title: 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.
authors.affiliation: Department of Pediatrics, Division of Neonatology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Herington
authors.affiliation: Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, United States of America.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Swale
authors.affiliation: Department of Pediatrics, Division of Neonatology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Naoko
authors.initials: N
authors.lastname: Brown
authors.affiliation: Department of Pediatrics, Division of Neonatology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Elaine L
authors.initials: EL
authors.lastname: Shelton
authors.affiliation: Department of Pediatrics, Division of Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Hyehun
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Charles H
authors.initials: CH
authors.lastname: Williams
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.|Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Hong
authors.affiliation: Department of Pediatrics, Division of Neonatology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Bibhash C
authors.initials: BC
authors.lastname: Paria
authors.affiliation: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.|Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.
authors.forename: Jerod S
authors.initials: JS
authors.lastname: Denton
authors.affiliation: Department of Pediatrics, Division of Neonatology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.|Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Jeff
authors.initials: J
authors.lastname: Reese
mesh_terms: D000818:Animals; D002118:Calcium / Q000819:agonists / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D009215:Myometrium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010121:Oxytocin / Q000494:pharmacology; D011247:Pregnancy; D061251:Primary Cell Culture; D015203:Reproducibility of Results; D014590:Uterine Contraction; D014599:Uterus / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology*
chemical_list: D002121:Calcium Channel Blockers; D010121:Oxytocin; D002118:Calcium