Development and optimization of a high-throughput bioassay for TRPM7 ion channel inhibitors

Abstract

TRPM7 is a ubiquitously expressed and constitutively active divalent cation channel essential for cell survival and proliferation because it provides a mechanism for Mg2+ entry. This makes the channel an attractive target for proliferative diseases. In keeping with its role in Mg2+ homeostasis, TRPM7 is inhibited by intracellular Mg2+ and Mg-ATP. TRPM7 has been implicated in anoxia-mediated cell death following brain ischemia. Despite its critical role in ischemic cell death and cell proliferation, there are no reports of selective inhibitors of TRPM7. The authors developed and optimized a fluorescent dye-based bioassay measuring the fluorescence quench of fura-2 by TRPM7-mediated Mn2+ influx in HEK293 cells that stably overexpress TRPM7. The following bioassay conditions were evaluated: (a) cell density, (b) dye loading conditions, (c) bioassay temperature, (d) concentration of the fura-2 quenching agent Mn2+, and (e) concentration of vehicle solvent. The bioassay was validated by measuring the effects of the known (nonselective) inhibitor 2-APB and La3+ on Mn2+ influx, and furthermore, the performance of the assay was evaluated by screening a subset of a marine bacteria-derived extract library. The quality of the bioassay window is excellent based on an established statistical parameter used to evaluate high-throughput screening window quality (Z and Z' factors > or =0.5).

Fig. 1

Ionic current development and Mn²⁺-induced fluorescent quench in HEK293-overexpressing mouse TRPM7. (A) Average time course of current development assessed in TRPM7-HEK293 using the whole-cell configuration of the patch-clamp technique (n = 10). Error bars indicate SEM. Note different scale for positive and negative currents. (B) Current-voltage (I/V) curve extracted from an example cell at 300 s into the whole-cell experiment. (C) Fura-2 quenching in TRPM7-HEK293 cells (positive control, blue circles) and noninduced TRPM7-HEK293 cells (negative controls, black circles) that were plated at 60,000 cells/well. The fura-2 loading buffer contained 2 µM fura-2-AM, 2 mM probenecid, and 0.1% pluronic F-127 in KRH, and incubation was at 37 °C for 60 min. Each data point is the mean ± SD of 48 replicate wells for TRPM7-HEK293 cells and 24 replicate wells for noninduced TRPM7-HEK293 cells, and Z′ factor values (red triangles) are plotted.

Fig. 2

Optimization of cell plating density. TRPM7-HEK293 cells (positive control, blue circles) and WT-HEK293 cells (negative controls, black squares) were plated at (A) 30,000, (B) 60,000, and (C) 120,000 cells/well. Each data point is the mean ± SD of 48 replicate wells for TRPM7-HEK293 cells and 24 replicate wells for WT-HEK293 cells, and Z′ factor values (red triangles) are plotted.

Fig. 3

Optimization of fura-2-AM concentration and loading time. TRPM7-HEK293 cells (positive control, blue circles) and WT-HEK293 cells (negative controls, black squares) were incubated with (A) 2 µM, (B) 1 µM, or (C–F) 0.5 µM fura-2-AM for (A–D) 60 min, (E) 45 min, or (F) 30 min prior to reading. Each data point is the mean ± SD of 48 replicate wells, and Z′ factor values (red triangles) are plotted.

Fig. 4

Effects of probenecid and pluronic F-127 on fura-2-AM loading. TRPM7-HEK293 cells (positive control, blue circles) and WT-HEK293 cells (negative controls, black squares) were loaded with fura-2 in the presence of (A) 2 mM probenecid and 0.1% pluronic F-127, (B) 0.1% pluronic F-127 but without probenecid, (C) 2 mM probenecid but without pluronic F-127, or (D) no probenecid and no pluronic F-127. Each data point is the mean ± SD of 24 replicate wells for TRPM7-HEK293 cells and 12 replicate wells for WT-HEK293 cells, and Z′ factor values (red triangles) are plotted.

Fig. 5

Validation of the TRPM7-mediated fura-2 Quench by Mn²⁺. TRPM7-HEK293 cells (positive control) and WT-HEK293 cells (negative controls) were preincubated with known nonselective inhibitors. (A) LaCl₃ was serial diluted 2:1 to give final concentrations of 7 mM to 9.6 µM, and each data point represents the mean ± SD of 8 replicate wells from 2 experiments. (B) 2-APB was serial diluted 2:1 to give final concentrations of 500 to 0.69 µM, and each data point represents the mean ± SD of 10 replicate wells from 2 experiments. The positive controls (n = 17) and negative controls (n = 16) on each plate, for each experimental day, yielded Z′ factors ≥0.5.

Fig. 6

Pilot screening of a marine-derived natural product library using the optimized TRPM7 assay. (A) TRPM7-HEK293 cells were preincubated with vehicle (positive control; n = 66) or microbial extracts (100 µg/mL; n = 420). WT-HEK293 cells were preincubated with vehicle (negative controls; n = 48). The Z′ factor was calculated from pooled normalized controls (± SD), and collective normalized extract responses were similar to positive controls and were used to calculate the Z factor. (B) Well-by-well pooled normalized extract responses (n = 6/well; ± SD).