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.

中文翻译：

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高通量筛选可识别调节心肌细丝激活的小分子效应物

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，并且与不希望的副作用有关。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器直接靶向收缩肌丝本身具有治疗潜力，并且可能会导致更高的药物功效和选择性。在这项研究中，我们开发了一种稳健且高度可重复的基于荧光偏振的高通量筛选 (HTS) 测定，该测定直接针对心肌肌钙蛋白 C (cTnC) 和心肌肌钙蛋白 I (cTnI _{SP ) 之间的钙依赖性相互作用})，目的是识别心脏细丝激活通路的小分子效应物。我们筛选了一个市售的小分子库，并确定了几种具有抑制和激活作用的热门化合物。我们使用一系列生物物理和生化方法来表征命中化合物，并确定芬戈莫德（一种 1-磷酸鞘氨醇受体调节剂）是一种新的基于肌钙蛋白的小分子效应物。芬戈莫德以剂量依赖性方式降低脱膜心肌纤维的 ATP 酶活性和钙敏感性，表明该化合物可作为钙脱敏剂。我们结合计算研究、生物物理方法和合成化学研究了芬戈莫德的作用机制，表明与 cTnC 结合的芬戈莫德排斥 cTnI_SP主要通过其带正电的尾部的静电排斥。这些结果表明，芬戈莫德是一种潜在的新先导化合物/支架，可用于开发以肌钙蛋白为导向的心力衰竭疗法。