Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters

Abstract

ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anti-cancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency

“…al. [39]. To assess the transport efficacy of 15Y mutant P-gp, we measured and compared the steady-state accumulation of seventeen fluorescent P-gp substrates in WT and 15Y mutant P-gp-expressing HeLa cells by flow cytometry (Fig.…”

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes

“…MTG and JC-1 have already been demonstrated as substrates of ABCB1, ABCC1 and ABCG2 [16–18]. Our previous cDNA microarray data for the expression of some ABC transporter genes (available in NCBI GEO repository #GSE72431) reveal that only ABCB1 gene is specifically upregulated in C6 glioma SP cells compared with MP cells approximately by 10.6-fold (SP:MP = 0.64:0.060), suggesting that the ability of SP cells to expel xenobiotics is mainly dependent on ABCB1 in C6 cells [25, 26].…”

“…In fact, some fluorescent probes are already known as substrates of ABC transporters [16–18], but a detailed examination of the appropriateness of their use for CSC research has not been made. In this study, with the use of C6 glioma SP cells as an established CSC model, we examined three fluorescent probes, CellROX, MTG and JC-1 for their applicability to the assessment of SP-defined CSC metabolism, and precisely compared the metabolic differences between CSCs and non-CSCs.…”

ABCC5 is required for cAMP-mediated hindgut invagination in sea urchin embryos

“…In most organisms, ABCC (MRP-type) transporters efflux a range of structurally diverse compounds, including signaling molecules (reviewed by Tiwari, 2011), toxicants (Bošnjak et al, 2009) and fluorescent dyes (Gökirmak et al, 2012(Gökirmak et al, , 2014Litman et al, 2001;Strouse et al, 2013). In blastula-stage embryos exposed to fluorescent compounds, we find that overexpression of C5a strongly reduces accumulation of fluorescein diacetate (FDA), but not 5-chloromethylfluorescein diacetate (CMFDA), calcein acetoxymethyl ester (calcein-AM, C-AM), 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM), bodipy-verapamil (b-Ver) or bodipy-vinblastine (b-Vin) (Fig.…”