Biochemistry. 2020 Oct 20;59(41):4029-4038. doi: 10.1021/acs.biochem.0c00511. Epub 2020 Oct 7.
The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery.
两种磷酯酶 C-γ（PLC-γ）同工酶是多种疾病的主要信号枢纽和新兴治疗靶点，但目前还没有针对这些酶的选择性抑制剂。我们开发了一种基于脂质体的高通量检测方法，该方法使用了一种荧光膜结合报告分子 XY-69，用于检测哺乳动物 PLC 同工酶。该检测方法使用 384 孔格式的药理学活性化合物库 1280（LOPAC）进行了初步筛选验证；它具有高度可重复性，并且有可能捕获正位和变构抑制剂。通过二次检测对选定的命中化合物进行了确认，进一步的分析揭示了一个有趣的发现，即三磷酸腺苷通过非竞争性抑制强烈抑制 PLC-γ 同工酶，这提出了这些磷脂酶内源性、核苷酸依赖性调节的有趣可能性。这些结果突出了该检测平台在大规模筛选化学文库以鉴定 PLC-γ 同工酶的变构调节剂作为化学探针和药物发现方面的优势。
