title: 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).
authors.affiliation: College of Natural Sciences, Hawaii Pacific University, Kaneohe, Hawaii 96813, USA.
authors.forename: Brandi
authors.initials: B
authors.lastname: Castillo
authors.forename: Peter
authors.initials: P
authors.lastname: Pörzgen
authors.forename: Reinhold
authors.initials: R
authors.lastname: Penner
authors.forename: F David
authors.initials: FD
authors.lastname: Horgen
authors.forename: Andrea
authors.initials: A
authors.lastname: Fleig
mesh_terms: D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D018408:Patch-Clamp Techniques; D020442:Poloxamer / Q000378:metabolism; D011339:Probenecid / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D015203:Reproducibility of Results; D013501:Surface-Active Agents / Q000378:metabolism; D050053:TRPM Cation Channels / Q000037:antagonists & inhibitors*; D014528:Uricosuric Agents / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D013501:Surface-Active Agents; D050053:TRPM Cation Channels; D014528:Uricosuric Agents; C049925:fura-2-am; D020442:Poloxamer; D008345:Manganese; D017346:Protein Serine-Threonine Kinases; C486583:TRPM7 protein, human; D008274:Magnesium; D011339:Probenecid; D016257:Fura-2