PLCγ2（P522R）的罕见编码变异体在微胶质细胞中表达，与野生型相比，它引起的酶活性轻微上升。报道称此突变体可预防晚发阶段阿尔茨海默病（LOAD）相关的认知能力退化，因此，建议激活野生型PLCγ2作为预防和治疗LOAD的潜在治疗靶点。此外，PLCγ2被发现与癌症和一些自身免疫性疾病有关，其中酶活性大幅上升的突变体已被确定。这时药理学抑制剂可能提供治疗效果。为了促进我们对PLCγ2活性的研究，我们开发了一种优化的荧光基底，以监测水溶液中的酶活性。首先，我们探索了各种“开启”荧光染料的光谱特性，最有前途的开启荧光染料被纳入到水溶性PLCγ2报道基质中。我们将其命名为C8CF3-香豆素。确认了PLCγ2能够酶解C8CF3-香豆素，并确定了反应的动力学。在优化反应条件的基础上，筛选出小分子激活剂，并对具有潜在PLCγ2激活剂的化合物库（LOPAC1280）进行试点筛选。优化的筛选条件允许识别出潜在的PLCγ2激活剂和抑制剂，从而证明了这种高通量筛选方法的可行性版权所有 © 2023 Elsevier Inc.

A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various "turn-on" fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.Copyright © 2023. Published by Elsevier Inc.