Identifying Small Molecules that Regulate Uterine Contractions

点击翻译按钮获取中文摘要

PROJECT SUMMARY The uterine myometrium is a primary therapeutic target for: 1) mitigation of preterm labor (PTL), 2) labor induction, and 3) control of postpartum hemorrhage (PPH). Current tocolytics used to inhibit uterine contractions, and uterotonics used for PPH, are limited by their undesirable off-target effects and short duration of benefit. Moreover, there is almost a complete lack of drug development for PTL, PPH and other obstetric indications. Thus, a substantial need exists for novel, safe tocolytic and uterotonic agents with improved efficacy and selectivity. High-throughput screening (HTS) provides a drug-discovery platform for researchers to identify and then optimize small-molecules with increased affinity, selectivity and efficacy/potency. Based on the central role of Ca2+-mobilization in uterine contractions, and potent effects of oxytocin (OT) on uterine intracellular Ca2+- release, exploitation of intracellular Ca2+-release from uterine myometrial (UT-myo) cells in the absence and/or presence of OT provides an excellent strategy to discover new uterotonics and/or tocolytics, respectively. Thus, we developed a dual-addition Ca2+-mobilization assay, using a fluorescent Ca2+-sensitive probe and primary mouse UT-Myo cells, to allow dual-detection of agonists (uterotonics) of Ca2+-mobilization and antagonists (tocolytics) of OT-induced Ca2+-mobilization in a single screen. This assay is robust, reproducible (Z´ =0.73), and DMSO tolerant. A pilot screen of 2,727 compounds from the Spectrum Collection, NIH Clinical I and II Collections demonstrated: 1) excellent assay performance, 2) feasibility of using primary mouse UT-myo cells for HTS and 3) identified compounds for immediate testing for ability to regulate ex vivo uterine contractions. The goal of this application is to employ our HTS-ready assay to identify small molecules that regulate uterine myometrial Ca2+-mobilization with high affinity and selectivity from a large-compound library. In Aim 1 we will: 1) implement a large-scale HTS-campaign against 100,978 compounds in the SelleckChem FDA- approved Drug Library and Vanderbilt Discovery Collection, to identify hit-compounds that stimulate Ca2+- mobilization or inhibit OT-induced Ca2+-mobilization in UT-myo cells; and 2) narrow hits to lead-compounds after confirmation and evaluation of "hits" undergoing a comparative screen and dose-response relationship analysis. In Aim 2 we will validate lead small-molecules by using an ex vivo functional isometric contractile assay to record the therapeutic-effects of lead-compounds on human uterine myometrial contractile activity. The successful completion of our studies will identify a series of new lead-compounds for pre-clinical development of selective regulators of uterine myometrial contractility.