Derivative of 5-cyano-6-phenylpyrimidin antagonizes ABCB1- and ABCG2-mediated multidrug resistance

European Journal of Pharmacology 863 (2019) 172611

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Derivative of 5-cyano-6-phenylpyrimidin antagonizes ABCB1- and ABCG2mediated multidrug resistance

T

Jing-Quan Wanga,1, Wang Bob,c,d,e,1, Zi-Ning Leia, Qiu-Xu Tenga, Jonathan Y. Lia,2, Wei Zhanga, Ning Jia, Chao-Yun Caia, Ma Li-Yingb,c,d,e, Hong-Min Liub,c,d,e,∗, Zhe-Sheng Chena,∗∗ a

Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY, 11439, USA Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, PR China c Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, PR China d Key Laboratory of Henan Province for Drug Quality and Evaluation, PR China e School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 450001, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: 5-Cyano-6-phenylpyrimidin derivative MDR ABCB1 ABCG2 Reversal

Multidrug resistance (MDR) lead to inadequate response to chemotherapy and cause failure in cancer treatment. One of the targeted approaches to overcome MDR in cancer cells is interfering or inhibiting ATP binding cassette (ABC) transporters. Among all members in ABC transporters superfamily, ABCB1 (ABC transporter subfamily B #1) and ABCG2 (ABC transporter subfamily G #2) play an important role in the development of cancer MDR. In this study, we synthesized a novel 5-cyano-6-phenylpyrimidin derivative 479, which exhibited selective dualactivity in reversing MDR mediated by ABCB1 and ABCG2, without affecting MDR mediated by ABCC1 (ABC transporter subfamily C #1) and ABCC10 (ABC transporter subfamily C #10). Further mechanism studies demonstrated that 479 increased the accumulation of paclitaxel and mitoxantrone in cancer cells by interrupting the efflux function of transporters and stimulating ABCB1/ABCG2 ATPase activity. In silico study provided evidence that 479 formed multiple physiochemical bonds with the drug-binding pocket of ABCB1 and ABCG2. Overall, our results provide a promising prototype in designing potent dual reversal agents targeting ABCB1- and ABCG2-meidated MDR.

1. Introduction Advancements in cancer chemotherapy decreased mortality in cancer patients. However, due to multidrug resistance (MDR) in cancer cells, the efficacy of a wide range of anticancer agents was significantly attenuated (Li et al., 2017; Wu et al., 2014). Multiple mechanisms have been reported causing MDR, including efflux transporters, reduced apoptosis, DNA damage/repair and autophagy regulation (Cui et al., 2018; Gillet and Gottesman, 2010; Goldman, 2003; Higgins, 2007; Li et al., 2017; Szakács et al., 2006; Zhang et al., 2015). The ATP-binding cassette transporters superfamily consists of subfamilies from ABCA to ABCG, amongst which ABCB1 (P-glycoprotein, P-gp), ABCG2 (Breast Cancer Resistance Protein, BCRP) and ABCCs (Multidrug Resistance-associated Proteins, MRPs) are major inducers in MDR cancer cells and have been deeply studied (Cui et al., 2019b; Dean

and Annilo, 2005; Gottesman and Ambudkar, 2001). ABCB1 is present in kidney, brain, liver, placenta, adrenal glands and blood-brain barrier (BBB) (Gottesman et al., 2002; Zhang et al., 2015). Typically, ABCB1 pumps out a broad spectrum of chemotherapeutic drugs including paclitaxel, vinblastine, methotrexate, vincristine and doxorubicin, thus overexpression of ABCB1 triggers MDR in cancer cells (Takara et al., 2006; Wu et al., 2014). Overexpression of ABCB1 played significant roles to miscellaneous types of cancer such as non-small cell lung cancer (NSCLC) and thyroid cancer (Han et al., 2007; Wang et al., 2017). ABCG2 is found in liver, colon epithelium and placenta (Zhang et al., 2018). Numerous studies revealed that overexpression of ABCG2 led to MDR in solid tumor and hematological cancers (Kawabata et al., 2003; Sargent et al., 2001) (Cai et al., 2019). The ABCG2 has been reported to induce resistance to a wide range of antineoplastic drugs including tyrosine kinase inhibitors, mitoxantrone, doxorubicin, flavopiridol and

∗

Corresponding author. School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China. Corresponding author. Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, New York, USA. E-mail addresses: [email protected] (H.-M. Liu), [email protected] (Z.-S. Chen). 1 These authors contributed equally to this work. 2 Present address: Loomis Chaffee, 4 Batchelder Road, Windsor, CT 06095. ∗∗

https://doi.org/10.1016/j.ejphar.2019.172611 Received 13 June 2019; Received in revised form 9 August 2019; Accepted 14 August 2019 Available online 30 August 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

European Journal of Pharmacology 863 (2019) 172611

J.-Q. Wang, et al.

Scheme. 1. Reagents and conditions: a: absolute ethanol, absolute K2CO3, reflux, 10 h; b: (i) (COCl)2, reflux, 4 h; (ii) arylamine, rt, 20min; c: (i) dioxane, reflux; (ii) phosphorous oxychloride, reflux, 1 h; d: 3-nitroaniline, absolute ethanol, reflux, 6 h.

Table 1 479 sensitizes ABCB1-substrate-selected resistant cells. Treatment

IC50 value ± S.D.a (nM, Resistance Foldb) SW620

SW620/Ad300

KB-3-1

KB-C2

Vincristine +479 1.0 μM +479 2.0 μM +479 4.0 μM +Verapamil 4.0 μM

37.09 43.03 38.39 41.11 30.49

± ± ± ± ±

10.21 (1.0) 12.33 (1.2) 2.14 (1.0) 18.24 (1.1) 16.40 (0.8)

6460.43 ± 371.23 (174.6) 1262.32 ± 94.12a (34.1) 200.49 ± 19.49a (5.4) 41.34 ± 3.41a (1.1) 43.16 ± 7.28a (1.2)

33.21 42.39 39.41 33.21 31.19

Paclitaxel +479 1.0 μM +479 2.0 μM +479 4.0 μM +Verapamil 4.0 μM

19.28 17.03 18.09 18.29 17.11

± ± ± ± ±

1.24 3.37 2.48 3.31 1.47

7230.44 ± 64.22 (380.5) 1775.23 ± 81.19a (93.4) 78.22 ± 6.43a (4.1) 18.09 ± 3.04a (0.9) 19.43 ± 1.18a (1.0)

9.34 9.45 8.22 6.37 7.39

Cisplatin +479 1.0 μM +479 2.0 μM +479 4.0 μM +Verapamil 4.0 μM

1793.33 1255.40 2331.05 2152.36 1614.14

2152.31 1972.04 1793.24 1076.01 1614.43

1222.04 ± 524.25 (1.0) 1589.36 ± 872.40 (1.3) 1344.44 ± 752.11 (1.1) 978.05 ± 238.12 (1.0) 1222.46 ± 347.24 (1.0)

± ± ± ± ±

(1.0) (0.9) (0.9) (0.9) (0.9)

239.43 (1.0) 112.10 (0.7) 309.44 (1.3) 199.28 (1.2) 84.33 (0.9)

± ± ± ± ±

239.23 648.43 242.44 936.04 713.27

(1.2) (1.1) (1.0) (0.6) (0.9)

± ± ± ± ±

± ± ± ± ±

8.41 (1.0) 14.11 (1.3) 18.17 (1.2) 11.12 (1.0) 9.15 (0.9)

2.01 3.31 5.08 1.48 3.21

(1.0) (1.0) (0.9) (0.7) (0.8)

8227.32 ± 83.19 (249.3) 1102.34 ± 28.27a (33.4) 218.38 ± 23.47a (6.6) 46.17 ± 8.19a (1.4) 43.43 ± 8.09a (1.3) 5251.36 ± 721.23 (583.4) 299.48 ± 31.27a (33.2) 73.31 ± 10.00a (8.1) 7.10 ± 2.04a (0.8) 8.42 ± 3.30a (0.9) 1589.36 1344.05 1100.38 1733.25 1466.18

± ± ± ± ±

836.21 777.00 384.25 811.06 671.29

(1.3) (1.1) (0.9) (0.6) (1.2)

P < 0.05. Group with 479 versus group without 479. a IC50 values were calculated from three independent experiments in triplicate. b Resistance folds were determined using division of the IC50 values of resistant cells by the IC50 values of parental cells with or without 497.

without reducing protein expression level and intracellular localization of ABCB1 and ABCG2. ATPase assay showed that 479could stimulate the ATP hydrolase activity of both, which suggested that 479may competitively bind to the drug-binding pocket by displacing conventional anticancer drugs and itself being pumped out. Overall, this study highlighted a ABCB1 and ABCG2 dual-modulator, which may be a lead scaffold for the design and development of dual MDR modulators.

topoisomerase inhibitors (Mao, 2005; Polgar et al., 2008, p. 2; Szakács et al., 2006). It has been widely mentioned that the expression level of ABC transporters in cancer cells is closely related to the efficacy of anticancer drugs as well as the progression of malignancy (Borst and Elferink, 2002; Ween et al., 2015). As a result, it is worth developing novel modulators to interfere with the function of ABC transporters in cancer cells so as to enhance the efficacy of chemotherapy (Ji et al., 2019b; Shukla et al., 2008). Following our previous success in identifying new reversal agents against ABCB1-mediated multidrug resistance (Wang et al., 2018a), we herein report another novel compound479, as a bifunctional reversal agent against MDR induced by ABCB1 and ABCG2 overexpression, without significantly affecting ABCC1- and ABCC10-mediated MDR. Further studies demonstrated that 479 could increase paclitaxel and mitoxantrone intracellular accumulation and attenuate drug efflux,

2. Material and methods 2.1. Chemicals Reagents and solvents were obtained from commercial sources and used directly without further purification. Melting point of compound was determined by an X-5 Micromelting apparatus, and 1H NMR and 2

European Journal of Pharmacology 863 (2019) 172611

J.-Q. Wang, et al.

mitoxantrone and verapamil were purchased from Sigma-Aldrich (St. Louis, MO). KO143 was purchased from Enzo life Sciences (Farmingdale, NY). [3H]-paclitaxel and [3H]-mitoxantrone were purchased from Moravek Biochemicals, Inc (Brea, CA).

Table 2 479 sensitizes ABCB1-transfected resistant cells. Treatment

IC50 value ± S.D.a (nM, Resistance Foldb) HEK293/pcDNA3.1

HEK293/ABCB1

Vincristine +479 1.0 μM +479 2.0 μM +479 4.0 μM +Verapamil 4.0 μM

22.23 28.01 27.04 23.34 26.26

± ± ± ± ±

5.21 2.21 1.40 5.42 4.44

1095.03 ± 78.32 (49.8) 249.45 ± 23.23 (11.3) 70.18 ± 3.12a (3.2) 20.08 ± 9.47a (0.9) 24.18 ± 2.09a (1.1)

Paclitaxel +479 1.0 μM +479 2.0 μM +479 4.0 μM +Verapamil 4.0 μM

42.33 34.11 29.19 38.10 46.23

± ± ± ± ±

9.29 (1.0) 12.08 (0.8) 3.24 (0.7) 10.16 (0.9) 9.32 (1.1)

Cisplatin +479 1.0 μM +479 2.0 μM +479 4.0 μM +Verapamil 4.0 μM

1113.33 ± 294.09 (1.0) 1447.23 ± 99.48 (1.3) 1670.09 ± 105.00 (1.5) 890.36 ± 94.82 (0.8) 1224.41 ± 34.32 (1.1)

(1.0) (1.3) (1.2) (1.0) (1.2)

2.3. Cell lines and cell culture Human epidermoid cancer cell KB-3-1 and colchicine-selected KBC2 cell, human colon cancer cell SW620 and doxorubicin-selected SW620/Ad300 cell, HEK293 cells transfected with empty pcDNA3.1 vector or pcDNA3.1-ABCB1 were used for ABCB1 reversal study. Human colon cancer cell S1 and mitoxantrone-selected S1-M1-80, HEK293 transfected with empty vector pcDNA3.1 or wild type ABCG2 or mutants of ABCG2 (R482G, R482T) were used for ABCG2 reversal study. HEK293 transfected with pcDNA3.1-ABCC1 and pcDNA3.1ABCC10 were used for ABCC1 and ABCC10 reversal studies respectively. Maintenance of KB-C2, SW620/Ad300 and S1-M1-80 follows the protocols described previously (Ji et al., 2018a, 2019a). HEK293 transfected cells were selected with 2 mg/ml G418. All cells grow at 37 °C in FBS-DMEM (10% FBS, 1% penicillin/streptomycin) in a water jacketed CO2 incubator (5% CO2) with thermal protection. All cell lines were cultured without anticancer drug for at least 2 weeks before use.

2339.18 ± 48.08 (55.7) 412.34 ± 91.14a (9.8) 88.03 ± 8.22a (2.1) 34.43 ± 2.13a (0.8) 45.04 ± 13.29a (1.1) 935.41 ± 41.08 (0.8) 1119.43 ± 44.08 (1.1) 1002.05 ± 88.36 (0.9) 1558.23 ± 23.22 (1.4) 1447.19 ± 28.42 (1.3)

P < 0.05. Group with 479 versus group without 479. a IC50 values were calculated from three independent experiments in triplicate. b Resistance folds were determined using division of the IC50 values of resistant cells by the IC50 values of parental cells with or without 497.

2.4. MTT assay Cytotoxicity of drugs in vitrowas determined using MTT assay as previously described (Wang et al., 2018a). For reversal study, cells were seeded evenly into 96-well plates at a final density of 5000 cells per well and final volume of 160 μl. After incubating overnight, 20 μl 479 at indicated concentrations was added 2 h before addition of anticancer drugs. After 72 h incubation, 4 mg/ml MTT reagent was added to the plates and further incubated for 4 h. Subsequently, the medium with MTT was removed followed by adding 100 μl DMSO to dissolve formazan crystals. Absorbance at 570 nm was determined by microplate reader (Dynex Technologies, Chantilly, VA). IC50 values were calculated after plotting survival curves using Bliss method (Zhang et al., 2018). Positive reversal agents were verapamil (4 μM), KO143 (1 μM), MK571 (25 μM) and cepharanthine (4 μM) in ABCB1, ABCG2, ABCC1 and ABCC10 respectively. Cisplatin was the negative control as it is not substrate of any transporters mentioned above.

Table 3 479 sensitizes ABCG2-substrate-selected resistant cells. Treatment

IC50 value ± S.D.a (μM, Resistance Foldb) S1

S1-M1-80

Mitoxantrone + 479 0.25 μM +479 0.5 μM +479 1.0 μM +KO143 1.0 μM

0.38 0.49 0.49 0.46 0.31

± ± ± ± ±

0.10 0.22 0.34 0.22 0.13

(1.0) (1.3) (1.3) (1.2) (0.8)

52.01 ± 12.84 (138.5) 3.85 ± 1.91a (10.2) 2.01 ± 1.12a (5.4) 0.65 ± 0.10a (1.7) 0.75 ± 0.14a (2.0)

SN-38 +479 0.25 μM +479 0.5 μM +479 1.0 μM +KO143 1.0 μM

0.29 0.39 0.25 0.31 0.35

± ± ± ± ±

0.12 0.02 0.04 0.11 0.15

(1.0) (1.3) (0.8) (1.0) (1.2)

55.00 ± 2.93 (183.9) 1.29 ± 0.33a (4.3) 0.65 ± 0.01a (2.2) 0.21 ± 0.11a (0.7) 0.37 ± 0.02a (1.2)

Cisplatin +479 0.25 μM +479 0.5 μM +479 1.0 μM +KO143 1.0 μM

6.79 7.82 4.90 5.69 7.85

± ± ± ± ±

2.52 3.63 1.15 0.95 0.64

(1.0) (1.2) (0.7) (0.8) (1.2)

5.83 7.82 7.31 8.72 7.45

± ± ± ± ±

1.92 0.93 0.54 1.84 1.11

2.5. Western blotting

(0.9) (1.1) (1.1) (1.2) (1.1)

The procedure of Western blotting has been described previously (Ji et al., 2019a). Briefly, cells were cultured with or without 479 of different time (0 h, 24 h, 48 h, 72 h), or with different concentrations of 479 (1, 2, 4 μM for ABCB1; 0.25, 0.5, 1 μM for ABCG2) for same time (72 h). After cell lysis, a BCA protein assay was performed to measure protein concentration using the Protein Assay Kit (Pierce, Rockford, IL, USA). Cell lysate was loaded equally to 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Afterwards, the membranes were soaked in 5% milk powder dissolved in TBST buffer (10 mmol/L Tris-HCl, 150 mmol/L NaCl and 0.1% Tween-20, adjust pH to 8.0). Target protein (ABCB1 or ABCG2) was probed with monoclonal antibody against ABCB1 (dilution 1:500) or ABCG2 (dilution 1:1000), respectively. GAPDH was used as loading control and was probed with corresponding antibody. Relative protein expression level was quantified in ImageJ (NIH, MD).

P < 0.05. Group with 479 versus group without 479. a IC50 values were calculated from three independent experiments in triplicate. b Resistance folds were determined using division of the IC50 values of resistant cells by the IC50 values of parental cells with or without 497. 13 C NMR spectrum were recorded by Bruker 400 and 100 MHz spectrometer, respectively. High-resolution mass spectra was recorded by Waters Micromass Q-T of Micromass spectrometer. The purity of compound was determined to be > 95% by reverse phase HPLC analysis.

2.2. Materials

2.6. Immunofluorescence assay

ABCB1 and GAPDH monoclonal antibodies, anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 594 were purchased from Thermo Fisher Scientific Inc (Rockford, IL). ABCG2 monoclonal antibody was purchased from Millipore (Billerica, MA). Dulbecco's modified Eagle's Medium (DMEM), fetal bovine serum (FBS), antibiotic (penicillin and streptomycin) and trypsin were purchased from Hyclone (Pittsburgh, PA). Paclitaxel, cisplatin, vincristine,

The procedure of immunofluorescence assay has been described previously (Ji et al., 2018a). Cells were cultured with 479 for 24, 48 and 72 h. Cells were seeded in a 24-well plate with a final density of 2 × 104 cells per well. After 72 h, cells were fixed with 4% paraformaldehyde for 15 min and treated by 0.1% Triton X-100 for 15 min to 3

European Journal of Pharmacology 863 (2019) 172611

J.-Q. Wang, et al.

Table 4 479 sensitizes ABCBG2-transfected resistant cells. IC50 value ± S.D.a (nM, Resistance Foldb)

Treatment

HEK293/pcDNA3.1

HEK/ABCG2-R482

HEK/ABCG2-R482G

HEK/ABCG2-R482T

Mitoxantrone +479 0.25 μM +479 0.5 μM +479 1.0 μM +KO143 1.0 μM

88.09 92.33 94.43 83.48 88.36

1179.49 ± 14.14 (13.4) 871.25 ± 80.33 (9.9) 278.04 ± 44.47a (3.2) 78.18 ± 9.25a (0.9) 86.33 ± 14.34a (1.0)

1522.23 ± 41.35 (17.3) 880.26 ± 24.28 (10.0) 405.09 ± 32.11a (4.6) 91.47 ± 20.14a (1.0) 77.22 ± 9.28a (0.9)

915.00 ± 8.23 (10.4) 766.33 ± 22.41 (8.7) 431.23 ± 8.44 (4.9) 73.25 ± 4.33a (0.9) 83.19 ± 8.09a (1.0)

SN-38 +479 0.25 μM +479 0.5 μM +479 1.0 μM +KO143 1.0 μM

9.43 ± 0.23 (1.0) 11.34 ± 2.21 (1.2) 8.19 ± 1.32 (0.8) 10.38 ± 3.33 (1.1) 8.09 ± 1.38 (0.9)

117.28 ± 4.38 (13.6) 72.39 ± 14.28 (8.4) 41.11 ± 4.09a (4.8) 7.47 ± 3.15a (0.8) 8.34 ± 1.13a (0.9)

316.27 ± 21.29 (36.9) 216.39 ± 34.22 (25.1) 58.41 ± 9.38a (6.7) 10.36 ± 7.22a (1.2) 19.41 ± 8.21a (2.2)

157.34 ± 14.32 (18.3) 79.43 ± 14.25 (9.2) 45.37 ± 2.19a (5.2) 7.38 ± 1.48a (0.8) 8.36 ± 1.46a (0.9)

Cisplatin +479 0.25 μM +479 0.5 μM +479 1.0 μM +KO143 1.0 μM

1694.47 1480.04 1259.23 1539.49 1564.14

1344.43 1363.32 1155.45 1476.10 1519.71

1290.16 1313.45 1671.38 1332.23 1531.07

1650.35 1236.40 1256.27 1428.44 1595.08

± ± ± ± ±

3.24 (1.0) 19.27 (1.1) 8.32 (1.1) 2.32 (1.3) 13.18 (1.0)

± ± ± ± ±

133.33 144.12 147.28 162.23 137.08

(1.0) (0.9) (0.7) (1.0) (1.0)

± ± ± ± ±

200.48 179.21 200.47 133.06 181.48

(0.8) (0.9) (0.7) (0.9) (0.9)

± ± ± ± ±

129.39 (0.8) 152.41 (0.8) 187.16 (1.0) 126.39 (0.8) .36,181 (1.0)

± ± ± ± ±

213.03 148.09 174.14 133.40 128.26

(1.0) (0.7) (0.7) (0.9) (1.0)

P < 0.05. Group with 479 versus group without 479. a IC50 values were calculated from three independent experiments in triplicate. b Resistance folds were determined using division of the IC50 values of resistant cells by the IC50 values of parental cells with or without 497. Table 5 479 does not sensitize ABCC1-transfected resistant cells. Treatment

Table 6 479 does not sensitize ABCC10-transfected resistant cells.

IC50 value ± S.D.a (nM, Resistance Foldb) HEK293/pcDNA3.1

HEK293/ABCC1

Vincristine +479 1.0 μM +479 2.0 μM +479 4.0 μM +MK571 25.0 μM

65.47 69.34 58.47 63.40 66.32

1287.00 ± 78.33 (19.8) 1053.38 ± 23.36 (16.2) 1229.34 ± 43.23 (18.9) 1196.34 ± 99.45 (18.4) 202.24 ± 27.28a (3.1)

Cisplatin +479 1.0 μM +479 2.0 μM +479 4.0 μM +MK571 25.0 μM

987.42 ± 101.34 (1.0) 888.42 ± 99.41 (0.9) 1184.44 ± 105.25 (1.2) 928.10 ± 71.16 (0.9) 1481.07 ± 194.13 (1.5)

691.32 ± 87.15 (0.7) 1283.16 ± 134.17 (1.3) 1382.35 ± 176.45 (1.4) 809.37 ± 194.02 (0.8) 908.09 ± 201.08 (0.9)

± ± ± ± ±

8.41 (1.0) 12.24 (1.1) 11.26 (0.9) 9.43 (1.0) 14.35 (1.0)

Treatment

HEK293/pcDNA3.1

HEK293/ABCC10

Vincristine +479 1.0 μM +479 2.0 μM +479 4.0 μM +Cepharanthine 4.0 μM

82.35 77.17 92.28 89.09 86.11

1648.25 ± 128.23 (20.1) 1501.19 ± 92.10 (18.3) 1607.28 ± 189.39 (19.6) 1451.31 ± 341.28 (17.7) 139.23 ± 44.16a (1.7)

Cisplatin +479 1.0 μM

1009.33 ± 87.18 (1.0) 1312.23 ± 333.28 (1.3) 1554.07 ± 298.35 (1.5) 707.18 ± 98.22 (0.7) 1039.28 ± 244.35 (1.0)

+479 2.0 μM +479 4.0 μM +Cepharanthine 4.0 μM

P < 0.05. Group with 479 versus group without 479. a IC50 values were calculated from three independent experiments in triplicate. b Resistance folds were determined using division of the IC50 values of resistant cells by the IC50 values of parental cells with or without 497.

IC50 value ± S.D.a (μM, Resistance Foldb)

± ± ± ± ±

38.17 92.15 34.11 19.05 31.17

(1.0) (0.9) (1.1) (1.1) (1.0)

807.33 ± 113.11 (0.8) 827.26 ± 79.38 (0.8) 1314.21 ± 83.94 (1.3) 1222.12 ± 317.48 (1.2) 948.30 ± 102.09 (0.9)

P < 0.05. Group with 479 versus group without 479. a IC50 values were calculated from three independent experiments in triplicate. b Resistance folds were determined using division of the IC50 values of parental cells by the IC50 values of resistant cells with or without 497.

increase cell membrane permeability. To locate ABCB1 and ABCG2, diluted monoclonal antibody were added and incubated with processed cells at 4 °C overnight. Alexa Fluor 488 and 594 conjugated secondary antibody (1:1000) were used to generate ABCB1- or ABCG2-specific fluorescence signal, respectively. DAPI was used to mark the nuclei Nikon TE-2000S fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) was used for final imaging.

2.7. Intracellular accumulation of [3H]-paclitaxel and [3H]-mitoxantrone [3H]-paclitaxel accumulation was determined in SW620 and SW620/Ad300 for ABCB1 reversal study, [3H]-mitoxantrone accumulation was measured in S1 and S1-M1-80 for ABCG2 reversal study. Procedure was similar to previously described (Ji et al., 2018b). After culturing cells in 24-well plates overnight, different concentrations of 479 were added 2 h before [3H]-paclitaxel or [3H]-mitoxantrone were added. After incubating at 37 °C for 2 h, cells were rinsed with cold PBS twice and resuspended using 0.25% trypsin. Cell suspension was transferred into scintillation vials, following the addition of 5 ml scintillation fluid. 3H Radioactivity was measured using Packard TRI-CARB

Fig. 1. Effect of 479 on thermal stability of ABCB1 and ABCG2. 479 engaged to ABCB1 and ABCG2 in different temperatures. Upper: ABCB1; Lower: ABCG2. DMSO was used as negative control.

4

European Journal of Pharmacology 863 (2019) 172611

J.-Q. Wang, et al.

Fig. 2. Protein expression of ABCB1 and ABCG2 after incubation with 479. (A) Relative ABCB1 expression level in SW620 and SW620/Ad300 after incubating with 4 μM 479 for different time. (B). Relative ABCBG2 expression level in S1 and S1-M180 after incubating with 1 μM 479 for different time. The error bar is calculated by the mean ± S.D. from three independent experiments in triplicate. *P < 0.05, compared with parental cells.

(Aller et al., 2009). The docking grid for human ABCG2 (PDB ID: 6ETI) was generated using ALA397, VAL401, LEU405, LEU539, ILE543 and F547 (Jackson et al., 2018). Glide v 7.4 XP docking default protocol (Schrödinger, LLC, New York, NY) and an induced-fit docking (IFD) were performed and we used the best scored binding pose of ligand and protein from Glide XP. Top scoring docked result was used in graphical analysis. The unit of docking scores were kcal/mol.

1900CA liquid scintillation analyzer (Packard Instrument, Downers Grove, IL, USA). 2.8. Efflux assay of [3H]-paclitaxel and [3H]-mitoxantrone To ascertain the effect of 479 on [3H]-paclitaxel and [3H]-mitoxantrone efflux, we used ABCB1- or ABCG2- overexpressing SW620/ Ad300 and S1-M1-80 cells, respectively. Following the accumulation assay, cells were incubated in fresh medium without [3H]-label for 0, 30, 60 and 120 min. Serial aliquots were taken at each time point and analyzed for efflux of [3H]-paclitaxel and [3H]-mitoxantrone in Packard TRI-CARB 1900CA liquid scintillation analyzer (Packard Instrument, Downers Grove, IL, USA).

2.12. Statistical analysis All data are presented as mean ± S.D. and analyzed using a oneway ANOVA. All experiments were repeated at least three times. Differences were considered significant when P < 0.05.

2.9. ATPase assay

3. Results

Vanadate-sensitive adenosine triphosphate hydrolase activity of ABCB1 and ABCG2 were measured using method as previously described (Ji et al., 2018b). ABCB1- and ABCG2-overexpressed membrane vesicles were obtained from BD Biosciences (San Jose, CA, USA). In brief, 10 μg membrane vesicles was incubated in assay buffer for 3 min. The mixture was incubated with or without 400 μM sodium orthovanadate (Na3VO4) 37 °C for 5 min then with 0–20 μM of 479 for 3 min 5 mM of Mg-ATP was used to initiate ATPase reaction in at 37 °C for 20 min. Then 100 μl of 5% SDS solution was added to terminate the reaction. The activity of ATP hydrolase was represented by inorganic phosphate (blue) release, and absorbance at 880 nm was measured using a spectrophotometer based on a standard curve.

3.1. Chemistry The general synthesis route of compound 479 is depicted in Scheme 1. As mentioned previously (Wang et al., 2018a), 3,4,5-trimethoxybenzaldehyde (compound 1), ethyl cyanoacetate (compound 2), thiourea (compound 3) were prolonged heated in ethanol containing potassium carbonate to obtain compound 4; compound 6 was synthesized from 5 and the corresponding arylamines following literature procedures and then reacted with compound 4 to obtain compound 7. Finally, compounds 8 (compound 479) was synthesized by the highly activated intermediate 7 reacting with appropriately 3-nitroaniline. Characterization of compound 479 is as follows: 2-((5-cyano-4-((3nitrophenyl)amino)-6-(3,4,5-trimethoxyphenyl)pyrimidin-2-yl)thio)N-(pyridin-2-ylcarbamoyl)acetamide (479) Yield 79.0%. Yellow solid. Mp: 213–214 °C. 1H NMR (400 MHz, DMSO-d6, δ, ppm)δ 11.12 (s, 1H, NH, D2Oexchangeable), 10.52 (s, 1H, NH, D2O exchangeable), 10.28 (s, 1H, NH, D2O exchangeable), 8.48 (s, 1H, ArH), 8.27 (s, 1H, ArH), 7.91 (ddd, J = 36.8, 22.0, 7.6 Hz, 4H, ArH), 7.64 (t, J = 7.9 Hz, 1H, ArH), 7.17 (d, J = 43.4 Hz, 3H, ArH), 4.21 (s, 2H,CH2), 3.79 (d, J = 35.6 Hz, 9H, OCH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): δ172.71, 170.46, 168.19, 160.67, 153.15, 151.30, 150.59, 148.56, 148.10, 140.63,139.36, 138.96, 130.96, 130.18, 130.06, 120.01, 119.70, 118.24, 116.37, 113.37,106.92, 86.18, 60.66, 56.49, 36.22. HRMS (ESI) calcd for C28H25N8O7S [M+H]+:617.1567, found: 617.1564.

2.10. Cellular thermal shift assay (CETSA) Generally, cells were lysed by repeating cycles of freezing-thawing using liquid nitrogen for three times. Subsequently, the mixture was centrifuged at 13,700 g for 10 min and supernatant was collected. The sample was then incubated with candidate compound or control solvent at 37 °C for 30 min, after which the lysate was equally loaded into 8 PCR tubes and heated to the temperatures of 40, 43, 46, 49, 52, 55, 58, and 61 °C for 3 min. Protein amount in lysates was determined using Western blotting. 2.11. ABCB1 and ABCG2 homology modeling with 479

3.2. Effect of 479 on the efficacy of anticancer drugs in parental and ABCB1- or ABCG2-overpressing cells

Molecular docking simulations were run in Maestro v11.1 platform (Schrödinger, LLC, Cambridge, MA). Ligand preparation and protein preparation followed default protocols as previously described (Wang et al., 2018a). The human ABCB1 homology model was established based on refined mouse ABCB1 protein (PDB ID: 4M1M) and was kindly provided by S. Aller (Aller et al., 2009). The docking grid (length: 25 Å) for human ABCB1 was refined by setting the centroid with the amino acid residues that are suggested to have interaction with substrates

Non-toxic concentration of 479 was determined by MTT, results were displayed in supplementary materials (Figs. S1 and S2). According to Table 1, the IC50 values of SW620/Ad300 and KB-C2 to vincristine and paclitaxel were significantly reduced when combining 2 μM or 4 μM 479 compared to control group. Such reduction showed a dosedependent manner as the resistant folds of SW620/Ad300 and KB-C2 5

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Fig. 3. Protein localization of ABCB1 in resistant cells and their parental cells after incubation with 479. (A) Effect of 4 μM 479 on subcellular localization of ABCB1 after incubated for 24, 48 and 72 h. (B) Effect of 1 μM 479 on subcellular localization of ABCG2 after incubated for 24, 48 and 72 h. Scale bar: 10 μm.

decreased with higher concentrations of 479. Furthermore, 479 showed similar manner in HEK293/ABCB1 cells in Table 2. Meanwhile, treatment with 479 did not show significant effect on the IC50 value of vincristine and paclitaxel in parental cells. Verapamil (4 μM) was used as a positive control inhibitor for ABCB1. Known polymorphisms in ABCG2 could alter its substrate-related function (Mao, 2005), thus we evaluated the effect of 479 in both ABCG2-overexpressing cells (wildtype R482 and mutant R482G, R482T). From Table 4, the data showed that 479 at 0.5 and 1 μM significantly decreased ABCG2-mediated multidrug resistance to mitoxantrone and SN-38 in a concentrationdependent manner in HEK293/ABCG2-R482, HEK293/ABCG2-R482G and HEK293/ABCG2-R482T. Additionally, 479 also significantly

sensitized S1-M1-80 cells to the substrates of ABCG2 based on Table 3. KO143 was a positive control of ABCG2 reversal agents.

3.3. Effect of 479 on the efficacy of anticancer drugs in parental and ABCC1- or ABCC10-overpressing cells We also evaluated the effect of 479 in sensitizing resistant cells which overexpress other transporters such as ABCC1 (MRP1) and ABCC10 (MRP7). Results were shown in Tables 5–6. Compound 479 at 1, 2 or 4 μM did not show significant alteration on the IC50 of vincristine in HEK293/ABCC1 or HEK293/ABCC10 cells. In these tests, MK571 and cepharanthine were used as positive control or ABCC1 and ABCC10 6

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Fig. 4. Effect of 479 on the intracellular drug accumulation in MDR and parental cells. (A) Effect of 479 on the accumulation of [3H]-paclitaxel in SW620 and ABCB1overexpressing SW620/Ad300 cells. (B) Effect of 479 on the accumulation of [3H]mitoxantrone in S1 and ABCG2-overexpressing S1-M1-80 cells. The error bar represents the mean ± S.D. from three independent experiments in triplicate. *P < 0.05, compared with control group.

Fig. 5. Effect of 479 on the efflux function in MDR and parental cancer cells. (A) Effect of 479 on [3H]-paclitaxel efflux in SW620 cells. (B) Effect of 479 on [3H]paclitaxel efflux in SW620/Ad300 cells. (C) Effect of 479 on [3H]-mitoxantrone efflux in S1 cells. (D) Effect of 479 on [3H]-mitoxantrone efflux in S1-M1-80 cells. The error bar represents the mean ± S.D. from three independent experiments in triplicate. *P < 0.05, compared with control group.

denaturation profile in cells without compound treatment. As is shown in Fig. 1, 479 could significantly stabilize both ABCB1 and ABCG2 at higher temperature compared to negative control DMSO. The results suggested that the reversal effect of 479 may be associated with its engagement with ABCB1 and ABCG2.

reversal agents, respectively. In all reversal tests, we used cisplatin as negative control (Tables 1–6), as it is not the substrate of ABCB1, ABCG2, ABCC1 or ABCC10. The results indicated that 479 showed reversal effect on ABCB1- and ABCG2-mediated multidrug resistance, but not on MDR mediated by ABCC1 or ABCC10. 3.4. Effect of 479 on the thermal stability of ABCB1 and ABCG2

3.5. Effect of 479 on ABCB1 and ABCG2 expression and subcellular localization

Celluar thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins, and it has become a valuable tool for the validation and optimization of drug target engagement (Molina et al., 2013). To measure the target engagement in cells, we measured the ability of 479 in penetrating cells and binding to ABCB1 and ABCG2. Once the target protein was bound to compound, it will remain stable after heating compared to the

Since we found 479 could antagonize multidrug resistance mediated by ABCB1 and ABCG2, the possible mechanism is then studied. Generally, ABCB1 and ABCG2 proteins anchored in cell membrane to transport out drugs and other molecules, thus it is possible that 479 down-regulates protein expression and/or varies the transporter subcellular localization. To determine this, we used immunofluorescence assay and Western blotting to investigate protein expression level and 7

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Fig. 6. Effect of 479 on ABCB1 and ABCG2 adenosine 5′-triphosphatase activity. (A) Effect of 479 on ABCB1 adenosine 5′-triphosphatase activity. (B) Effect of 479 on ABCG2 adenosine 5′-triphosphatase activity. The error bar represents the mean ± S.D.

Fig. 7. Docking analysis of 479 with homology model of ABCB1 and ABCG2. (A) Compound 479 docked in the ligand-binding cavity of ABCB1. (B) Compound 479 docked within the ligand-binding cavity of ABCG2. Atoms of 479 are displayed as balls and sticks, and are colored as follows: carbon-orange, oxygen-red, nitrogen-blue, sulfur-yellow. Residues forming direct chemical linkage are displayed and colored in a different scheme: carbon-grey, oxygen-red, nitrogen-blue. π-π stacking interactions are displayed as cyan dash line and hydrogen bonds are yellow dash line. (C) A 2-D diagram of the binding position of 479 to ABCB1 homology model. (D) A 2-D diagram of the binding position of 479 to ABCG2 homology model. Colored bubbles represented amino acids within 3 Å and cyan indicates polar residues while green indicates non-polar residues. Green and purple solid lines indicate π-π stacking interactions and hydrogen bonds, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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3.9. 479-ABCB1 and 479-ABCG2 induced-fit docking (IFD) analysis

subcellular localization of ABCB1 and ABCG2. Results were shown in Figs. 2 and 3. According to Fig. 2, ABCB1 (170 kDa) and ABCG2 (72 kDa) expression level were not altered significantly after incubating with 479 for 24, 48 and 72 h in SW620/Ad300 and S1-M1-80. As is shown in Fig. 3, ABCB1 and ABCG2 were found on the membrane of SW620/Ad300 and S1-M1-80 respectively. For ABCB1, cells were incubated with 4 μM 479 for 24, 48 and 72 h. For ABCG2, cells were incubated with 1 μM 479 for 24, 48 and 72 h. Results showed that the subcellular localization of neither ABCB1 nor ABCG2 were altered by 479.

To better understand reversal effect of 479 on ABC transporters, a molecular docking simulation was performed. Parameters such as centroid have been described in previous section. The IFD results showed that 479 had a docking score of -12.078 kcal/mol to ABCB1 and -11.440 kcal/mol to ABCG2, which indicated that 479 had good affinity to both ABCB1 and ABCG2. Fig. 7A showed the best-scored docked position of 479 within the drug-binding pocket in the homology model of human ABCB1. Similarly, Fig. 7B showed the interaction of 479 and the drug-binding pocket in cryo-EM structure of human ABCG2. Fig. 7A showed hydrogen bonds and π-π stacking interactions that could stabilize the binding between 479 and ABCB1 drug-binding pocket. As is shown in Fig. 7C, 479 could form three hydrogen bonds with GLN725 and GLN90 and four π-π stacking interactions with TYR307, PHE343, PHE728 and PHE983. The binding position of the best-scored 479 in ABCG2 was displayed in Figs. 7B, 9D. Similarly, from the 2-D diagram, we found 479 could form a hydrogen bond with ASN436 in chain A. Moreover, 479 was found to fit well with high degree of shape complementarity in a drug-binding cavity of ABCG2, which is formed by several polar and non-polar residues (Fig. 7B).

3.6. Effect of 479 on the intracellular accumulation of anticancer drugs in ABCB1- and ABCG2-overexpressing cancer cells The MTT results showed that 479 could significantly sensitize ABCB1- and ABCG2-mediated MDR cells to corresponding substrates. However, according the Western blotting and immunofluorescence results, incubation with compound 479 did not affect transporter expression level or subcellular localization. To further investigate the reason why 479 could reverse ABCB1- and ABCG2-mediated MDR, we measured the intracellular level of [3H]-paclitaxel and [3H]-mitoxantrone in ABCB1- and ABCG2-mediated MDR cells respectively with or without incubation with 479. As is shown in Fig. 4A, incubation with 479 greatly increased the intracellular accumulation of [3H]-paclitaxel in SW620/Ad300 cells but not in parental SW620 cells. Similarly, the intracellular amount of [3H]-mitoxantrone in S1-M1-80 was also significantly elevated with the addition of 479 (Fig. 4B). Moreover, such effects of 479 showed a concentration-dependent manner in both ABCB1- and ABCG2-overexpressing cells. These results provided evidence for the conclusion that 479 sensitized MDR cells by affecting the function of ABCB1 and ABCG2 transporters. Verapamil and KO143 were used as positive control for ABCB1 and ABCG2 respectively.

4. Discussion MDR has been a major barrier in successful chemotherapy. And overexpressing ABC transporters on cancer cell membrane is an important factor in forming MDR (Lepper et al., 2005). Among the members of ABC transporters family, ABCB1 and ABCG2 are well documented and have been proved closely related to MDR in various cancer including non-small cell lung cancer (NSCLC) (Burger et al., 2005) and colon cancer (Sun et al., 2012). Numerous in vitroand in vivostudies have showed that co-administration of ABC transporters inhibitors could antagonize MDR in cancer cells (Cui et al., 2019a; Fan et al., 2018; Wu et al., 2019). Clinically, cancer MDR could be induced by multiple types of ABC transporters, thus it is clinically important to develop ABC transporter inhibitors to further lift the efficacy of anticancer drugs. Numerous studies have been conducted to develop modulators which can bind and block the transportation function of ABC-transporters. To date, ABCB1 inhibitors could be classified into three generations. First-generation inhibitors include verapamil, cyclosporine A and quinidine. Due to the low clinical effect and high toxicity, the second generation was quickly introduced by modifying the chemical structure of the first generation inhibitors, in order to obtain high potency and low toxicity (Wilson et al., 1995). Examples include dexverapamil, valspodar and biricodar citrate (Li et al., 2016). The third generation inhibitors, such as tariquidar, mitotane and laniquidar, have even higher efficacy and affinity to transporters than second generation (Bates et al., 1991; Fox and Bates, 2007; Luurtsema et al., 2009), however, no satisfactory results have been reported in clinical trials because of safety concerns, drug interaction or insignificant clinical outcome (Shukla et al., 2008). Thus, it is an urgent requirement to develop more controllable and effective ABC transporter inhibitors to overcome cancer MDR and increase chemotherapy efficiency. In our previous study, a potent triazole contained pyrimidine-based MDR mediator PRA-1 was designed and shown to significantly antagonize ABCB1-mediated MDR by enhancing paclitaxel accumulation (Wang et al., 2018b). Inspired by the structure of PRA-1, we modified the structure to further improve its resersal activity. Compared to PRA1, 479 has more interactive sites such as amide bonds which promote strong hydrogen bonding interactions with ABCB1 hydrophobic pocket (Raub, 2006). Also, its potency in reversing ABCB1 and ABCG2-mediated MDR is comparable to positive inhibitor verapamil/KO143 as well as to most reported dual modulators such as bafetinib reversal concentration 3 μM for ABCB1 and 3 μM for ABCG2 (Zhang et al., 2016), sildenafil (reversal concentration 10 μM for ABCB1 and 50 μM for

3.7. Effect of 479 on the efflux activity of chemotherapeutic drugs in MDR cells The protocal has been described in our previous study (Ji et al., 2019b). In brief, in order to delve deeper into the mechanism of 479 in sensitizing ABCB1- and ABCG2-overexpressing cells, efflux activity of ABCB1 and ABCG2 was measured to ascertain that the increased accumulation was due to the alteration of efflux function of transporters. As is shown in Fig. 5A and B, compound 479 at 4 μM significantly inhibited [3H]-paclitaxel efflux in SW620/Ad300 cells, compared to control group without 479. Furthermore, after incubated with 1 μM 479, S1-M1-80 cells showed similar reducing trend in drug efflux (Fig. 5C and D). These reductions were comparable to the decrease caused by incubating with 4 μM verapamil for ABCB1 and 1 μM KO143 for ABCG2. 3.8. Effect of 479 on ABCB1- and ABCG2- adenosine 5′-triphosphatase activity To further explorer the mechanism of 479 increasing intracellular accumulation and decreasing efflux of anticancer drugs, we performed ATPase assay of ABCB1 and ABCG2, to measure the effect of different concentrations of 479 (0–20 μM) in ABCB1- and ABCG2- ATP hydrolase activity. As is shown in Fig. 6A, compound 479 stimulated ABCB1 ATPase with a fold-stimulation of 3.6, in a dose-dependent manner. Moreover, when the concentration of 479 reached 0.5 μM, ATPase activity got 50% maximum stimulation (EC50). For ABCG2, 479 showed stimulating effect on its ATPase activity in a dose-dependent manner. Meanwhile, the concentration of 479 to reach 50% maximum stimulation of ABCG2 ATPase (EC50) is 0.4 μM, and the maximum stimulation fold was 2.1 (Fig. 6B). 9

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ABCG2) (Shi et al., 2011) and nilotinib (reversal concentration 2.5 μM for ABCB1 and 2.5 μM for ABCG2) (Tiwari et al., 2009). Molecular docking results showed that enlongated amide chain also enables the drug molecule to fold and form a more planar structure, which has been reported to be important in binding to ABCG2 and inhibit its function (Pick et al., 2008). Cytotoxicity assay showed that 479 presented similar IC50 in both parental and resistant cells, thus 479 has potential to be an inhinitor of ABCB1 or ABCG2. Actually, our MTT results showed that the novel compound 479 (reversing paclitaxel resistance by 380 folds in SW620/Ad300 cells) has better reversal effect on ABCB1-mediated MDR than PRA-1(reversing paclitaxel resistance by 136 folds in SW620/Ad300 cells) (Wang et al., 2018a). Moreover, 479 also showed more significant effect on reversing ABCG2-mediated MDR than previous compounds (data not shown), such antagonization was observed on MDR mediated by both wild-type ABCG2 and its mutants. The reversal effect of 479 against ABCB1 and ABCG2 is comparable to their positive reversal agents verapamil (ABCB1) and KO143 (ABCG2), respectively. To determine the reversal mechanism of 479, we first performed Western blotting and immunofluorescence assay to see whether the reversal effect was caused by protein down-regulation or translocation. The results showed that 479 did not alter protein expression level or subcellular distribution of either ABCB1 or ABCG2 after incubating for up to 72 h. Such findings suggested that the reversal effect of 479 on ABCB1- and ABCG2-related MDR is not associated with protein level modulation or subcellular localization. As ABCB1 and ABCG2 were both drug efflux pumps, we performed an efflux assay using [3H]-labeled drugs and the results showed that ABCB1- and ABCG2-overexpression cells had accelerated drug efflux, which was significantly decreased by adding 479. The efflux result does not guarantee intracellular drug accumulation, beause drugs could be metabolized via modified pathways in cancer cells (Herling et al., 2011). Thus an accumulation assay was performed to directly observe intracellular drug accumulation quantitatively. Results indicated that 479 significantly elevated the intracellular accumulation of [3H]-paclitaxel and [3H]-mitoxantrone in ABCB1-overexpressing and ABCG2-overexpressing cells, respectively, leading to increased intracellular drug concentration and cell death. The transportation function of ABC transporters majorly relies on ATP hydrolysis by ATPase (Li et al., 2016). By our observation that 479 stimulated ABCB1 ATPase by 3.6-fold and ABCG2 ATPase by 2.1-fold, 479 might competitively interact with the substrate binding site of ABCB1 and ABCG2 then affect their functions. It has been revealed that ABC transporters have two distinct binding sites—the M-site (binds modulators) and H/R site (binds substrates) (Ferreira et al., 2013; Zhang et al., 2017). In our results, both ABCB1 and ABCG2 ATPase activity exihibited an Michaelis-Menten kinetics curve, which indicated different affinities of ATPase binding sites. So that we could hypothesize that 479 binds stronger to the modulator site of ABCB1 and ABCG2 (Ferreira et al., 2013). Furthermore, the cellular thermal shift assay showed that 479 efficiently stabilized ABCB1 and ABCG2, which provided evidence that 479 could bind with ABCB1 and ABCG2. In silico study further supported the conclusion that 479 could bind to transporters directly by interacting with amino acid residues. Specifically, 479 could form multiple π-π interactions and hydrogen bonds with human ABCB1, and fit in a drug-binding cavity with high degree of shape complementarity in ABCG2.

highlighted a ABCB1 and ABCG2 modulators with new structure, which may be a lead scaffold for the design and development of dual-targeting MDR modulators. Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements We would like to thank Dr. Stephen Aller (The University of Alabama at Birmingham, Birmingham) for kindly providing the human ABCB1 homology model. We thank Tanaji T. Talele (St. John's University, New York, NY) for providing the computing resources for the docking analysis. We thank Drs. Susan E. Bates and Robert W. Robey (NCI, NIH, Bethesda, MD) for providing the cell lines. Furthermore, this work was also supported by National Natural Science Foundation of China (Project No. 81430085, No. 21372206, No. 81773562, and No. 81703328). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejphar.2019.172611. References Aller, S.G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P.M., Trinh, Y.T., Zhang, Q., Urbatsch, I.L., Chang, G., 2009. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323, 1718–1722. https://doi. org/10.1126/science.1168750. Bates, S.E., Shieh, C.Y., Mickley, L.A., Dichek, H.L., Gazdar, A., Loriaux, D.L., Fojo, A.T., 1991. Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/P-glycoprotein) which is also expressed by adrenocortical carcinomas. J. Clin. Endocrinol. Metab. 73, 18–29. https://doi.org/10. 1210/jcem-73-1-18. Borst, P., Elferink, R.O., 2002. Mammalian ABC transporters in health and disease. Annu. Rev. Biochem. 71, 537–592. https://doi.org/10.1146/annurev.biochem.71.102301. 093055. Burger, H., van Tol, H., Brok, M., Wiemer, E.A.C., de Bruijn, E.A., Guetens, G., de Boeck, G., Sparreboom, A., Verweij, J., Nooter, K., 2005. Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps. Cancer Biol. Ther. 4, 747–752. https://doi.org/10.4161/cbt.4.7.1826. Cai, C.-Y., Zhai, H., Lei, Z.-N., Tan, C.-P., Chen, B.-L., Du, Z.-Y., Wang, J.-Q., Zhang, Y.-K., Wang, Y.-J., Gupta, P., Wang, B., Chen, Z.-S., 2019. Benzoyl indoles with metabolic stability as reversal agents for ABCG2-mediated multidrug resistance. Eur. J. Med. Chem. 179, 849–862. https://doi.org/10.1016/j.ejmech.2019.06.066. Cui, Q., Cai, C.-Y., Gao, H.-L., Ren, L., Ji, N., Gupta, P., Yang, Y., Shukla, S., Ambudkar, S.V., Yang, D.-H., Chen, Z.-S., 2019a. Glesatinib, a c-MET/SMO dual inhibitor, antagonizes P-glycoprotein mediated multidrug resistance in cancer cells. Front. Oncol. 9. https://doi.org/10.3389/fonc.2019.00313. Cui, Q., Wang, J.-Q., Assaraf, Y.G., Ren, L., Gupta, P., Wei, L., Ashby, C.R., Yang, D.-H., Chen, Z.-S., 2018. Modulating ROS to overcome multidrug resistance in cancer. Drug Resist. Updates 41, 1–25. https://doi.org/10.1016/j.drup.2018.11.001. Cui, Q., Yang, Y., Ji, N., Wang, J.-Q., Ren, L., Yang, D.-H., Chen, Z.-S., 2019b. Gaseous signaling molecules and their application in resistant cancer treatment: from invisible to visible. Future Med. Chem. 11, 323–336. https://doi.org/10.4155/fmc-2018-0403. Dean, M., Annilo, T., 2005. Evolution of the ATP-binding cassette (ABC) transporter superfamily in vertebrates. Annu. Rev. Genom. Hum. Genet. 6, 123–142. https://doi. org/10.1146/annurev.genom.6.080604.162122. Fan, Y.-F., Zhang, W., Zeng, L., Lei, Z.-N., Cai, C.-Y., Gupta, P., Yang, D.-H., Cui, Q., Qin, Z.-D., Chen, Z.-S., Trombetta, L.D., 2018. Dacomitinib antagonizes multidrug resistance (MDR) in cancer cells by inhibiting the efflux activity of ABCB1 and ABCG2 transporters. Cancer Lett. 421, 186–198. https://doi.org/10.1016/j.canlet.2018.01. 021. Ferreira, R.J., Ferreira, M.-J.U., dos Santos, D.J.V.A., 2013. Molecular docking characterizes substrate-binding sites and efflux modulation mechanisms within P-glycoprotein. J. Chem. Inf. Model. 53, 1747–1760. https://doi.org/10.1021/ci400195v. Fox, E., Bates, S.E., 2007. Tariquidar (XR9576): a P-glycoprotein drug efflux pump inhibitor. Expert Rev. Anticancer Ther. 7, 447–459. https://doi.org/10.1586/ 14737140.7.4.447. Gillet, J.-P., Gottesman, M.M., 2010. Mechanisms of multidrug resistance in cancer. Methods Mol. Biol. Clifton NJ 596, 47–76. https://doi.org/10.1007/978-1-60761416-6_4.

5. Conclusions In this study, we identified compound 479 from a novel 5-cyano-6phenylpyrimidin derivative as a bifunctional reversal agent against ABCB1- and ABCG2- mediated multidrug resistance. We found that MDR reversal by compound 479 was not due to the expression reduction or cellular localization of target protein, but caused by inhibiting the efflux function of ABC transporters and increasing intracellular accumulation of anticancer agents. Taken together, our work 10

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