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

Abstract

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 (IC₅₀ 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.

Keywords: Traditional Medicine; Allosteric modulator; Confertifolin; Digallic acid; Dioscin; Flavonoids; P2X7; Scopafungin.

Fig. 1

Purified Natural Product Set IV chemicals with action at hP2X7. A HEK-hP2X7 cells were pre-treated with diversity set IV compounds or ginsenoside CK (10 µM) for 10 min. ATP-dependent YO-PRO-1 iodide dye uptake was measured over 300 s following administration of ATP (1 mM). B Concentration–response curve for ATP in the presence of ginsenoside CK (10 µM) or scopafungin (10 µM) using the YO-PRO-1 dye uptake assay. C Inhibitory effect of AZ10606120 (10 µM) on responses elicited by ATP in the presence of either CK or scopafungin. D HEK-hP2X7 cells were pre-treated with diversity set IV compounds or AZ10606120 (10 µM) for 10 min. ATP-dependent YO-PRO-1 iodide dye uptake was measured over 300 s following administration of ATP (1 mM). E Concentration–response curve for ATP in the presence of digallic acid, confertifolin, or acronine (10 µM) using the YO-PRO-1 dye uptake assay. F Dose inhibition curve for confertifolin and digallic acid at hP2X7. The symbol “*” represents statistical significance (P < 0.05) from one-way ANOVA with Sidak’s multiple comparison post hoc test

Fig. 2

Extracts exhibiting inhibition or potentiation of hP2X7. HEK-hP2X7 cells were pre-treated with 30 µg/mL of TCM extract for 60 s prior to the administration of 1 mM ATP. YO-PRO-1 uptake was measured for an additional 210 s, and the YO-PRO-1 uptake between 200 and 300 s was quantified as a percentage of the ATP response in the absence of extracts. The addition of 10 µM AZ10606120 was added as a control for inhibition, 10 µM CK was used as a control for potentiation, and DMSO was used as a vehicle control. A Extracts exhibiting more than 75% inhibition were considered inhibitory. B Extracts enhancing ATP responses to > 150% of the original ATP response were considered potentiators. Data are representative of two technical replicates but one independent experiment due to the availability of the extracts. Red line indicates the control response in each case

Fig. 3

Inhibitory effect of purified chemicals on hP2X7 responses. HEK-hP2X7 cells were pre-treated with purified compounds for 10 min, and ATP-dependent YO-PRO-1 iodide dye uptake was measured over 300 s following administration of ATP (1 mM). Concentration–response curve for A berberine and emodin; B quercetin and kaempferol; C ECG and EGCG; D bergapten and scopoletin; E palmitic acid, methyl syringate, and genistein; and F AZ11645373. Induced fit docking for G berberine (green), H quercetin (cyan), and I EGCG (yellow) at hP2X7 NAM site using a homology model built on zfP2X4. AZ10606120 is shown in red as a comparison

Fig. 4

Cell-free YO-PRO-1 assay to measure compounds with interference. Compounds were diluted in a low divalent cation buffer containing 2 µM YO-PRO-1 iodide and 90 µL plated per well. Calf thymus DNA (1 µg/mL) was injected at 30 s using a Flexstation 3 and fluorescence measured over a further 60 s. A A typical response to DNA under control conditions. B Lack of interference by AZ11645373. Dose–response curves show the effects of C bergapten, berberine, and emodin; D genistein, palmitic acid, scopoletin, and methyl syringate; E quercetin and kaempferol; and F ECG and EGCG over the concentration range 100 nM–100 µM

Fig. 5

Plant extracts that potentiate hP2X7 dye uptake responses can enhance the secretion of IL-1β and cell death responses. A THP-1 cells were treated with LPS (100 ng/mL) for 4 h followed by the addition of TCM plant extract (30 µg/mL) and ATP (500 µM) for the final 30 min of incubation. IL-1β release into supernatants was quantified and compared to the secretion of IL-1β in the absence of plant extract. Ginsenoside CK (10 µM) was used as the positive control and 3 mM ATP was used as a control to demonstrate P2X7-induced IL-1β secretion. B HEK-hP2X7 cells were treated with ATP (500 µM) in the presence or absence of selected TCM plant extracts (30 µg/mL) for 24 h prior to quantification of viable cells by using AlamarBlue assay. Ginsenoside CK (10 µM) was used as a positive control for the enhancement of cell death. Data are representative of two technical replicates from one independent experiment due to the limited availability of the TCM plant extracts

Fig. 6

Dioscin enhances P2X7-dependent pore formation and potentiates channel opening. A Dioscorea nipponica (organic) plant extract (30 µg/mL) increased ATP-dependent YO-PRO-1 iodide uptake. AZ10606120 (10 µM, grey bars) was added to block hP2X7. B YO-PRO-1 uptake following a co-stimulation of HEK-hP2X7 cells with 200 µM ATP ± the named compounds in the presence or absence of AZ10606120. YO-PRO-1 uptake was quantified between 50 and 300 s (n = 5). C Whole-cell patch-clamp recordings were performed at room temperature. Cells were voltage-clamped at − 60 mV, and responses to a 5-s stimulation of ATP (100 µM) followed by a 5-s stimulation of ATP (100 µM) + dioscin/diosgenin (10 µM) were measured from HEK-hP2X7 cells. Representative traces are shown. D Summary of normalised current amplitudes represented as a fold change compared to ATP responses in the absence of dioscin/diosgenin (n = 10–18 cells). E Concentration response to ATP in the presence of dioscin (10 µM, green) or vehicle (DMSO, black) using YO-PRO-1 uptake assay in HEK-hP2X7 and F HEK-293 cells. G Cell viability assay using AlamarBlue shows dioscin (10 µM) is toxic over 24 h in HEK-293 cells. 1 mM ATP does not induce cell death. H HEK-293 cells were treated with dioscin or CK (10 µM) for 10 min and stained with propidium iodide to reveal permeabilised cells. Images taken with EVOS microscope