Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA; IUSM-Purdue TREAT-AD Center, West Lafayette IN 47907, USA; Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA; IUSM-Purdue TREAT-AD Center, West Lafayette IN 47907, USA.
SLAS Discov. 2023 Jun;28(4):170-179. doi: 10.1016/j.slasd.2023.03.003. Epub 2023 Mar 17.
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 the 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 (LOPAC) 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.
在微胶质细胞中表达的 PLCγ2（P522R）罕见编码变异与野生型相比，其酶活性的轻度激活。据报道，这种突变可预防与迟发性阿尔茨海默病（LOAD）相关的认知能力下降，因此，激活野生型 PLCγ2 已被提议作为预防和治疗 LOAD 的潜在治疗靶点。此外，PLCγ2 还与其他疾病有关，例如癌症和一些自身免疫性疾病，在这些疾病中已经发现了 PLCγ2 活性增加幅度更大的突变。在这里，药理学抑制可能提供治疗效果。为了方便我们研究 PLCγ2 的活性，我们开发了一种优化的荧光底物，以在水溶液中监测酶活性。这是通过首先探索各种“开启”荧光团的光谱特性来实现的。最有前途的开启荧光团被掺入水溶性 PLCγ2 报告底物中，我们将其命名为 C8CF3-香豆素。证实了 PLCγ2 酶促处理 C8CF3-香豆素的能力，并确定了反应的动力学。优化了反应条件以鉴定小分子激活剂，并对药理学活性化合物库 1280（LOPAC）进行了初步筛选，目的是鉴定 PLCγ2 的小分子激活剂。优化的筛选条件允许鉴定潜在的 PLCγ2 激活剂和抑制剂，从而证明了这种方法用于高通量筛选的可行性。
