Comparison of 3 assay systems using a common probe substrate, calcein AM, for studying P-gp using a selected set of compounds.
The multidrug resistance protein 1 (MDR1) transporter is the most abundantly investigated adenosine triphosphate (ATP)-Binding Cassette (ABC) transporter protein. Multiple assay systems were developed to study MDR1-mediated transport and possible drug-drug interactions. Yet, as different probe substrates are used in these assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 assay systems developed to study MDR1: the cellular dye efflux assay, the ATPase assay, and the vesicular transport assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 assay systems. The vesicular transport assay proved most sensitive, detecting 18 of 22 interactions with the protein. The ATPase assay detected 15 interactions, whereas the cellular dye efflux assay was the least sensitive with only 10 hits. A correlation was found between the hydrophobicity of the compound and the ratio of cellular and vesicular transport IC(50) values, indicating the effect of passive permeability on the result. Based on hydrophobicity, the current study provides guidelines on applying the most correct tool for studying MDR1 interactions.
SOLVO Biotechnology, Szeged, Hungary.
Péter
P
Szerémy
Akos
A
Pál
Dóra
D
Méhn
Beáta
B
Tóth
Ferenc
F
Fülöp
Péter
P
Krajcsi
Krisztina
K
Herédi-Szabó
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001681:Biological Assay; D001693:Biological Transport, Active / Q000187:drug effects*; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020128:Inhibitory Concentration 50; D010539:Permeability / Q000187:drug effects*; D011485:Protein Binding
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM