Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery

Siricilla, Shajila; Knapp, Kelsi M.; Rogers, Jackson H.; Berger, Courtney; Shelton, Elaine L.; Mi, Dehui; Vinson, Paige N.; Condon, Jennifer C.; Paria, Bibhash C.; Reese, Jeff; Sheng, Quanhu; Herington, Jennifer L.

doi:10.1016/j.phrs.2019.104268

Cited by 6 publications

(17 citation statements)

References 61 publications

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“…Primary myometrial cells were isolated, selectively enriched for smooth muscle cells and cultured as previously described [3,23]. Primary myometrial cells were unpassaged and became near-confluent 10-15 days postisolation, with media (DMEM supplemented with 10% fetal bovine serum (FBS), 25 mM HEPES, 100 U/ml penicillin-streptomycin) changed every 3 days.…”

Section: Primary Human Uterine Myometrial Tissue and Cellsmentioning

confidence: 99%

“…Primary myometrial cells were unpassaged and became near-confluent 10-15 days postisolation, with media (DMEM supplemented with 10% fetal bovine serum (FBS), 25 mM HEPES, 100 U/ml penicillin-streptomycin) changed every 3 days. The cells were dissociated at near-confluency using 0.25% Trypsin-EDTA, and then plated at 4,000 cells/well in black-walled 384-well plates (Grenier Bio-One) for Ca 2+mobilization assays exactly as previously described [23].…”

Section: Primary Human Uterine Myometrial Tissue and Cellsmentioning

confidence: 99%

“…Mundulone and MA were assayed at 10-point three-fold titrations starting at 60μM in triplicate, as previously described [3], to compare the Emax and IC50 values between myometrial and VSMCs. A dual-addition Ca 2+mobilization assay was performed exactly as previously described using the automated equipment within the Vanderbilt University High-Throughput Screening Facility [3,23]. Briefly, Fluo-4AM (2μM) was loaded into cells and a Panoptic (WaveFront Biosciences) integrated pipettor was used to add the titrations of mundulone or MA to cells.…”

Section: Comparative Concentration-dependent Ca 2+ -Mobilizationmentioning

confidence: 99%

“…To account for position bias and edge effects, the matrices were repeated at different positions of the 384-well plate. Also, the z`-factor determined during the optimization of the Ca 2+ assay [3,23] ensures lack of position effects. Raw data was first analyzed using Waveguide, as described above and previously reported [23].…”

Section: Comparative Concentration-dependent Ca 2+ -Mobilizationmentioning

confidence: 99%

“…Also, the z`-factor determined during the optimization of the Ca 2+ assay [3,23] ensures lack of position effects. Raw data was first analyzed using Waveguide, as described above and previously reported [23]. After calculating the %Response for each compound concentration examined, we determined synergistic combinations using Combenefit software [24], which provided synergy scores and heat-maps to visualize models of Bliss independence, Highest Single Agent and Loewe additivity.…”

Section: Comparative Concentration-dependent Ca 2+ -Mobilizationmentioning

confidence: 99%

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Identification of mundulone and mundulone acetate as natural products with tocolytic efficacy in mono- and combination-therapy with current tocolytics

Siricilla

et al. 2021

Preprint

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Currently, there are a lack of FDA-approved tocolytics for the management of preterm labor. We previously observed that the isoflavones mundulone and mundulone acetate (MA) inhibit intracellular Ca2+-regulated myometrial contractility. Here, we further probed the potential of these natural products to be small molecule leads for discovery of novel tocolytics by: (1) examining uterine-selectivity by comparing concentration-response between human primary myometrial cells and a major off-target site, aortic vascular smooth muscle cells (VSMCs), (2) identifying synergistic combinations with current clinical tocolytics to increase efficacy or and reduce off-target side effects, (3) determining cytotoxic effects and (4) investigating the efficacy, potency and tissue-selectivity between myometrial contractility and constriction of fetal ductus arteriosus (DA), a major off-target of current tocolytics. Mundulone displayed significantly greater efficacy (Emax = 80.5% vs. 44.5%, p=0.0005) and potency (IC50 = 27 μM and 14 μM, p=0.007) compared to MA in the inhibition of intracellular-Ca2+ from myometrial cells. MA showed greater uterine-selectivity, compared to mundulone, based on greater differences in the IC50 (4.3 vs. 2.3 fold) and Emax (70% vs. 0%) between myometrial cells compared to aorta VSMCs. Moreover, MA demonstrated a favorable in vitro therapeutic index of 8.8, compared to TI = 0.8 of mundulone, due to its significantly (p<0.0005) smaller effect on the viability of myometrial (hTERT-HM), liver (HepG2) and kidney (RPTEC) cells. However, mundulone exhibited synergism with two current tocolytics (atosiban and nifedipine), while MA only displayed synergistic efficacy with only nifedipine. Of these synergistic combinations, only mundulone + atosiban demonstrated a favorable TI = 10 compared to TI=0.8 for mundulone alone. While only mundulone showed concentration-dependent inhibition of ex vivo mouse myometrial contractions, neither mundulone or MA affected mouse fetal DA vasoreactivity. The combination of mundulone and atosiban yielded greater tocolytic efficacy and potency on term pregnant mouse and human myometrial tissue compared to single-drugs. Collectively, these data highlight the difference in uterine‐selectivity of Ca2+‐mobilization, effects on cell viability and tocolytic efficacy between mundulone and MA. These natural products could benefit from medicinal chemistry efforts to study the structural activity relationship for further development into a promising single- and/or combination-tocolytic therapy for management of preterm labor.

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Section: Primary Human Uterine Myometrial Tissue and Cellsmentioning

confidence: 99%

Section: Primary Human Uterine Myometrial Tissue and Cellsmentioning

confidence: 99%

Section: Comparative Concentration-dependent Ca 2+ -Mobilizationmentioning

confidence: 99%

Section: Comparative Concentration-dependent Ca 2+ -Mobilizationmentioning

confidence: 99%

Section: Comparative Concentration-dependent Ca 2+ -Mobilizationmentioning

confidence: 99%

See 3 more Smart Citations

Identification of mundulone and mundulone acetate as natural products with tocolytic efficacy in mono- and combination-therapy with current tocolytics

Siricilla

et al. 2021

Preprint

Self Cite

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CD38/cADPR‐mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1

Dogan,

Walseth,

Guvenc Tuna

et al. 2024

IUBMB Life

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Cyclic ADP‐ribose (cADPR) has emerged as a calcium‐regulating second messenger in smooth muscle cells. CD38 protein possesses ADP‐ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP‐ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET‐1 elicited [Ca2+]i responses, and 8‐Br‐cADPR, a cADPR antagonist significantly attenuated agonist‐induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.

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Drug discovery strategies for the identification of novel regulators of uterine contractility

Siricilla

Iwueke

Herington

2020

Current Opinion in Physiology

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Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery

Cited by 6 publications

References 61 publications

Identification of mundulone and mundulone acetate as natural products with tocolytic efficacy in mono- and combination-therapy with current tocolytics

Identification of mundulone and mundulone acetate as natural products with tocolytic efficacy in mono- and combination-therapy with current tocolytics

CD38/cADPR‐mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1

Drug discovery strategies for the identification of novel regulators of uterine contractility

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