The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

中文翻译：

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心脏兰尼碱受体 N 末端区域生物传感器通过基于 FRET 的高通量筛选识别新型抑制剂。

兰尼碱受体 (RyR) 通道的 N 末端区域 (NTR) 对于肌肉兴奋-收缩 (EC) 耦合过程中 Ca2+ 释放的调节至关重要。NTR 包含许多与骨骼 (RyR1) 和心脏 (RyR2) 肌病相关的突变，突出了其作为治疗靶点的潜力。在这里，我们通过用 FRET 对在域 A、B 和 C 处标记小鼠 RyR2 NTR，构建了两个生物传感器。使用分子内 FRET 信号的荧光寿命 (FLT) 检测，我们使用这些生物传感器开发了高通量筛选 (HTS) 测定，以识别小分子 RyR 调节剂。然后，我们筛选了一个小型验证库并确定了几个命中。使用 [3H] 兰尼碱与分离的肌质网囊泡结合来进一步测试具有可饱和 FRET 剂量反应曲线和以前未报告的对 RyR 影响的命中，以确定对其天然膜中完整 RyR 开口的影响。我们鉴定了三种新的 RyR1 和 RyR2 抑制剂以及两种对纳摩尔 Ca2+ 有效的 RyR1 选择性抑制剂。其中两个命中仅在微摩尔 Ca2+ 处激活 RyR1，突出显示它们是激发-收缩耦合的潜在增强剂。为了确定此类命中是否可以抑制肌肉中的 RyR 泄漏，我们进一步关注一种 FDA 批准的天然抗生素夫西地酸 (FA)。在皮肤骨骼肌纤维和透化心肌细胞中，FA 抑制 RyR 渗漏，对骨骼肌纤维兴奋-收缩耦合没有不利影响。然而，在完整的心肌细胞中，FA 诱导致心律失常 Ca2+ 瞬变，这是对具有其他可靠安全记录的化合物的警示观察。这些结果表明，使用 NTR 生物传感器的 HTS 活动可以识别具有治疗潜力的化合物。