Purpose: Preterm birth (PTB) rate continues to rise with over 15 million PTB/year and contributes to >80% neonatal deaths. Postpartum hemorrhage (PPH) is the leading cause of maternal death, occurring at rates 100 times higher in developing countries compared to developed countries. Novel tocolytic and uterotonic agents to treat PTB and PPH, respectively, are urgently needed due to the detrimental off-target side effects (for both infant and mother) and short duration of benefit of current therapeutics. Repurposing existing FDA-approved drugs for new indications offers a better risk-versus-benefit trade-off compared to traditional drug development process. The repurposing strategy bypasses extensive and expensive drug development stages, allowing accelerated and more cost-effective progress to clinical stage.
The uterine myometrium (UT-myo) is a therapeutic target for regulating uterine contractions. A final pathway controlling myometrial contractions involves stimulation of intracellular calcium [Ca2+]i-release. To this respect, we have developed an assay for high-throughput screening (HTS) of small-molecules to regulate Ca2+-mobilization in UT-myo cells. The goal of this work was to identify potent, selective regulators of uterine contractility to treat PTB and PPH by: 1) using our high-throughput, dual addition Ca2+-mobilization assay to screen 1,180 small-molecules within the SelleckChem library of FDA-approved drugs and 2) examining the dose-response of hit-agonists and antagonists and counterscreening against vascular smooth muscle cells—the major off-targets limiting the use of current tocolytics and uterotonics. Finally, we removed uterine-selective hit-drugs that have reported reproductive toxicity, included adverse pregnancy outcome and fetal anomalies.
Methods: Primary human UT-myo cells were isolated from tissue collected at the time of cesarean delivery from women at term (≥37 weeks) pregnancy. After 24hr attachment of UT-myo cells (4000 cells/well) in 384-well plates, a fluorescent-based high-throughput Ca2+-mobilization assay was performed. On each day of screening, a dose-response analysis of oxytocin (OT), the most potent endogenous agonist uterine contractility, was performed to calculate its EC80. The SelleckChem FDA compound library of 1,180 compounds was accessed from Vanderbilt HTS facility and screened at 10M identify agonists and antagonists of Ca2+-mobilization before and after the addition of OT at submaximal EC80, respectively. The % response, % stimulation and % inhibition of all compounds were analyzed using an in house software, Waveguide, to identify hit-compounds. A robust Z` -score calculation was used to identify hit-compounds based on whether a test compound`s value was > 3 times the mean absolute standard deviations (MADs) away from the median of all the vehicle (0.1% DMSO) wells before or after the OT addition. After retesting of hits, validated hit FDA-approved drugs were counterscreened against human primary aorta and pulmonary vascular smooth muscle cells, utilizing the same Ca2+-mobilization assay and U46619 (an agonist of the thromboxane A2 receptor), instead of OT, to induce intracellular Ca2+-release from VSMCs. Dose-response analysis of uterine-selective hit-agonists and antagonists were generated using thirteen-point, 3-fold dilutions of each compound to determine IC50 or EC50, respectively. Finally, we searched the Reprotox database for reports of known reproductive toxicity of drugs during pregnancy.
Results: We identified 70 hit-agonists and 60 hit-anatgonists of Ca2+-mobilization from the screen of 1,180 compounds in SelleckChem FDA library (Table 1 and Figure 1). The hits identified were from diverse pharmacological classes. Specifically, the majority of hit-agonists were identified as: Dopamine antagonist, adrenergic beta-agonists and histamine agonists. Moreover, the pharmacological classes of hit-antagonists included: antimicrobial, anti-asthmatic, cathartic, antihypertensive. A total of 27 drugs were identified as uterine selective, with greater than 5-fold potency towards UT-myo cells. In total, 2 hit-agonists and 14 hit-antagonist drugs were identified as potent with an IC50 ≤10μM. We identified 0 hit-agonists and 2 hit-antagonists that are reported to cause reproductive toxicity.
Conclusion: Phenotypic high-throughput screening of FDA-approved drugs against human primary UT-myo cells and VSMCs has allowed us to identify uterine-selective modulators of myometrial contractions. Based on potency and known reproductive toxicity, 14 hit-antagonists and 2 hit-agonists show promise for their potential to be repurposed for the treatment of PTB and PPH, respectively.
Jackson Rogers– Research Assistant, Vanderbilt University Medical Center, NASHVILLE, Tennessee
Lauren Lambert– Research Assistant, Vanderbilt University Medical Center, NASHVILLE, Tennessee
Dehui Mi– Drug Discovery Scientist, Vanderbilt University, Nashville, Tennessee
Paige Vinson– Director, High-throughput screening facility, Vanderbilt University, Nashville, Tennessee
Bibhash Paria– Associate Professor, Vanderbilt University Medical Center, NASHVILLE, Tennessee
Jeff Reese– Professor of Pediatrics, Vanderbilt University Medical Center, NASHVILLE, Tennessee
Jennifer Herington– Assistant Professor, Vanderbilt University Medical Center, NASHVILLE, Tennessee