Elucidation of the structural basis of interaction of the ...

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Nilotinib potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, and ABCC10-multidrug resistance xenograft models. Cancer Lett 2013; 328:.
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961 bone marrow blasts, 2 with splenomegaly, 7 with diploid cytogenetics and 1 with JAK2-V617F mutation. Whether these patients share a specific genetic phenotype is conceivable, and molecular characterization of this subgroup is ongoing. Our analysis validates the diagnosis of MDS/MPN-U as a unique pathologic entity, with distinctive features such as an increased (15%) incidence of isolated trisomy 8. Despite the relative frequency of trisomy 8 in myeloid malignancies, remarkably little is known about the pathogenic basis of this abnormality.13 Similar ongoing molecular analysis will investigate whether trisomy 8 associates with particular somatic mutations in this cohort. Despite statistical significance, the MDS-IPSS model is not ideal, as the majority (68%) of MDS/MPN-U patients had lower-risk scores according to the MDS-IPSS, and yet had poorer survival than their lower-risk MDS counterparts. Furthermore, the improved survival seen in the Int-1 category compared with the low-risk category is contrary to expectation. One prognostic model of clinicopathologic variables has recently been developed from a cohort (n ¼ 92) of patients with either MDS-U or MDS/MPN-U; interestingly, this cohort did not find platelet count to be of prognostic importance.14 The MDA global model also provided a significant tool for the MDS/MPN-U cohort (P ¼ 0.004). It is noteworthy that the MDA model was originally validated within 176 patients with CMML and leukocytosis, suggesting that this may be an appropriate prognostic model to use in MDS/MPN patient populations. No treatment regimen significantly improved response. Given their relative novelty, only two patients received JAK2-inhibitor therapy. In view of the JAK2-V617 mutations present among the MDS/ MPN-U patients signifying overactivity of JAK/STAT signaling, JAK2inhibitor therapy may ultimately prove effective, and indeed a clinical trial incorporating the combination of ruxolitinib and azacitidine for patients with MDS/MPN-U is ongoing at our institution.

CONFLICT OF INTEREST The authors declare no conflict of interest.

CD DiNardo1, N Daver1, N Jain1, N Pemmaraju1, C Bueso-Ramos2, CC Yin2, S Pierce1, E Jabbour1, JE Cortes1, HM Kantarjian1, G Garcia-Manero1 and S Verstovsek1 1 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 2 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA E-mail: [email protected]

REFERENCES 1 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th edn. International Agency for Research on Cancer (IARC): Lyon, 2008. 2 Orazi A, Germing U. The myelodysplastic/myeloproliferative neoplasms: myeloproliferative diseases with dysplastic features. Leukemia 2008; 22: 1308–1319. 3 Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114: 937–951. 4 Wang SA, Hasserjian RP, Loew JM, Sechman EV, Jones D, Hao S et al. Refractory anemia with ringed sideroblasts associated with marked thrombocytosis harbors JAK2 mutation and shows overlapping myeloproliferative and myelodysplastic features. Leukemia 2006; 20: 1641–1644. 5 Atallah E, Nussenzveig R, Yin CC, Bueso-Ramos C, Tam C, Manshouri T et al. Prognostic interaction between thrombocytosis and JAK2 V617F mutation in the WHO subcategories of myelodysplastic/myeloproliferative disease-unclassifiable and refractory anemia with ringed sideroblasts and marked thrombocytosis. Leukemia 2008; 22: 1295–1298. 6 Cannella L, Breccia M, Latagliata R, Frustaci A, Alimena G. Clinical and prognostic features of patients with myelodysplastic/myeloproliferative syndrome categorized as unclassified (MDS/MPD-U) by WHO classification. Leuk Res 2008; 32: 514–516. 7 Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89: 2079–2088. 8 Cervantes F, Dupriez B, Pereira A, Passamonti F, Reilly JT, Morra E et al. New prognostic scoring system for primary myelofibrosis based on a study of the International Working Group for Myelofibrosis Research and Treatment. Blood 2009; 113: 2895–2901. 9 Greenberg PL, Tuechler H, Schanz J, Sanz G, Garcia-Manero G, Sole F et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012; 120: 2454–2465. 10 Kantarjian H, O’Brien S, Ravandi F, Cortes J, Shan J, Bennett JM et al. Proposal for a new risk model in myelodysplastic syndrome that accounts for events not considered in the original International Prognostic Scoring System. Cancer 2008; 113: 1351–1361. 11 Kantarjian H, Giles F, List A, Lyons R, Sekeres MA, Pierce S et al. The incidence and impact of thrombocytopenia in myelodysplastic syndromes. Cancer 2007; 109: 1705–1714. 12 Germing U, Hildebrandt B, Pfeilstocker M, Nosslinger T, Valent P, Fonatsch C et al. Refinement of the international prognostic scoring system (IPSS) by including LDH as an additional prognostic variable to improve risk assessment in patients with primary myelodysplastic syndromes (MDS). Leukemia 2005; 19: 2223–2231. 13 Mertens F, Johansson B, Heim S, Kristoffersson U, Mitelman F. Karyotypic patterns in chronic myeloproliferative disorders: report on 74 cases and review of the literature. Leukemia 1991; 5: 214–220. 14 Liu Y, Tabarroki A, Visconte V, Hasrouni E, Bupathi M, Hamilton BK et al. A Prognostic Scoring System for Unclassifiable MDS and MDS/MPN. ASH Annual Meeting Abstracts 2012 2012; 120: 1701.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Elucidation of the structural basis of interaction of the BCR-ABL kinase inhibitor, nilotinib (Tasigna) with the human ABC drug transporter P-glycoprotein Leukemia (2014) 28, 961–964; doi:10.1038/leu.2014.21 Nilotinib, imatinib (structures are shown in Supplementary Figure S1) and other tyrosine kinase inhibitors (TKIs) have been shown to be transported by the ATP-binding cassette (ABC) drug

transporters P-glycoprotein (P-gp) and ABCG2.1,2 This is clinically important as the transporters not only hamper the bioavailability of these TKIs but may also cause the emergence of drug resistance in patients. We have previously shown that imatinib and nilotinib interact at the substrate-binding pocket of ABC transporters, but do not interact at the adenosine triphosphate (ATP)

Accepted article preview online 14 January 2014; advance online publication, 7 February 2014

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962 sites of these transporters.3 Identification of the key structural features of nilotinib and similar TKIs is essential for understanding their interaction with P-gp. Toward this goal, molecular docking, mutational mapping and quantitative structure–activity relationship (SAR) were used to identify nilotinib’s binding site on P-gp. Nilotinib was docked in a human P-gp homology model that was developed on the basis of the mouse P-gp crystal structure4 using the XP-Glide docking method to understand the orientation and the complementarity of pharmacophore features of nilotinib with respect to the residues in the drug-binding pocket of P-gp (Figure 1a). Comparison of binding energy data for the docked poses of nilotinib at sites 1–4 (ref. 5) suggested site-1 (QZ59-RRR site)4,6 as the most favorable site (binding energy score of  9.52 kcal/mol). The binding pocket is lined by residues that form electrostatic and hydrophobic contacts with a pyridine, a pyrimidine, a methyl-substituted phenyl ring, the carbonyl oxygen atom of the amide linker and the trifluoromethylphenyl ring of nilotinib (Figure 1a). Among these, the Y307 residue showed significant interaction through hydrogen bonding to the pyridine ring (–N—HO–Y307, 2.4 Å), whereas A985 had hydrophobic contact with the CF3 group (3.3 Å), phenyl ring (3.2 Å) and imidazole ring (4.1 Å) of nilotinib. Furthermore, M949 also showed hydrophobic contact with the imidazole ring (5.1 Å) of nilotinib (highlighted in red in Figure 1a). Therefore, the residues (Y307, M949 and A985) that interact with three major functional groups (pyridine, CF3 and imidazole) of nilotinib were selected for further analysis. The docking studies indicated these residues might determine the orientation and stabilization of nilotinib within the substrate-binding site of P-gp. These residues were mutated to Cys residue in a Cys-less P-gp to verify their role in interaction with nilotinib. Control Cys-less wild-type (WT) P-gp, Y307C, M949C and A985C P-gp mutants were expressed in HeLa cells (Supplementary Figure S2; mutants exhibited similar expression and transport of fluorescent substrates as Cys-less WT P-gp) and High-Five insect cells, as described in Supplementary Materials

and Methods. Crude membranes from High-Five insect cells expressing similar levels of mutant proteins (Figure 1b) were used to determine the interaction of these mutant P-gps with nilotinib. The effect of nilotinib was evaluated on ATPase activity and on photolabeling of mutant P-gps with [125I]-IAAP binding (Figure 1c and Supplementary Table S1), as these approaches can be used to determine the interaction of substrates at the substrate-binding pocket of P-gp.7,8 Nilotinib’s ability to stimulate the ATPase activity of Y307C-, M949C- and A985C-mutant P-gps was significantly reduced or abolished compared with Cys-less WT P-gp (Supplementary Table S1). Similarly, nilotinib’s ability to compete for [125I]-IAAP photolabeling was significantly reduced for Y307Cand almost completely lost for M949C- and A985C-mutant P-gps (Figure 1c and Supplementary Table S1). These observations provided experimental support to the in silico docking studies. The residues Y307, M949 and A985 contribute to nilotinib binding, indicating that site-1 may be the primary binding site for nilotinib on P-gp. In silico introduction of these mutations in the homology model helped to visualize the local changes in the binding pocket (Supplementary Figure S3). In the nilotinib-docked model of P-gp, pyridine nitrogen was present at a position 2.4 Å from the side chains of Y307; M949 was 5.1 Å from the imidazole ring, while A985 was 4.1 Å from the imidazole ring of nilotinib (Figure 1). In the triple mutant, the pyridine nitrogen atom lost one critical hydrogen bonding interaction with the Y307 residue, increasing the distance to 5.9 Å (Supplementary Figure S3). Similarly, the hydrophobic interactions with the imidazole ring and the trifluoromethyl aniline moiety were lost when M949 and A985 were mutated to a hydrophilic cysteine residue (Supplementary Figure S3). These data, taken together, provide clear evidence that site-1 is indeed the primary site of nilotinib binding on P-gp, with Y307 interacting with the pyridine ring, A985 interacting with the trifluoromethylphenyl group and M949 interacting with the imidazole ring of nilotinib. To further validate the importance of functional groups of nilotinib for interacting with P-gp, five structural derivatives of

Figure 1. Docking of nilotinib in the drug-binding pocket of human P-gp and analyses of mutant proteins. (a) Glide-predicted binding pocket of nilotinib in a homology model of human P-gp. Nilotinib was docked in a human P-gp homology model using Glide, as described in Supplementary Materials and Methods. The amino acids that contribute to nilotinib’s binding site are shown here. Three residues (Y307, M949 and A985) used for mutational analyses are highlighted by red boxes. The predicted distance of these residues from the closest functional group of nilotinib is marked. (b) Expression of mutant P-gps. Colloidal blue stain of crude membrane protein (10 mg/lane) from Cys-less WT, Y307C, M949C and A985C P-gps expressed in High-Five insect cells. (c) Nilotinib does not inhibit the labeling of mutant P-gps with [125I]-IAAP. A representative autoradiogram from three independent experiments with Cys-less WT, Y307C-, M949C- and A985C-mutant P-gps photocrosslinked with [125I]-IAAP in the absence or presence of 5 mM nilotinib is shown. Leukemia (2014) 935 – 979

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963

Figure 2. Synthesis of nilotinib derivatives and characterization of their interaction with P-gp. (a) Chemical structures of nilotinib and its derivatives used in this study. Nilotinib and derivatives 1, 2, 3, 4 and 5 were synthesized as described in Supplementary Materials and Methods. (b) A representative autoradiogram from three independent experiments with Cys-less WT P-gps photo-crosslinked with [125I]-IAAP in the absence or presence of 5 mM nilotinib or derivative 3 and 5 is shown. (c) The histogram shows accumulation of rhodamine 123 in the presence and absence of 5 mM of nilotinib or derivative 3 or 5 in BacMam P-gp virus-transduced HeLa cells (additional details are given in the legend to Supplementary Figure S2).

nilotinib (Figure 2a) that lacked critical functional groups such as the pyridine ring, pyrimidine ring, CF3 group and imidazole ring were synthesized (as described in Supplementary Materials and Methods). These derivatives were evaluated for their interaction with P-gp by testing their ability to inhibit rhodamine 123 efflux from HeLa cells. Compound 1 (CF3 replaced by CH3) showed inhibition similar to that of nilotinib (data not shown), suggesting that substitution of fluorine with hydrogen at the CF3 group is not a critical determinant of nilotinib’s binding to the P-gp. Derivative 2 (no pyrimidine–pyridine ring system) was comparable to 3 (no pyridine ring), and 4 (imidazole replaced with a benzoic acid) was comparable to 5 (no imidazole ring) with respect to their ability to inhibit P-gp transport activity (data not shown). Therefore, 5 and 3, derivatives lacking the key imidazole or pyridine/pyrimidine rings, respectively, were further tested for interaction with P-gp. Compared with nilotinib, 3 completely lost the ability to inhibit photolabeling of P-gp with [125I]-IAAP, but 5 was still able to inhibit B30–35% of [125I]-IAAP photolabeling (Figure 2b and Supplementary Table S2). Similarly, 3 did not stimulate the ATPase activity but 5 was equally effective as nilotinib in stimulating the ATPase activity of Cys-less WT P-gp (Supplementary Table S3). In addition, 5 partially inhibited the rhodamine 123 efflux by P-gp, whereas 3 had no effect (Figure 2c). These results show that the interaction of nilotinib at the substrate-binding pocket of P-gp is significantly affected when the pyridine and/or pyrimidine ring is absent, whereas loss of the imidazole rings only slightly perturbs this interaction. Taken together, the results with derivatives corroborate the docking & 2014 Macmillan Publishers Limited

conformation and the mutational mapping data. Whereas the pyridine and pyrimidine moieties of nilotinib are important for interaction at the drug-binding pocket, the imidazole group is not critical for this interaction. Nilotinib and imatinib were also compared for their binding orientation in the substrate-binding pocket of P-gp (Supplementary Figure S4). As described in Supplementary Results, the observed affinity differences between nilotinib and imatinib for P-gp can be explained on the basis of the above docking analysis and its comparison with the known crystal structures of imatinib and nilotinib bound to BCR-ABL kinase.9,10 Several studies have used a docking-based approach to identify the substrate-binding pocket in P-gp, but most of those studies relied on either SAR or mutagenesis alone (reviewed in Palmeira et al.11). We used a two-pronged approach, where the docked orientation of nilotinib was not only validated by directed mutagenesis of selected residues but also was verified using the structural derivatives of nilotinib. Although the data derived from modeling and mutational studies with nilotinib and its derivatives corroborate well with the docked conformation of nilotinib, there is still a possibility that nilotinib may bind to a secondary site because of the chemical and structural flexibility of a large drugbinding pocket that can accommodate more than one ligand simultaneously.12–16 In recent years, multidrug resistance-linked transporters have gained considerable attention as potential targets to improve cancer chemotherapy and to increase bioavailability/tissue penetration of drugs. Therefore, the interaction of these transporters with targeted therapeutic drugs such as nilotinib at the molecular level needs further elucidation. To our knowledge, this is the first report that provides an understanding of the interaction of nilotinib with human P-gp through molecular modeling, mutational mapping and SAR studies. We identified residues that are crucial for binding of nilotinib to the primary site on P-gp and by using derivatives, we defined the molecular determinants of nilotinib for binding to P-gp. We believe these findings will help to synthesize novel TKIs that do not interact with P-gp, thus minimizing the possibility of development of resistance in cancer cells. EDITOR’S NOTE This report is important to identify the primary binding site of nilotinib in the drug-binding pocket of P-gp. It is promising in view of exploiting the designing of novel tyrosine kinase inhibitors that will not be recognised by ABC drug transporters. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Mr George Leiman for editorial assistance. This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, Center for Cancer Research and National Center for Advancing Translational Sciences at the NIH.

S Shukla1, EE Chufan1, S Singh2, AP Skoumbourdis3, K Kapoor1, MB Boxer3, DY Duveau3, CJ Thomas3, TT Talele2 and SV Ambudkar1 1 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 2 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St John’s University, Queens, NY, USA and 3 NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, Rockville, MD, USA E-mail: [email protected] Leukemia (2014) 935 – 979

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964 REFERENCES 1 Shukla S, Chen ZS, Ambudkar SV. Tyrosine kinase inhibitors as modulators of ABC transporter-mediated drug resistance. Drug Resist Updat 2012; 15: 70–80. 2 Bro´zik A, Hegedu¨s C, Erdei Z, Hegedu+s T, O¨zvegy-Laczka C, Szaka´cs G et al. Tyrosine kinase inhibitors as modulators of ATP binding cassette multidrug transporters: substrates, chemosensitizers or inducers of acquired multidrug resistance? Expert Opin Drug Metab Toxicol 2011; 7: 623–642. 3 Shukla S, Sauna ZE, Ambudkar SV. Evidence for the interaction of imatinib at the transport-substrate site(s) of the multidrug-resistance-linked ABC drug transporters ABCB1 (P-glycoprotein) and ABCG2. Leukemia 2008; 22: 445–447. 4 Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R et al. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 2009; 323: 1718–1722. 5 Shi Z, Tiwari AK, Shukla S, Robey RW, Singh S, Kim IW et al. Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance. Cancer Res 2011; 71: 3029–3041. 6 Tiwari AK, Sodani K, Dai C-l, Abuznait AH, Singh S, Xiao Z-J et al. Nilotinib potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, and ABCC10-multidrug resistance xenograft models. Cancer Lett 2013; 328: 307–317. 7 Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 1999; 39: 361–398.

8 Sauna ZE, Ambudkar SV. About a switch: how P-glycoprotein (ABCB1) harnesses the energy of ATP binding and hydrolysis to do mechanical work. Mol Cancer Ther 2007; 6: 13–23. 9 Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J. Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase. Science 2000; 289: 1938–1942. 10 Weisberg E, Manley P, Mestan J, Cowan-Jacob S, Ray A, Griffin JD. AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL. Br J Cancer 2006; 94: 1765–1769. 11 Palmeira A, Sousa E, Vasconcelos MH, Pinto M, Fernandes MX. Structure and ligand-based design of P-glycoprotein inhibitors: a historical perspective. Curr Pharm Des 2012; 18: 4197–4214. 12 Dey S, Ramachandra M, Pastan I, Gottesman MM, Ambudkar SV. Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc Natl Acad Sci USA 1997; 94: 10594–10599. 13 Loo TW, Bartlett MC, Clarke DM. Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein. J Biol Chem 2003; 278: 39706–39710. 14 Lugo MR, Sharom FJ. Interaction of LDS-751 and rhodamine 123 with P-glycoprotein: evidence for simultaneous binding of both drugs. Biochemistry 2005; 44: 14020–14029. 15 Ambudkar SV, Kim IW, Sauna ZE. The power of the pump: mechanisms of action of P-glycoprotein (ABCB1). Eur J Pharm Sci 2006; 27: 392–400. 16 Marcoux J, Wang SC, Politis A, Reading E, Ma J, Biggin PC et al. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc Natl Acad Sci USA 2013; 110: 9704–9709.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Philadelphia chromosome-negative very high-risk acute lymphoblastic leukemia in children and adolescents: results from Children’s Oncology Group Study AALL0031 Leukemia (2014) 28, 964–967; doi:10.1038/leu.2014.29

The Children’s Oncology Group (COG) AALL0031 study included very high-risk (VHR) pediatric acute lymphoblastic leukemia (ALL) patients who had an expected 5-year event-free survival p45%. The chemotherapy regimen was based on previous strategies; eligible patients received 4 weeks of standard induction chemotherapy and then were enrolled on AALL0031, which included an intensive consolidation followed by a continuation regimen (Supplementary Figure 1).1 COG AALL0031 enrolled patients aged 1–21 years with VHR ALL from 14 October 2002 to 20 October 2006. Induction therapy was limited to a combination of vincristine, prednisone or dexamethasone, and asparaginase with or without daunomycin. VHR features included the following: (a) Philadelphia chromosome [t(9;22)(q34;q11.2)]; (b) hypodiploidy: defined as p44 chromosomes or DNA index o0.81; (c) any rearrangement of the MLL gene in conjunction with a slow early response X5% marrow blasts at day 15 and/or X0.1% minimal residual disease (MRD) at the end of induction as detected by multiparameter flow cytometry;2,3 and (d) induction failure (IF) defined as either 425% blasts (M3 marrow status) by histology at the end of 4 weeks of induction therapy or an M2 marrow (5–25% blasts) or MRD X1% by flow cytometry at the end of induction followed by an M2 (or M3) marrow or MRDX1% after receiving two additional weeks of induction therapy (M2/M2 IFs). The therapy was identical to that presented in a previous publication on outcomes for Ph þ ALL patients,1 except

that the Ph  patients received no imatinib (see Supplementary Figure 1). Prior approval was obtained from the National Cancer Institute and the Institutional Review Boards of the COG member institutions. Informed consent was obtained in accordance with the Federal guidelines. Sixty-three hypodiploid (41) and IF (22) patients were enrolled in AALL0031 after 4 weeks of a three- or four-drug induction regimen for National Cancer Institute standard and high-risk ALL, respectively. Data on adverse events and clinically significant abnormal laboratory findings were collected using National Cancer Institute Common Terminology Criteria version 2.0. MRD was assessed by multiparameter flow cytometry.2 Samples were available from 46 of 63 (73%) patients at study entry. MRD high was defined as 40.01% and low as p0.01%. The primary outcome in this report is disease-free survival (DFS). Overall survival (OS), DFS and event-free survival were all defined as the time from the end of consolidation to the first event or last contact. An event was defined as relapse at any site, secondary malignancy or death in remission. A historical control data set of hypodiploid patients included patients enrolled on the Pediatric Oncology Group 8602, 9005, 9006, 9201, 9405, 9406 and 9605 protocols for B-ALL (January 1986–November 1999).3 The percentage of patients undergoing bone marrow transplant (BMT) in these comparator studies is unknown. IF patients were excluded from post-induction therapy in the historical control trial. Estimates of DFS, event-free survival and OS were computed using the Kaplan–Meier method4 and s.e. of the estimates according to Peto and Peto.5 The log-rank test was used for comparison of survival curves

Accepted article preview online 17 January 2014; advance online publication, 11 February 2014

Leukemia (2014) 935 – 979

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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 Pgps were generated in pDest-625 and E. coli DH10Bac cells, as previously described (1). Cys-

less wild-type and mutant P-gps were expressed in HeLa cells using a BacMam-based expression system, as described earlier (1). 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 (1). 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 (1). 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 NBDcyclosporine 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 (2). The protein content was estimated using the amido black Bdye binding assay, as described earlier (3). Synthesis of nilotinib and derivatives Nilotinib and the derivatives were synthesized using a series of published methods as described earlier (4), (5). 4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5(trifluoromethyl)phenyl)-3-(4-(pyridine-3-yl)pyrimidin2-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-1yl)-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)benzamide (1): 1H NMR (400 MHz, MeOH-d4)  9.41 (d, J = 1.6

3

Hz, 1H), 9.33 (d, J = 2.0 Hz,1H), 8.89 (ddd, J = 1.6, 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)-5trifluoromethyl)phenyl)-3-(pyrimidin-2ylamino)benzamide (3): 1H NMR (400 MHz, MeOHd4)  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-2ylamino)benzoic acid (4): 1H NMR (400 MHz, DMSOd6)  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-(3trifluoromethyl)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 (6). 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 (7). 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 (8, 9). 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 (8, 9). 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. (10). 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 (1113). While the mechanistic explanation for the affinity differences between the two TKIs for inhibiting BCR-ABL kinase has been reported (14, 15), 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) (15) and imatinib (PDB ID: 2HYY) (16) 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

8

F382 in the Abl kinase. Therefore, the binding orientation of nilotinib and imatinib in nilotinibAbl 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.

9

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BacMam vectors for expression of ABC drug transporters in mammalian cells. Drug Metab Dispos. 2012;40(2):304-12. Epub 2011/11/02. 2.

Kerr KM, Sauna ZE, Ambudkar SV. Correlation between steady-state ATP hydrolysis

and vanadate-induced ADP trapping in Human P-glycoprotein. Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates. J Biol Chem. 2001;276(12):8657-64. Epub 2000/12/31. 3.

Schaffner W, Weissmann C. A rapid, sensitive, and specific method for the determination

of protein in dilute solution. Anal Biochem. 1973;56(2):502-14. 4.

Wei-Sheng H, William CS. An Efficient Synthesis of Nilotinib (AMN107). Synthesis.

2007;14:2121-4. Epub 03.07.2007. 5.

Duveau DY, Hu X, Walsh MJ, Shukla S, Skoumbourdis AP, Boxer MB, et al. Synthesis

and biological evaluation of analogues of the kinase inhibitor nilotinib as Abl and Kit inhibitors. Bioorg Med Chem Lett. 2013;23(3):682-6. 6.

Sauna ZE, Ambudkar SV. Evidence for a requirement for ATP hydrolysis at two distinct

steps during a single turnover of the catalytic cycle of human P-glycoprotein. Proc Natl Acad Sci U S A. 2000;97(6):2515-20. 7.

Ambudkar SV. Drug-stimulatable ATPase activity in crude membranes of human

MDR1-transfected mammalian cells. Methods Enzymol. 1998;292:504-14. Epub 1998/08/26. 8.

Shi Z, Tiwari AK, Shukla S, Robey RW, Singh S, Kim IW, et al. Sildenafil reverses

ABCB1- and ABCG2-mediated chemotherapeutic drug resistance. Cancer Res. 2011;71(8):3029-41. Epub 2011/03/16.

10

9.

Tiwari AK, Sodani K, Dai C-l, Abuznait AH, Singh S, Xiao Z-J, et al. Nilotinib

potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, and ABCC10-multidrug resistance xenograft models. Cancer Lett. 2013;328(2):307-17. 10.

Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, et al. Structure of P-

Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding. Science. 2009;323(5922):1718-22. 11.

Shukla S, Sauna ZE, Ambudkar SV. Evidence for the interaction of imatinib at the

transport-substrate site(s) of the multidrug-resistance-linked ABC drug transporters ABCB1 (Pglycoprotein) and ABCG2. Leukemia. 2008;22(2):445-7. Epub 2007/08/11. 12.

Dohse M, Scharenberg C, Shukla S, Robey RW, Volkmann T, Deeken JF, et al.

Comparison of ATP-binding cassette transporter interactions with the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib. Drug Metab Dispos. 2010;38(8):1371-80. Epub 2010/04/29. 13.

Weisberg E, Manley P, Mestan J, Cowan-Jacob S, Ray A, Griffin JD. AMN107

(nilotinib): a novel and selective inhibitor of BCR-ABL. Br J Cancer. 2006;94(12):1765-9. Epub 2006/05/25. 14.

Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J. Structural

mechanism for STI-571 inhibition of abelson tyrosine kinase. Science. 2000;289(5486):1938-42. Epub 2000/09/16. 15.

Weisberg E, Manley PW, Breitenstein W, Bruggen J, Cowan-Jacob SW, Ray A, et al.

Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell. 2005;7(2):129-41. Epub 2005/02/16.

11

16.

Cowan-Jacob SW, Fendrich G, Floersheimer A, Furet P, Liebetanz J, Rummel G, et al.

Structural biology contributions to the discovery of drugs to treat chronic myelogenous leukaemia. Acta Crystallogr D Biol Crystallogr. 2007;63(Pt 1):80-93. Epub 2006/12/14.

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1.

Shukla S, Chen ZS, Ambudkar SV. Tyrosine kinase inhibitors as modulators of ABC

transporter-mediated drug resistance. Drug Resist Updat. 2012;15(1-2):70-80. Epub 2012/02/14. 2.

Brózik A, Hegedüs C, Erdei Z, Hegedűs T, Özvegy-Laczka C, Szakács G, et al. Tyrosine

kinase inhibitors as modulators of ATP binding cassette multidrug transporters: substrates, chemosensitizers or inducers of acquired multidrug resistance? Expert Opin Drug Metab Toxicol. 2011;7(5):623-42. 3.

Shukla S, Sauna ZE, Ambudkar SV. Evidence for the interaction of imatinib at the

transport-substrate site(s) of the multidrug-resistance-linked ABC drug transporters ABCB1 (Pglycoprotein) and ABCG2. Leukemia. 2008;22(2):445-7. Epub 2007/08/11. 4.

Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, et al. Structure of P-

Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding. Science. 2009;323(5922):1718-22. 5.

Shi Z, Tiwari AK, Shukla S, Robey RW, Singh S, Kim IW, et al. Sildenafil reverses

ABCB1- and ABCG2-mediated chemotherapeutic drug resistance. Cancer Res. 2011;71(8):3029-41. Epub 2011/03/16. 6.

Tiwari AK, Sodani K, Dai C-l, Abuznait AH, Singh S, Xiao Z-J, et al. Nilotinib

potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, and ABCC10-multidrug resistance xenograft models. Cancer Lett. 2013;328(2):307-17. 7.

Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM.

Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol. 1999;39:361-98.

13

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Sauna ZE, Ambudkar SV. About a switch: how P-glycoprotein (ABCB1) harnesses the

energy of ATP binding and hydrolysis to do mechanical work. Mol Cancer Ther. 2007;6(1):1323. Epub 2007/01/24. 9.

Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J. Structural

mechanism for STI-571 inhibition of abelson tyrosine kinase. Science. 2000;289(5486):1938-42. Epub 2000/09/16. 10.

Weisberg E, Manley P, Mestan J, Cowan-Jacob S, Ray A, Griffin JD. AMN107

(nilotinib): a novel and selective inhibitor of BCR-ABL. Br J Cancer. 2006;94(12):1765-9. Epub 2006/05/25. 11.

Palmeira A, Sousa E, Vasconcelos MH, Pinto M, Fernandes MX. Structure and ligand-

based design of P-glycoprotein inhibitors: a historical perspective. Curr Pharm Des. 2012;18(27):4197-214. Epub 2012/07/26. 12.

Dey S, Ramachandra M, Pastan I, Gottesman MM, Ambudkar SV. Evidence for two

nonidentical drug-interaction sites in the human P- glycoprotein. Proc Natl Acad Sci U S A. 1997;94(20):10594-9. 13.

Loo TW, Bartlett MC, Clarke DM. Simultaneous binding of two different drugs in the

binding pocket of the human multidrug resistance P-glycoprotein. J Biol Chem. 2003;278(41):39706-10. Epub 2003/08/12. 14.

Lugo MR, Sharom FJ. Interaction of LDS-751 and rhodamine 123 with P-glycoprotein:

evidence for simultaneous binding of both drugs. Biochemistry. 2005;44(42):14020-9. Epub 2005/10/19. 15.

Ambudkar SV, Kim IW, Sauna ZE. The power of the pump: mechanisms of action of P-

glycoprotein (ABCB1). Eur J Pharm Sci. 2006;27(5):392-400. Epub 2005/12/15.

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reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc Natl Acad Sci U S A. 2013;110(24):9704-9. Epub 2013/05/22.

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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

125

I-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

a

Basal (no addition) activity taken as 1.0

b125

I-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 125

Cysless-WT-P-gp

a 125

I-IAAP labeling (% inhibition)a

Imatinib

Nilotinib

5

3

(25-50 µM)

(5 µM)

(5 µM)

(5 µM)

60 ± 7

72 ± 5

33 ± 11

0-5

I-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 hydrolysis by P-gp ________________________________________________________________ ATP hydrolysis (fold-stimulation)a

Cysless-WT-P-gp

Imatinib

Nilotinib

5

3

(5 µM)

(1 µM)

(1 µM)

(1 µM)

1.4 ± 0.2

1.5 ± 0.2

1.6 ± 0.1

1.1 ± 0.1

a

Basal (no addition) activity taken as 1.0

The values represent mean ± SD from three independent experiments.

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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.

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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.

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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.

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