High Throughput Screen Identifies Small Molecule Effectors That Modulate Thin Filament Activation in Cardiac Muscle
High Throughput Screen Identifies Small Molecule Effectors That Modulate Thin Filament Activation in Cardiac Muscle
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DOI：
10.1021/acschembio.0c00908
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发表时间：
2021-01-15
影响因子：
4
通讯作者：
Kampourakis, Thomas
中科院分区：
文献类型：
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作者：
Parijat, Priyanka;Kondacs, Laszlo;Kampourakis, Thomas
目前对心脏病和心力衰竭的治疗干预在很大程度上是不够的，并且与不期望的副作用有关。生物医学研究强调了肌节蛋白功能对心脏正常性能和能量效率的作用，这表明使用小分子效应物直接靶向收缩肌丝本身具有治疗潜力，并且可能导致更大的药物功效和选择性。在这项研究中，我们开发了一个强大的和高度可重复的荧光偏振为基础的高通量筛选（HTS）测定，直接靶向心肌肌钙蛋白C（cTnC）和心肌肌钙蛋白I（cTnI（SP））的开关区之间的钙依赖性相互作用，目的是确定心脏细丝激活途径的小分子效应。我们筛选了市售的小分子文库，并鉴定了几种具有抑制和活化作用的命中化合物。我们使用了一系列生物物理和生物化学方法来表征命中化合物，并确定芬戈莫德，鞘氨醇-1-磷酸受体调节剂，作为一种新的肌钙蛋白为基础的小分子效应。芬戈莫德以剂量依赖性方式降低去膜心肌纤维的ATP酶活性和钙敏感性，表明该化合物作为钙脱敏剂。我们使用计算研究、生物物理方法和合成化学的组合研究了芬戈莫德的作用机制，表明与cTnC结合的芬戈莫德主要通过其带正电荷的尾部的静电排斥来排斥cTnI（SP）。这些结果表明，芬戈莫德是一种潜在的新的先导化合物/支架，用于开发肌钙蛋白导向的心力衰竭治疗。
Current therapeutic interventions for both heart disease and heart failure are largely insufficient and associated with undesired side effects. Biomedical research has emphasized the role of sarcomeric protein function for the normal performance and energy efficiency of the heart, suggesting that directly targeting the contractile myofilaments themselves using small molecule effectors has therapeutic potential and will likely result in greater drug efficacy and selectivity. In this study, we developed a robust and highly reproducible fluorescence polarization-based high throughput screening (HTS) assay that directly targets the calcium-dependent interaction between cardiac troponin C (cTnC) and the switch region of cardiac troponin I (cTnI(SP)), with the aim of identifying small molecule effectors of the cardiac thin filament activation pathway. We screened a commercially available small molecule library and identified several hit compounds with both inhibitory and activating effects. We used a range of biophysical and biochemical methods to characterize hit compounds and identified fingolimod, a sphingosin-1-phosphate receptor modulator, as a new troponin-based small molecule effector. Fingolimod decreased the ATPase activity and calcium sensitivity of demembranated cardiac muscle fibers in a dose-dependent manner, suggesting that the compound acts as a calcium desensitizer. We investigated fingolimod's mechanism of action using a combination of computational studies, biophysical methods, and synthetic chemistry, showing that fingolimod bound to cTnC repels cTnI(SP) via mainly electrostatic repulsion of its positively charged tail. These results suggest that fingolimod is a potential new lead compound/scaffold for the development of troponin-directed heart failure therapeutics.