Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.

中文翻译：

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用于发现小分子抑制剂的高通量甲基转移酶分析的优化。

甲基转移酶 (MTases) 在细胞过程中发挥着不同的作用。异常甲基化水平与许多疾病有关，这表明需要鉴定和开发每种 MTase 的小分子抑制剂。特定抑制剂可以作为探针来研究功能并验证相应 MTase 的治疗潜力。高通量筛选 (HTS) 是一种强大的方法，可以识别初始命中以进行进一步优化。在这里，我们报告了基于荧光的 MTase 检测的开发，并将这种格式与最近开发的 MTase-Glo 发光检测进行了比较，以在 HTS 中应用。使用蛋白质 N 端甲基转移酶 1 (NTMT1) 作为模型系统，我们将其小型化为 1536 孔定量 HTS 格式。通过对 1428 种药理活性化合物的初步筛选和随后的验证，我们发现 MTase-Glo 产生的假阳性率低于基于荧光的 MTase 测定。尽管如此，这两种测定都显示出强大的性能和低试剂要求，并且有可能用作发现任何 MTase 抑制剂的通用 HTS 格式。