A Selective ATP-Binding Cassette Subfamily G Member 2 Efflux Inhibitor Revealed via High-Throughput Flow Cytometry

Abstract

Chemotherapeutics tumor resistance is a principal reason for treatment failure and clinical and experimental data indicate that multidrug transporters such as ATP-binding Cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate we identified a piperazine substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused SAR-driven chemistry effort we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2 over-expressing tumor model. At least two analogs significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

A high throughput flow cytometric assay platform targeting transporter inhibition

“…Along with data uploaded to PubChem (http://pubchem.ncbi.nlm.nih.gov/), an ABCG2 efflux inhibition probe (ML230) was found with 36 fold selectivity over ABCB1 [42]. The probe reversed chemoresistance in a mouse model of ovarian cancer [43]. …”

A High-Content Assay Strategy for the Identification and Profiling of ABCG2 Modulators in Live Cells

“…For this reason, we investigated the use of the JC-1 dye as an alternative fluorescent ABCG2 substrate. 9,12 In contrast to Hoechst, the significant difference in JC-1 intracellular accumulation between U87MG and U87MG-ABCG2 (three-to eightfold) remains constant for *22 h (Fig. 3B-D), thus being more amenable to our assay and automated screening.…”

“…For flow cytometry-based assays, the readout is the efflux of a fluorescent dye, and such an assay cannot discriminate between toxic compounds, fluorescent compounds, compounds competing with the fluorogenic substrate, and compounds affecting the expression of the transporter. 12 Another approach that is used for the identification of ABC transporters inhibitors are cytotoxicity-based assays. 39,40 This type of assay identifies compounds that reverse cell resistance to cytotoxic drugs.…”

“…Despite significant efforts, suitable ABCG2 inhibitors are still lacking. Several in vitro assays have been established for the identification of new ABCG2 modulators, such as drug-efflux activity using FACS, [8][9][10][11][12] transport assays measuring the net flux across the monolayer, 13 bioluminescence imaging, 14 and ATPase assays. 15 All these assays measure only one parameter and provide hits based on a single criterion: the ABCG2 function, as measured by the efflux of a fluorescent substrate.…”

Novel ABCG2 Antagonists Reverse Topotecan-Mediated Chemotherapeutic Resistance in Ovarian Carcinoma Xenografts

“…We synthesized and identified three ABCG2-selective efflux inhibitors, CID44640177, CID1434724, and CID46245505, by high-throughput flow cytometry as previously reported. (28,29) For the purposes of this manuscript we will refer to the ABCG2 inhibitors CID44640177, CID1434724, and CID46245505 as 177, 724, and 505, respectively.…”