Department of Cellular & Molecular Medicine, University of Arizona, Tucson Arizona, USA.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
J Biol Chem. 2021 Jul;297(1):100840. doi: 10.1016/j.jbc.2021.100840. Epub 2021 May 28.
Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.
心肌肌球蛋白结合蛋白 C（cMyBP-C）与肌动蛋白和肌球蛋白相互作用，调节心肌收缩力。这些相互作用不利于 cMyBP-C 的磷酸化。心力衰竭患者常表现出 cMyBP-C 磷酸化减少，而在模型系统中观察到的磷酸化对心力衰竭具有心脏保护作用。因此，cMyBP-C 是一种潜在的心力衰竭药物靶点，这些药物可以模拟磷酸化或干扰其与肌动蛋白/肌球蛋白的相互作用。在这里，我们使用了一种新的基于荧光寿命的测定法来鉴定肌动蛋白-cMyBP-C 结合的小分子抑制剂。肌动蛋白在其与 cMyBP-C 结合的部位附近用荧光染料（Alexa Fluor 568，AF568）标记；当与 cMyBP-C N 端片段 C0-C2 结合时，AF568-肌动蛋白的荧光寿命会降低。使用这种寿命的减少作为肌动蛋白结合的读出，对 1280 种化合物文库进行高通量筛选，鉴定出三种可重复的活性化合物（苏拉明、NF023 和金精三羧酸），这些化合物以微摩尔范围抑制 C0-C2 与肌动蛋白的结合。这些化合物也阻止了磷酸化的 C0-C2 的结合。通过肌动蛋白-C0-C2 时间分辨荧光共振能量转移（TR-FRET）结合测定证实了它们特异性地阻断结合。等温滴定量热法（ITC）和瞬态磷光各向异性（TPA）证实这些化合物与 cMyBP-C 结合，但不与肌动蛋白结合。TPA 结果也与这些化合物抑制 C0-C2 与肌动蛋白的结合一致。我们得出结论，肌动蛋白-cMyBP-C 荧光寿命测定法可检测到影响 cMyBP-C-肌动蛋白结合的具有药理活性的化合物。我们现在首次拥有了针对 cMyBP-C 的经过验证的高通量筛选，cMyBP-C 是心肌收缩力的调节剂，也是心力衰竭的关键因素。
