells made from agar and despite identifying five target genes capable of altering Ca 2 + , this did not translate to a strong phenotype of altered synaptic vesicle trafficking (Wabnig et al, 2015). It is likely that some GECIs are already part of biodiscovery projects within pharmaceutical companies and have not yet been described in journal articles, as has been done with small molecule-based probes (Herington et al, 2015). However, it is clear that further application of GECIs in academia and industry will help accelerate the discovery of a new generation of pharmacological agents capable of modulating calcium signaling in disease.…”