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.

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to TCDD from an industrial accident in Seveso, Italy did not Previous studies have demonstrated that 2,3,7,8-tetrachlororesult in increased incidence of breast cancer (4), while other dibenzo-p-dioxin (TCDD) increases cell recovery in the reports suggest that exposure to environmental contaminants human mammary epithelial cell line MCF-10A grown may increase risk of breast cancer (7). under growth factor-restricted conditions. TCDD was also Activation of growth factor receptors and their cognate found to mimic growth factor signaling pathways by stimusignaling pathways is a potential mechanism of mammary lating the tyrosine phosphorylation of numerous effector tumor promotion and progression (reviewed in ref. 8). Previous molecules, and increased phosphatidylinositol 3-kinasestudies have demonstrated that TCDD mimics growth factor (PI3K) activity in the absence of exogenously added growth stimulation and cell growth in the human mammary epithelial factors. In the present studies, we have expanded on these cell line, MCF-10A (9). In the absence of insulin, TCDD initial results to show that TCDD (3-30 nM) increases cell increased total tyrosine phosphorylation, as well as tyrosine recovery on days 2-6 by as much as 80% when insulin or phosphorylation of the insulin-like growth factor-I receptor β epidermal growth factor (EGF) was removed from the (IGF-IRβ), insulin receptor substrate-1 and Shc. In addition, media. The mechanism for this effect appears to be complex TCDD treatment led to an increase in phosphatidylinositol 3-as TCDD inhibited apoptosis stimulated by EGF, or EGF kinase (PI3K) activity, and TCDD's growth stimulatory effects and insulin, withdrawal by almost 80% as determined by upon co-treatment with the PI3K inhibitor LY29004 were Annexin V binding. However, withdrawal of insulin alone attenuated. Finally, Shc tyrosine phosphorylation is not unique did not induce apoptosis even though TCDD did increase to IGF signaling. Shc can also bind to phosphotyrosine residues cell number in its absence. These results were corroborated on the epidermal growth factor receptor (EGFR) resulting in by immunoblot analysis of poly(ADP-ribose) polymeraseRas activation through Grb2-Sos interactions and stimulation cleavage. Since TCDD stimulates PI3K activity, the of the mitogen activated protein kinase pathways (10). These phosphorylation status of Akt, a serine/threonine kinase results argue that TCDD could act as a mammary tumor that mediates PI3K-dependent inhibition of apoptosis, was promoter by over-stimulating epidermal growth factor (EGF) examined. Immunoblot analysis revealed that TCDD causes and insulin-like growth factor

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.