New functional assay of P-glycoprotein activity using Hoechst 33342.
In this study we describe a simplified, HTS-capable functional assay for the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) based on its substrate Hoechst 33342. The physicochemical properties of Hoechst 33342 and the enormous milieu dependency of its fluorescence intensity allowed performing the assay in a homogeneous manner. This new assay served as an effective tool to estimate the potency of 10 well recognized P-gp substrates and modulators. Further, the potency of these compounds was also estimated in the calcein AM assay. The Hoechst 33342 and calcein AM assays yielded significantly comparable results for all compounds tested. Principal component analysis (PCA) applied to literature data on inhibition of P-gp activity and our results obtained in the Hoechst 33342 and calcein AM assay indicated similarity of compared functional transport assays. However, no correlation could be detected between these functional assays and the ATPase activity assay.
Müller
Henrik
H
Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany.
Klinkhammer
Werner
W
Globisch
Christoph
C
Kassack
Matthias U
MU
Pajeva
Ilza K
IK
Wiese
Michael
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects; D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism; D001562:Benzimidazoles / Q000737:chemistry* / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D015394:Molecular Structure; D025341:Principal Component Analysis; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001562:Benzimidazoles; D005452:Fluoresceins; C085925:calcein AM; D000251:Adenosine Triphosphatases; C017807:bisbenzimide ethoxide trihydrochloride