Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen.
Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen.
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DOI：
10.1016/j.jbc.2021.100840
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发表时间：
2021-07
期刊：
影响因子：
--
通讯作者：
Colson BA
中科院分区：
文献类型：
--
作者：
Bunch TA;Guhathakurta P;Lepak VC;Thompson AR;Kanassatega RS;Wilson A;Thomas DD;Colson BA
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。这些相互作用不受cMyBP-C磷酸化的影响。心力衰竭患者通常表现出cMyBP-C磷酸化降低，并且模型系统中的磷酸化已被证明对心力衰竭具有心脏保护作用。因此，cMyBP-C是模拟磷酸化或干扰其与肌动蛋白/肌球蛋白相互作用的心力衰竭药物的潜在靶标。在这里，我们使用了一种新的荧光寿命为基础的测定，以确定小分子抑制剂的肌动蛋白-cMyBP-C结合。肌动蛋白在其cMyBP-C结合位点附近用荧光染料（Alexa Fluor 568，AF 568）标记;当与cMyBP-C N-末端片段C 0-C2结合时，AF 568-肌动蛋白的荧光寿命降低。使用这种寿命的减少作为肌动蛋白结合的读数，1280-化合物库的高通量筛选鉴定了三种可再现的命中化合物（苏拉明、NF 023和金精三羧酸），其在微摩尔范围内减少C 0-C2与肌动蛋白的结合。磷酸化的C 0-C2的结合也被这些化合物阻断。通过肌动蛋白-C 0-C2时间分辨FRET（TR-FRET）结合试验证实了它们特异性阻断结合。等温滴定量热法（ITC）和瞬时磷光各向异性（TPA）证实，这些化合物结合cMyBP-C，但不肌动蛋白。TPA结果也与这些化合物抑制C 0-C2与肌动蛋白结合一致。我们得出结论，actin-cMyBP-C荧光寿命测定允许检测影响cMyBP-C-肌动蛋白结合的生物活性化合物。我们现在第一次有了一个经过验证的高通量筛选，重点是cMyBP-C，一种心肌收缩力的调节剂和心力衰竭的已知关键因素。
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.
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DOI：
10.1085/jgp.202012707
发表时间：
2021-03-01
期刊：
The Journal of general physiology
影响因子：
--
作者：
Bunch TA;Lepak VC;Bortz KM;Colson BA
通讯作者：
Colson BA
影响因子：
--
作者：
Cornea RL;Gruber SJ;Lockamy EL;Muretta JM;Jin D;Chen J;Dahl R;Bartfai T;Zsebo KM;Gillispie GD;Thomas DD
通讯作者：
Thomas DD
影响因子：
--
作者：
Gruber SJ;Cornea RL;Li J;Peterson KC;Schaaf TM;Gillispie GD;Dahl R;Zsebo KM;Robia SL;Thomas DD
通讯作者：
Thomas DD
影响因子：
16.8
作者：
Nag S;Trivedi DV;Sarkar SS;Adhikari AS;Sunitha MS;Sutton S;Ruppel KM;Spudich JA
通讯作者：
Spudich JA
影响因子：
5.6
作者：
Gruen, M;Gautel, M
通讯作者：
Gautel, M