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 structure-activity relationship (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-overexpressing tumor model. At least two analogues 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.

Figure 1

Structures of small molecules chosen for direct experimental comparison. Probe compound CID44640177 (1), ABCB1 inhibitor XR9051 (2), ABCG2 inhibitors FTC (3) and Ko143 (4), ABCC1 inhibitor MK571 (5), and the pyrazolopyrimidine reversan (6).

Figure 2

Representative synthetic route for compound 1. (A) methyl 3-(furan-2-yl)-3-oxopropanoate, AcOH, 100°C, 2 hr (65% yield). (B) POCl₃, BnEt₃NCl, PhNMe₂, CH₃CN, 80°C, 16 hr (84% yield). (C) furan-3-yl(piperazin-1-yl)methanone, DIPEA, CH₃CN, 100 °C, 16 hr (99% yield). (D) diethylmalonate (21% yield) NaOEt, EtOH, 80°C, 3 hr (75% yield). (E) POCl₃, N,N-dimethylaniline, 115°C, 16 hr (42% yield). (F) potassium aryltrifluoroborate salt, Pd(OAc)₂, RuPhos, Na₂CO₃, EtOH, MWI, 90 °C, 6 hr.

Figure 3

General scheme for the duplex HTS flow cytometric screening campaign. (A) In 384 well format, 1 μM JC-1 in PBS is added to the assay wells. (B) A volume of 100 nL of test compound is added to each well via pintool transfer (final concentration of 6.6 μM). (C) A 3 × 10⁶ cells mL⁻¹ mixture of both cell lines is added to each well. The ABCB1 cell line was previously color-coded with CellTrace™ Far Red DDAO-SE prior to mixture with the unlabeled ABCG2 line. (D) Flow cytometric data of light scatter and fluorescence emission at 530 +/− 20 nm (488 nm excitation, FL1) and 665 +/− 10 nm (633 nm excitation, FL8) are then collected via HyperCyt®. Each population is gated in FL8 allowing for analysis of FL1 in individual time bins for each cell line. The JC-1 retention can then be quantified as an indication of efflux inhibition by test compound(s). The FL1 versus time excerpt shown represents 24 binned wells of a 384 well plate.

Figure 4

Scaffold modification summary from primary hit to probe compound. (A) Screening hit compound 7 (CID1434724) and regions of targeted SAR optimization (shaded areas). (B) Compound 8 (CID1441553) obtained from first-generation SAR optimization. (C) SAR refinement of ABCG2 selectivity leading to compound 1 (CID44640177).

Figure 5

Efflux inhibition and chemotherapeutic potentiation of 1 (CID 44640177). (A) A representative curve showing efflux inhibition of ABCB1 in Jurkat-DNR cells (closed circles). The average IC₅₀ (n = 3) is 4.65 ± 0.74 μM. (B) A representative curve showing efflux inhibition of ABCG2 in Ig-MXP3 cells (open circles). The average IC₅₀ (n = 2) is 0.13 ± 0.03 μM (C) Potentiation of daunorubicin (DNR) mediated killing in Jurkat-DNR cells with 1 (n = 2 per data point). The CR₅₀ (closed triangles) is 0.55 μM while the TD₅₀ (closed squares) is 5.52 μM. Minimum allowable toxicity is set at 15 μM, thus the toxicity here is below the cut-off. (D) Potentiation of mitoxantrone (MTX) mediated killing in Ig-MXP3 cells (n = 2 per data point). The CR₅₀ (open triangles) is 0.31 μM while the TD₅₀ (open squares) is 18.30 μM. The minimum toxicity and the CR₅₀/TD₅₀ ratio (equal to 59) meet the cut-off criteria for a desirable compound in the chemoreversal secondary ABCG2 assay.

Figure 6

Response of ABCG2 resistant Igrov1/T8 derived tumors in mice to combination therapy of 150 nM topotecan (TPT). The tumor size at 0, 24, 48, 72, and 96 hr is indicated (n = 3) along with the standard error of the mean (SEM). The significant difference between the mean values from 0 to 96 hr is indicated by an asterisk (p < 0.001). Inhibitor concentration was selected based on potentiation efficacy balanced with apparent cellular toxicity. Significant tumor reduction was noted in both cases. (A) Compound 7 (original MLSMR hit) at 500 nM in conjunction with TPT. (B) Probe compound 1 at 100 nM with TPT.