Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Montalbetti, Nicolas; Simonin, Alexandre; Dalghi Gens, Marianela Gisela; Kovacs, Gergely; Hediger, Matthias (2014). Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra. Journal of biomolecular screening, 19(6), pp. 900-908. Sage 10.1177/1087057114521663

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Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

Item Type:	Journal Article (Original Article)
Division/Institute:	04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine
UniBE Contributor:	Montalbetti, Nicolas, Simonin, Alexandre, Dalghi Gens, Marianela Gisela, Kovacs, Gergely, Hediger, Matthias
Subjects:	500 Science > 570 Life sciences; biology 600 Technology > 610 Medicine & health
ISSN:	1552-454X
Publisher:	Sage
Language:	English
Submitter:	Patrizia Catucci
Date Deposited:	23 Jul 2014 16:10
Last Modified:	05 Dec 2022 14:29
Publisher DOI:	10.1177/1087057114521663
PubMed ID:	24505080
Uncontrolled Keywords:	Calcium-5 FLIPR, Tetra SLC11A2 cell-based assay
URI:	https://boris.unibe.ch/id/eprint/43300