A new insight into purinergic pharmacology: Three fungal species as natural P2X7R antagonists

Abstract

P2X7 is a purinergic receptor involved in important physiological functions and pathological processes, such as inflammation, neurodegeneration, and pain. Despite its relevance, there is no selective antagonist useful in the treatment of diseases related to this receptor. In this context, research for a selective, safe, and potent antagonist compound that can be used in clinical therapy has been growing. In this work, we evaluated the potential antagonistic activity of three fungal extracts, namely, Vishniacozyma victoriae, Metschnikowia australis, and Ascomycota sp., which were discovered in a high-throughput screening campaign to search for new antagonists for P2X7R from natural products. First, the IC₅₀ values of these fungal extracts were determined in J774.G8 (murine macrophage cell line) and U937 (human monocyte cell line) cells through dye uptake assays. The IC₅₀ values of V. victoriae were 2.6 and 0.92 μg/mL, M. australis has IC₅₀ values of 3.8 and 1.5 μg/mL, and Ascomycota sp. showed values of 2.1 and 0.67 μg/mL in J774.G8 and U937 cells, respectively. These extracts also significantly inhibited propidium iodide and Lucifer yellow uptake via P2X7R pore, P2X7R currents in electrophysiology, IL-1β release, and the production of oxide nitric and reactive oxygen species. The extracts did not cause cytotoxicity within a period of 24 h. The results showed the promising antagonistic activity of these extracts toward P2X7R, thereby indicating that they can be future candidates for phytomedicines with potential clinical applicability.

Keywords: P2X7R; antagonist; drug discovery; fungi; natural products.

Figure 1

Effect of candidate extracts on dye uptake. J774.G8 cells were pretreated with extracts (100 μg/mL) or BBG (100 nM) for 15 min. Then, the cells were stimulated with ATP (5 mM) for a further 10 min. (a) Cationic (PI [375 nM]) or (b) anionic (LY [3 mM]) fluorescent dyes were added to the wells, and the fluorescence of the cells was measured using a spectrophotometer. Data are presented as the mean ± SDM of three independent experiments performed in triplicate. The results were analyzed by ANOVA and Tukey's post hoc test, p < .05. Asterisks indicate a significant difference in relation to ATP. RFU means relative fluorescence units

Figure 2

Concentration‐response curves of the extracts in murine and human cell lines. J774.G8 and U937 cells were pretreated with increasing concentrations of the extracts (2 to 100 μg/mL) for 15 min and then were stimulated with ATP (5 mM). PI was added to the wells, and after 5 min, the cell fluorescence was measured using a spectrophotometer. (a) Concentration‐response curve of V. victoriae extract in the J774.G8 cell line. (b) Concentration‐response curve of M. australis extract in the J774.G8 cell line. (c) Concentration‐response curve of Ascomycota sp. extract in the J774.G8 cell line. (d) Concentration‐response curve of V. victoriae extract in the U937 cell line. (e) Concentration‐response curve of M. australis extract in the U937 cell line. (f) Concentration‐response curve of Ascomycota sp. extract in the U937 cell line. Data are presented as the mean ± SEM of three independent experiments performed in triplicate

Figure 3

Effect of the candidate extracts on P2X7R ionic currents. J774.G8 cells were treated with increasing concentrations of each fungal extract (according to their IC₅₀) or BBG (100 nM, 500 nM or 1 µM) for 15 min and then were stimulated with ATP (1 mM) to evaluate the P2X7R ionic currents. The relative currents of (a) V. victoriae, (b) M. australis, and (c) Ascomycota sp. extracts were quantified, and the data are presented as the mean ± SDM of three independent experiments performed in triplicate. The results were analyzed by ANOVA and Tukey's post hoc test, p < .05. Asterisks indicate a significant difference in relation to ATP currents. (d) Representative electrophysiology measurements of fungal extracts at their IC₅₀ concentration and of BBG as an antagonist control of P2X7R

Figure 4

Cytotoxicity assay of the candidate extracts in murine and human cells. (a) J774.G8 and (b) U937 cells were treated with the fungal extracts at four times their IC₅₀ or with BBG (100 nM) for 24 h and then were incubated for a further 1 h in the presence or absence of ATP (5 mM). Cell supernatants were collected to perform the LDH assay. The concentrations of extracts used were V. victoriae (10.4 and 3.68 μg/mL), M. australis (15.2 and 6.0 μg/mL), and Ascomycota sp. (8.4 and 2.76 μg/mL) for J774.G8 and U937 cells, respectively. Data are presented as the mean ± SDM of three independent experiments performed in triplicate. The results were analyzed by ANOVA and Tukey's post hoc test, p < .05. Asterisks indicate a significant difference in relation to the control cells (without treatment). ns means “not significant,” when compared with the control cells

Figure 5

Effects of the candidate extracts on the physiological functions of P2X7R. (a) J774.G8 and (b) U937 cells were treated with extracts at their IC₅₀ concentrations or with BBG (100 nM) for 1 h and then were stimulated with ATP (5 mM) for a further 15 min. Cell supernatants were collected to perform an ELISA assay for IL‐1β detection. (c) J774.G8 and (d) U937 cells were treated with extracts at their IC₅₀ concentrations or with BBG (100 nM) for 15 min and then were stimulated with ATP (5 mM) for a further 10 min. Supernatants were collected to perform a Griess reaction to measure nitrite production. (e) J774.G8 and (f) U937 cells were treated with extracts at their IC₅₀ concentrations or with BBG (100 nM) for 15 min and then were stimulated with ATP (5 mM) for a further 10 min. DHE dye (10 nM) was added to the wells of plates containing cells to indicate ROS production. Data are presented as the mean ± SDM of three independent experiments performed in triplicate. The results were analyzed by ANOVA and Tukey's post hoc test, p < .05. Asterisks indicate a significant difference in relation to ATP