Identifying Small Molecules that Regulate Uterine Contractions

项目总结 子宫肌层是一个主要的治疗目标：1)减轻早产(PTL)，2)分娩 3)控制产后出血(PPH)。目前用于抑制子宫肌瘤的宫缩药物 宫缩和子宫强直用于产后出血，由于其不良的非靶点效应和持续时间较短而受到限制。 利益的问题。此外，几乎完全缺乏针对PTL、PPH和其他产科疾病的药物开发 有迹象表明。因此，对新型、安全的宫缩和子宫紧张剂的需求非常大。 功效和选择性。 高通量筛选(HTS)为研究人员提供了一个药物发现平台，以识别并随后 优化小分子，提高亲和力、选择性和功效/效力。基于核心角色 子宫收缩中的钙动员及催产素对子宫细胞内钙离子的影响 子宫肌层细胞(UT-myo)细胞内钙释放的释放和利用 OT的存在分别为发现新的宫缩和/或宫缩提供了一个很好的策略。 因此，我们开发了一种双加成钙动员实验，使用荧光钙敏感探针和 原代小鼠UT-Myo细胞，以允许双重检测钙动员和肌张力激动剂 OT诱导的钙动员的拮抗剂(产酵剂)在单一筛选中。该分析方法可靠，重现性好 (Z‘=0.73)，耐DMSO。来自NIH临床I光谱集合的2727种化合物的初步筛选 和II收集品证明：1)出色的检测性能，2)使用原代小鼠UT-myo的可行性 用于HTS的细胞和3)确定的化合物，用于立即测试调节体外子宫的能力 宫缩。 这个应用程序的目标是使用我们的HTS就绪分析来识别调节 从大化合物文库中高亲和力和选择性地动员子宫肌层钙离子。在目标1中 我们将：1)针对SelleckChem FDA中的100,978种化合物实施大规模HTS-活动- 批准的药物文库和Vanderbilt发现收藏，以确定刺激钙离子的热门化合物- 动员或抑制OT诱导的UT-myo细胞内钙离子的动员；2)对铅化合物的窄冲击 在确认和评估经历了比较筛选和剂量-反应关系的“命中”之后 分析。在目标2中，我们将通过使用体外功能等长收缩来验证铅小分子 记录铅化合物对人子宫肌层收缩活动的治疗效果的实验。 我们的研究的成功完成将为临床前确定一系列新的先导化合物 子宫肌层收缩选择性调节剂的研究进展。

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.