Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays.
Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays.
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DOI：
10.1038/s41598-023-32476-w
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发表时间：
2023-03-30
影响因子：
4.6
通讯作者：
Kampourakis, Thomas
中科院分区：
文献类型：
--
作者：
Parijat, Priyanka;Ponnam, Saraswathi;Attili, Seetharamaiah;Campbell, Kenneth S.;El-Mezgueldi, Mohammed;Pfuhl, Mark;Kampourakis, Thomas
人们普遍认识到，对新的心力衰竭治疗药物存在大量未满足的需求。在过去的几十年中，收缩性肌丝本身已经成为开发收缩性和舒张性心力衰竭新疗法的有吸引力的靶点。然而，肌纤维定向药物的临床应用受到限制，进一步的进展受到阻碍，因为在分子水平上对肌丝功能的理解不完全，以及在体外准确再现这种功能的小分子筛选技术。在这项研究中，我们设计，验证和表征了新的高通量筛选平台的小分子效应靶向之间的相互作用的肌钙蛋白C和肌钙蛋白I亚基的心肌肌钙蛋白复合物。使用基于荧光偏振的测定来筛选市售化合物文库，并且使用二级筛选和正交测定来验证命中。使用等温滴定量热法和NMR光谱表征命中化合物-肌钙蛋白相互作用。我们确定NS 5806为稳定活性肌钙蛋白的新型钙增敏剂。NS 5806显著增加了去膜供体心肌的钙敏感性和最大等长力。我们的研究结果表明，肌节蛋白导向的筛选平台适合于开发调节心肌肌丝功能的化合物。
The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function.
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影响因子：
2.9
作者：
Hoffman, RMB;Li, MX;Sykes, BD
通讯作者：
Sykes, BD
影响因子：
4
作者：
Parijat, Priyanka;Kondacs, Laszlo;Kampourakis, Thomas
通讯作者：
Kampourakis, Thomas
影响因子：
2.9
作者：
Li, MX;Spyracopoulos, L;Sykes, BD
通讯作者：
Sykes, BD
影响因子：
2.9
作者：
Gasmi-Seabrook, GMC;Howarth, JW;Rosevear, PR
通讯作者：
Rosevear, PR
影响因子：
2.9
作者：
Li, Monica X.;Hoffman, Ryan M. B.;Sykes, Brian D.
通讯作者：
Sykes, Brian D.