Screening herbal and natural product libraries to aid discovery of novel allosteric modulators of human P2X7.

 P2X7 is an emerging therapeutic target for several disorders and diseases due to its role in inflammatory signalling. This study aimed to exploit the unique chemical libraries of plants used in traditional medicinal practices to discover novel allosteric modulators from natural sources. We identified several compounds from the NCI Natural Product library as P2X7 antagonists including confertifolin and digallic acid (IC(50) values 3.86 µM and 4.05 µM). We also identified scopafungin as a novel positive allosteric modulator of hP2X7. Screening a traditional medicinal plant extract library revealed 39 plant species with inhibitory action at hP2X7 and 17 plant species with positive allosteric modulator activity. Using computational docking to filter identified components from these plant species and determine potential antagonists, we investigated nine purified chemicals including flavonoids quercetin, kaempferol, ECG, and EGCG. These were shown to inhibit ATP-induced YO-PRO-1 uptake into HEK-hP2X7 cells; however, we also showed that all four flavonoids demonstrated significant assay interference using a cell-free DNA YO-PRO-1 fluorescence test. One plant extract, Dioscorea nipponica, demonstrating positive modulator activity was investigated, and dioscin was identified as a glycoside with PAM activity in ATP-induced YO-PRO-1 uptake assay and whole-cell patch-clamp recordings. However, membrane permeabilisation was observed following application > 10 min limiting the use of dioscin as a pharmacological tool. This work describes a useful workflow with multiple assays for the identification of novel allosteric modulators for human P2X7.