Pharmacophore Modeling of Nilotinib as an Inhibitor of ATP-Binding Cassette Drug Transporters and BCR-ABL Kinase Using a Three-Dimensional Quantitative Structure–Activity Relationship Approach

Nilotinib (Tasigna) is a tyrosine kinase inhibitor approved by the FDA to treat chronic phase chronic myeloid leukemia patients. It is also a transport substrate of the ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-glycoprotein, P-gp) and ABCG2 (BCRP), which may have an effect on the pharmacokinetics and toxicity of this drug. The goal of this study was to identify pharmacophoric features of nilotinib in order to potentially develop specific inhibitors of BCR-ABL kinase with minimal interactions with ABC drug transporters. Three-dimensional pharmacophore modeling and quantitative structure–activity relationship (QSAR) studies were carried out on a series of nilotinib analogues to identify chemical features that contribute to inhibitory activity of nilotinib against BCR-ABL kinase activity, P-gp, and ABCG2. Twenty-five derivatives of nilotinib were synthesized and were then tested to measure their activity to inhibit BCR-ABL kinase and to inhibit the function of ABC drug transporters. A set of <i>in vitro</i> experiments including kinase activity and cell-based transport assays and photolabeling of P-gp and ABCG2 with a transport substrate, [¹²⁵I]-iodoarylazido-prazosin (IAAP), were carried out in isolated membranes to evaluate the potency of the derivatives to inhibit the function of ABC drug transporters and BCR-ABL kinase. Sixteen, fourteen, and ten compounds were selected as QSAR data sets, respectively, to generate PHASE v3.1 pharmacophore models for BCR-ABL kinase, ABCG2, and P-gp inhibitors. The IC₅₀ values of these derivatives against P-gp, ABCG2, or BCR-ABL kinase were used to generate pharmacophore features required for optimal interactions with these targets. A seven-point pharmacophore (AADDRRR) for BCR-ABL kinase inhibitory activity, a six-point pharmacophore (ADHRRR) for ABCG2 inhibitory activity, and a seven-point pharmacophore (AADDRRR) for P-gp inhibitory activity were generated. The derived models clearly demonstrate high predictive power for test sets of BCR-ABL, ABCG2, and P-gp inhibitors. In aggregate, these results should aid in the development of specific inhibitors of BCR-ABL kinase that exhibit no or minimal interaction with ABC drug transporters

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

<div><p>ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.</p></div

Analysis of fluorescence data from cell imaging-based efflux assay.

<p><b>A.</b> Calcein fluorescence in KB-V1 and KB-3-1 cells. Calcein AM (1 µM) was added to KB-V1 and KB-3-1 cells. One hour later, phase-contrast and fluorescent images were taken by the IncuCyte^TMFLR. Phase-contrast, fluorescent, and segmentation mask images are displayed (bar = 100 µm). <b>B.</b> Calcein AM dose-dependent fluorescence of KB-V1 and KB-3-1 cells. KB-3-1 and KB-V1 cells were loaded with various concentrations of calcein AM for 1 hour. The mean fluorescence intensities and the object intensities derived from the fluorescent images were exported from the IncuCyte^TMFLR and graphed. * indicates the <i>p</i> value is less than 0.05; **** indicates the <i>p</i> value is less than 0.0001. <b>C.</b> Correlation between normalized florescence mean intensities and the object intensities. The data sets in Fig. 2B from KB-V1 and KB-3-1 cells were normalized within each data set and plotted. The red lines in the graph are the best fit lines for each data set analyzed for linear correlation. All data presented are mean ± SD (n = 3).</p

The fluorescent cell imaging-based ABCB1-mediated efflux assay is reproducible.

<p>Three independent experiments were performed to screen the inhibitor library in the cell imaging-based ABCB1-mediated calcein AM efflux assay in 384-well plates. The final concentration of each compound was 10 µM. The object intensities (A) and the relative inhibitory activities (B) of each compound were calculated. 3D scatter graphs were plotted using SigmaPlot. The correlation coefficients between any two data sets were calculated in MS Excel.</p

BEZ235, BI 2536, IKK 16, and ispinesib are inhibitors of ABCB1-mediated efflux.

<p><b>A.</b> KB-V1 cells were treated with BEZ235, BI 2536, IKK 16, ispinesib, and bryostatin-1 and then calcein AM (1 µM). Doses of each compound were generated from 1∶2 dilutions of the highest concentration of 20 µM. The object intensities were plotted. Data presented are mean ± SD (n = 3). <b>B.</b> Flow cytometry-based efflux assays were performed to examine KB-V1 cells incubated with either calcein AM (1 µM, red line) or calcein AM plus BEZ235, BI 2536, IKK 16, ispinesib, or bryostatin-1 (5 µM, blue line).</p

BEZ235, BI 2536, and IKK 16 interfered with binding of ABCB1 and substrate; and inhibition of ABCB1 enhanced drug sensitivities of HCT-15-Pgp cells to BI 2536.

<p><b>A</b>. BEZ235, BI 2536, and IKK 16 inhibited [¹²⁵I]IAAP binding to ABCB1. Plasma membranes from Hifive cells overexpressing human ABCB1 were incubated with 20 µM BEZ235, BI 2536, IKK 16, ispinesib, and 5 µM XR9576, then [¹²⁵I]IAAP was added for 10 minutes. The incorporation of [¹²⁵I]IAAP into ABCB1 was shown by autoradiograph. <b>B</b>. HCT-15-Pgp cells plated in 96-well plates were treated with BI 2536 in the presence or absence of XR9576 (0.2 µM) and cyclosporin A (5 µM). After 48 hours, the cell viability was evaluated with CCK-8. The relative cell viabilities are presented as mean ±SD (n = 3).</p

Cell imaging-based ABCB1-mediated calcein AM efflux by KB-V1 and KB-3-1 cells.

<p><b>A.</b> Time-and dose-dependent fluorescent calcein accumulation in KB-V1 cells. Various concentrations of calcein AM were added to KB-V1 cells. The phase-contrast and fluorescent images were taken at the indicated times by the IncuCyte^TMFLR. The mean fluorescence intensities were obtained from the IncuCyte^TMFLR and plotted. Data presented are mean ± SD (n = 3). <b>B.</b> KB-V1 cells overexpress ABCB1. Particulate fractions of KB-3-1 and KB-V1 cells were subjected to western blotting with an anti-ABCB1 antibody. For the flow cytometry efflux assay, KB-V1 cells were treated with 5 µM of each inhibitor followed by 1 µM calcein AM. Red is for calcein AM; green is for XR9576; blue is for FTC; orange is for MK-571. <b>C.</b> XR9576 enhances calcein fluorescence in KB-V1 cells but not KB-3-1 cells. KB-V1 and KB-3-1 cells were treated with XR9576 before addition of calcein AM (1 µM). The phase and fluorescent images were taken 1 hour later by the IncuCyte^TMFLR. The mean fluorescence intensities were obtained from the IncuCyte^TMFLR. Data presented are mean ± SD (n = 3). <b>**</b> indicates <i>p</i><0.05.</p

Compounds from the kinase inhibitior library are identified as ABCB1 inhibitors by the high-throughput imaging-based efflux assay.

*<p>Compounds that have been previously reported to interact with ABCB1. References are listed separately (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060334#pone.0060334.s004" target="_blank">Text S1</a>).</p><p>KB-V1 cells treated with the kinase inhibitor library of 193 compounds at 10 µM and 1 µM calcein AM were examined by the IncuCyte^TMFLR. The object intensities of each sample were normalized to XR9576 treated cells and expressed as the percentage of inhibition. Compounds that displayed at least 50% inhibition of ABCB1-mediated efflux in all three independent experiments are listed.</p

Quizartinib increases uptake of fluoroquinolones in cells expressing ABCG2.

<p>K562/ABCG2 (<b>A</b>) and 8226/MR20 (<b>B</b>) cells, overexpressing ABCG2, were incubated with ciprofloxacin at a fixed concentration (25µg/ml) in the absence and presence of quizartinib at a range of concentrations for 30 minutes, and with the ABCG2 transport inhibitor fumitremorgin C (FTC) as a positive control. Cellular fluorescence was measured on a microplate reader. *p<0.05, **p<0.01 and ***p<0.001.</p

K562/ABCB1 and K562/ABCG2 cells exhibit collateral sensitivity to

<p><b>quizartinib.</b> K562, K562/ABCB1 and K562/ABCG2 cells were plated in the presence of quizartinib in increasing concentrations for 96 hours and viable cells were measured using the WST-1 assay.</p