Supplementary Information For:
Elucidation of the structural basis of interaction of the BCR-ABL kinase inhibitor, nilotinib
(Tasigna®) with the human ABC drug transporter P-glycoprotein
Suneet Shukla, Eduardo E. Chufan, Satyakam Singh, Amanda P. Skoumbourdis, Khyati Kapoor,
Matthew B. Boxer, Damien Y. Duveau, Craig J. Thomas, Tanaji T. Talele, Suresh V. Ambudkar
Supplementary Materials and Methods
Chemicals
Imatinib and nilotinib were obtained from SelleckChem Inc. (Houston, TX). Calcein-AM and
Rhodamine 123 were purchased from Invitrogen Corporation (Carlsbad, CA) and Sigma
Chemical (St. Louis, MO), respectively. Tariquidar (XR 9576) was kindly provided by Susan
Bates, National Cancer Institute, NIH. [125I]-iodoarylazidoprazosin (IAAP) (2200 Ci/mmol) was purchased from PerkinElmer Life Sciences (Wellesley, MA). NBD-cyclosporine A was a generous gift from Drs. Anika Hartz and Bjoern Bauer, University of Minnesota, Duluth, MN).
Cell lines
HeLa cells were cultured in DMEM media supplemented with 10% FBS, 1% Glutamine and 1% penicillin.
Expression of Cys-less wild-type and mutant P-gp in HeLa and High Five cells
The expression clones and bacmid DNA for wild-type, Cys-less wild-type (WT) and mutant P- gps were generated in pDest-625 and E. coli DH10Bac cells, as previously described . Cys-less wild-type and mutant P-gps were expressed in HeLa cells using a BacMam-based expression system, as described earlier . Briefly, HeLa cells were transduced with BacMam Cys-less WT or mutant P-gp virus, which was added at a titer of 50-60 viral particles per cell. DMEM medium was added after an hour and the cells were incubated for 3-4 hrs after which 20 mM butyric acid was added and the cells were grown overnight at 37 °C. The cells were trypsinized, washed, counted and analyzed by flow cytometry.
For biochemical studies, Cys-less wild-type and mutant P-gps were expressed in High Five insect cells using Bac-to-Bac® Baculovirus Expression from Life Technologies (Grand Island,
NY).
Detection of cell surface expression of P-gp by MRK-16 antibody
Cell surface expression of Cys-less WT or mutant P-gp was examined using the P-gp specific monoclonal MRK16 antibody. 250,000 Bacmam P-gp virus-transduced cells were incubated with
MRK16 antibody (1 µg per 100,000 cells) for 60 min. Cells were subsequently washed and incubated with FITC-labeled IgG2a anti-mouse secondary antibody (1 µg per 100,000 cells) for
30 min at 37°C. The cells were washed with cold PBS and analyzed as described earlier .
Determination of transport function of Cys-less WT and mutant P-gps using fluorescent substrates
Transport function of Cys-less WT and mutant P-gps in transduced HeLa cells was determined by flow cytometry, as previously described . Briefly, cells were trypsinized and incubated with calcein-AM (Cal-AM, 0.5 µM) for 10 min, rhodamine 123 (Rh123, 0.5 µg/ml) or NBD- cyclosporine A (NBD-CsA, 0.5 µM) for 45 min. Cells were washed with cold PBS before analysis. Nilotinib and its derivatives were used for reversal of transport function in each of these samples. Cys-less WT and the non-functional mutant E556Q/E1201Q were used as positive and
2 negative controls, respectively. Fluorescence of substrates was measured on a FACSort flow cytometer equipped with a 488 nm argon laser and 530 nm bandpass filter.
Isolation of crude membranes
Crude membranes from High Five insect cells expressing Cys-less WT or mutant P-gps were prepared as described elsewhere . The protein content was estimated using the amido black B- dye binding assay, as described earlier .
Synthesis of nilotinib and derivatives
Nilotinib and the derivatives were synthesized using a series of published methods as described earlier , (.
4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)phenyl)-3-(4-(pyridine-3-yl)pyrimidin-
2-ylamino)benzamide (nilotinib): 1H NMR (400 MHz,
DMSO-d6) 10.6 (s, 1H), 9.28 (d, J = 1.6 Hz, 1H), 9.16
(s, 1H), 8.67 (d, J = 3.5 Hz, 1H), 8.54 (d, J = 5.5 Hz,
1H), 8.44 (ddd, J = 1.6, 2.0, 8.2 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.29 (t, J = 2.0 Hz, 1H), 8.20
(s, 1H), 8.15 (s, 1H), 7.76 (dd, J = 1.6, 7.8 Hz, 1H), 7.71 (s, 1H), 7.53-7.45 (m, 4H), 2.36 (s,
+ 3H), 2.17 (s, 3H); LC-MS: RT (min) = 4.27; [M + H] 530.0; HRMS calcd for C19H23F3N7O (M
+ H) 530.1838, found 530.1917.
4-methyl-N-(3-(4-methyl-5-(4-methyl-1H-imidazol-1-
yl)-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)benzamide
1 (1): H NMR (400 MHz, MeOH-d4) 9.41 (d, J = 1.6
Hz, 1H), 9.33 (d, J = 2.0 Hz,1H), 8.89 (ddd, J = 1.6,
3 1.8, 8.4 Hz, 1H), 8.78 (d, J = 5.5 Hz, 1H), 8.56 (d, J = 5.1 Hz, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.11
(t, J = 1.8 Hz, 1H), 7.85 (dd, J = 5.1 Hz, 7.8, 1H), 7.79 (s, 1H), 7.73 (dd, J = 2.0, 7.8 Hz, 1H),
7.56 (s, 1H), 7.48 (m, 2H), 7.30 (s, 1H), 2.47 (s, 3H), 2.45 (s, 3H), 2.42 (s, 3H); LC-MS: RT
+ (min) 3.95; [M + H] 476.1; HRMS calcd for C28H26N7O (M + H) 476.2121, found 476.2195.
4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)phenyl)-3-aminobenzamide (2): 1H
NMR (400 MHz, DMSO-d6) 8.74 (t, J = 2.2 Hz, 2H),
8.51 (d, J = 1.6 Hz, 4H), 8.37 (m, 2H), 7.82 (d, J = 1.2
Hz, 2H), 3.31 (s, 1H), 2.18 (s, 3H), 2.16 (s, 3H); LC-MS: RT (min) = 3.64; [M + H]+ 375.1;
HRMS calcd for C19H18F3N4O (M + H) 375.1354, found 375.1431.
4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-
trifluoromethyl)phenyl)-3-(pyrimidin-2-
ylamino)benzamide (3): 1H NMR (400 MHz, MeOH-
d4) 8.35 (d, J = 4.8 Hz, 2H), 8.22 (d, J = 1.6 Hz, 2H),
8.10 (d, J = 12 Hz, 2H), 7.69 (dd, J = 2.0, 8.0 Hz, 1H), 7.60 (s, 1H), 7.41 (d, 8.0 Hz, 1H), 7.36 (s,
1H), 6.79 (t, J = 4.8 Hz, 1H), 2.36 (s, 3H), 2.26 (s, 3H); LC-MS: RT (min) = 4.34; [M + H]+
453.1; HRMS calcd for C23H20F3N6O (M + H) 453.1572, found 453.1651.
4 4-methyl-3-(4-(pyridine-3-yl)pyrimidin-2-
ylamino)benzoic acid (4): 1H NMR (400 MHz, DMSO-
d6) 9.26 (d, J = 1.6 Hz, 1H), 9.01 (s, 1H), 8.68 (dd, J =
1.6, 4.7 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.44 (dt, 1.8,
8.1 Hz, 1H), 8.23 (s, 1H), 7.62 (dd, J = 1.6, 7.8 Hz, 1H),
7.52 (dd, J = 4.9, 7.6 Hz, 1H), 7.46 (d, J = 5.5 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 2.31 (s, 3H);
+ LC-MS: RT (min) = 3.72; [M + H] 307.1; HRMS calcd for C17H15N4O2 (M + H) 307.1117, found 307.1195.
4-methyl-3-(4-(pyridine-3-yl)pyrimidin-2-ylamino)-N-(3-
trifluoromethyl)phenyl)benzamide (5): 1H NMR (400
MHz, DMSO-d6) 10.48 (s, 1H), 9.33 (d, J = 1.6 Hz, 1H),
9.24 (s, 1H), 8.80 (dd, J = 1.6, 5.1 Hz, 1H), 8.68 (dt, J =
1.8, 8.2 Hz, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.28 (d, J = 1.6
Hz, 1H), 8.24 (s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.76 (dd, J = 2.0, 7.8 Hz, 1H), 7.72 (dd, J = 5.1,
7.8 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.53 (d, J = 5.1 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 2.35 (s,
+ 3H); LC-MS: RT (min) = 5.17; [M + H] 450.1; HRMS calcd for C24H19F3N5O (M + H)
450.1463, found 450.1542.
Samples were analyzed for purity on a an Agilent 1200 series LC/MS using a Zorbax Eclipse
XBD-C8 reverse phase (5 micron, 4.6 x 150 mm) column and a 1.1 mL/min flow rate. A gradient was performed using an acetonitrile/water mobile phase (each containing 0.1% trifluoroacetic acid). The gradient was 4% to 100% acetonitrile over 7 minutes. Purity of final compounds was determined to be >95%, using a two microliter injection with quantitation by
5 AUC at 220 and 254 nanometers. High-res mass spectrometry was preformed by the MSU Mass
Spectrometry Facility and by the NIH Chemical Genomics Center Analytical Chemistry group.
Photoaffinity labeling of P-gp with [125I]-Iodoarylazidoprazosin (IAAP)
Crude membranes (50 µg protein) from Cys-less WT or mutant P-gp-expressing High Five cells were incubated with 5 µM nilotinib or indicated derivatives for 10 min at 21-23°C in 50 mM
Tris-HCl, pH 7.5. 3-6 nM [125I]-IAAP (2200 Ci/mmole) (Perkin Elmer Life Sciences, Wellesley,
MA) was added and samples were incubated for an additional 5 min under subdued light. The samples were illuminated with a UV lamp (365 nm) for 10 min at room temperature. They were separated on a 7% Tris-acetate gel at constant voltage and gels were dried and exposed to X-ray film for 12-24 h at -80 °C. The incorporation of [125I]-IAAP into the P-gp band was quantified using the STORM 860 phosphor imager system (Molecular Dynamics, Sunnyvale, CA) and the software ImageQuaNT, as described previously .
ATPase assay
Crude membrane protein (10 µg) from High Five cells expressing Cys-less WT or mutant P-gp was incubated at 37°C with indicated concentrations of imatinib, nilotinib or its derivatives in the presence and absence of 0.3 mM sodium orthovanadate in ATPase assay buffer (50 mM KCl, 5 mM NaN3, 2 mM EGTA, 10 mM MgCl2, 1 mM DTT pH 6.8) for 10 min. The reaction was started by the addition of 5 mM ATP and incubated for 20 min at 37°C. SDS solution (0.1 ml of
5% SDS) was added to terminate the reaction and the amount of inorganic phosphate released was quantified with a colorimetric reaction, as described previously . The specific activity was recorded as vanadate-sensitive ATPase activity.
6 Docking of nilotinib and imatinib in wild-type and mutant P-gp and model generation using GLIDE
The homology model of human P-gp was generated according to previous reports . An energy minimized human P-gp homology model was used to generate receptor grids for sites 1–4 as well as the ATP-binding site, as described previously . Based on docking scores generated at each of the sites and analysis of clustered similar multiple poses, it was found that the most favorable site of nilotinib binding is site-1 (QZ59-RRR site) for mouse P-gp as described by
Aller et al. . The Glide v5.0 docking protocol was followed with its default functions
(Schrödinger, LLC, New York, NY). The top scoring docked conformations of nilotinib and imatinib at site-1 of P-gp were used for graphical analysis.
To evaluate the effect of single (Y307C, M949C and A985C) and triple (Y307C:M949C:A985C) mutant residues of the drug-binding site of P-gp, the best docked conformation of nilotinib-P-gp site-1 complex was exported as a single file in .PDB molecular format. The single, double, and triple mutations of various residues to cysteine were generated in silico. These efforts led to one pdb file per mutant protein in complex with nilotinib. Since the mutations at one or more positions in the drug-binding site of P-gp may lead to some conformational change in the protein structure, each mutant protein was subjected to Prime v2.1 (Schrodinger, LLC, New York, NY) energy minimization using default functions to relieve the strain. Glide energy grids were generated for each of the resulting refined mutant protein-nilotinib complexes and scores for docking nilotinib were determined for each of the mutant derivatives described in this study. All computations were carried out on a Dell Precision 470n dual processor with the Linux OS (Red
Hat Enterprise WS 4.0).
7 Supplementary Results
Docking of imatinib and nilotinib in the drug-binding site of P-gp
Nilotinib and imatinib were also compared for their binding orientation in the substrate-binding pocket of P-gp (Figure S3). The experimental data from both BCR-ABL kinase and ABC transporter studies suggest that imatinib has lower affinity to kinases and ABC transporters .
While the mechanistic explanation for the affinity differences between the two TKIs for inhibiting BCR-ABL kinase has been reported , differences in their interactions with ABC drug transporters previously have not been determined. In silico docking analysis of both drugs in the homology model of P-gp suggested that the piperazinylmethylphenyl moiety in imatinib is oriented differently than the imidazole moiety in nilotinib in this binding pocket (Figure S4). It should be noted that the residues that interact with the imidazole and trifluoromethyl groups in nilotinib (labeled in red) are absent in the docked model of imatinib. The imatinib binding model represents an inverted conformation resulting in orientation of the piperazinylmethylphenyl moiety away from the nilotinib binding residues, which may explain imatinib’s lower affinity for
P-gp. Furthermore, data from derivatives indicates that the lack of an imidazole ring in nilotinib
(i.e. derivative 5) has a negligible effect on the ability of the drug to interact with the transporter
(Supplementary tables 1 and 2). The crystallographic structure of nilotinib (PDB ID: 3CS9) and imatinib (PDB ID: 2HYY) with Abl kinase also shows binding characteristics that can be compared with their binding orientation with P-gp. For example, there is hydrogen bonding between the pyridine ring and the backbone –NH of M318, between aniline –NH- and the side chain of T315, and between the amide carbonyl oxygen atom and the backbone –NH of D381.
Apart from these electrostatic interactions, nilotinib and imatinib are also stabilized by hydrophobic interactions with the side chains of F317, M318 and F382 in the Abl kinase.
8 Therefore, the binding orientation of nilotinib and imatinib in nilotinib-Abl and imatinib-Abl appears similar to nilotinib-Pgp and imatinib-Pgp complexes, suggesting that the docked models of imatinib and nilotinib on P-gp presented here (Figure S4) provide a rationalized view of the experimentally observed affinity differences between imatinib and nilotinib for binding to P-gp.
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15 Supplementary Table 1. Decreased effect of nilotinib on stimulation of ATP hydrolysis and inhibition of 125I-IAAP photo labeling of mutant P-gps
Mutation(s) ATP hydrolysis (fold-stimulation)a 125I-IAAP labeling (% inhibition) b
Nilotinib Nilotinib
(1 µM) (5 µM)
Cys-less WT-P-gp 1.5 ± 0.2 72 ± 5
Y307C 0.7 ± 0.2 44 ± 5
M949C 1.1 ± 0.1 17 ± 4
A985C 1.2 ± 0.3 15 ± 5
aBasal (no addition) activity taken as 1.0 b125I-IAAP labeling in the presence of DMSO (solvent) taken as 100%.
The values represent mean ± SD from three independent experiments.
Supplementary Table 2. Effect of imatinib, nilotinib and its derivatives on 125I-IAAP labeling of P-gp
125I-IAAP labeling (% inhibition)a
Imatinib Nilotinib 5 3
(25-50 µM) (5 µM) (5 µM) (5 µM)
Cysless-WT-P-gp 60 ± 7 72 ± 5 33 ± 11 0-5
a 125I-IAAP incorporation in the presence of DMSO (solvent) was taken as 100%.
16 The values represent mean ± SD from three independent experiments.
Supplementary Table 3. Effect of imatinib and nilotinib and its derivatives on ATP hydrolys is by P-gp
______
ATP hydrolysis (fold-stimulation)a
Imatinib Nilotinib 5 3
(5 µM) (1 µM) (1 µM) (1 µM)
Cysless-WT-P-gp 1.4 ± 0.2 1.5 ± 0.2 1.6 ± 0.1 1.1 ± 0.1 aBasal (no addition) activity taken as 1.0
The values represent mean ± SD from three independent experiments.
17 Supplementary Figure S1. Chemical structures of imatinib and nilotinib.
18 Supplementary Figure S2. Y307C, M949C and A985C mutant P-gps are functionally expressed at the cell surface of HeLa cells. (a) BacMam-P-gp-transduced HeLa cells expressing Cys-less
WT control, Y307C, M949C and A985C cells were incubated with MRK16 antibody (1
μg/100,000 cells) for 1 h at 37°C, followed by incubation with FITC-conjugated anti-mouse
IgG2a secondary antibody. (b, c) For functional studies, the cells were incubated with 0.5 μM calcein-AM or NBD-cyclosporine A for 10 or 45 min, respectively. The cells were washed and subsequently analyzed by flow cytometry, as described in Supplementary Materials and
Methods. The histogram shows fluorescence (x-axis) representing surface expression (in panel a) or efflux function (in panels b and c) of Cys-less WT, Y307C, M849C and A985C mutant P-gps as detected by MRK16 labeling (in panel a) or calcein (panel b) or NBD-Cs A accumulation
(panel c) plotted as a function of the number of cells (y-axis). In panel b and c, accumulation in non-functional EQ (E556Q/E1201Q) mutant P-gp is also shown. Individual histograms are labeled as shown and represent a single experiment that was done independently at least three times.
19 Supplementary Figure S3. Nilotinib binding interactions in Y307C/M949C/A985C triple mutant of human P-gp. Nilotinib was docked on a human P-gp homology model using Glide as described in Supplementary Materials and Methods. Relative change in the distance of three amino acids Y307, M949 and A 985 (when changed to cysteine) from functional groups of nilotinib is shown here. These amino acids are depicted as stick models whereas nilotinib is shown as a ball and stick model.
20 Nilotinib Imatinib
Supplementary Figure S4. Differences in nilotinib and imatinib binding orientation when docked in the drug-binding pocket of human P-gp. Important amino acids are depicted schematically along with nilotinib (orange) and imatinib (cyan) as ball and stick models. The amino acid residues that interact with both imatinib and nilotinib, with only imatinib and with only nilotinib are shown in black, blue and red colors, respectively.
21