Over expression of ATP-binding cassette transporters is considered one of the major reasons for non-responsiveness to antiepileptic drugs. Carbamazepine (CBZ), one of first line antiepileptic drug is known to influence ABCC2 expression but its exact molecular mechanism is unknown.
We investigated the effect of CBZ on expression of ABCC2 and pregnane X receptor (PXR) in HepG2 cell line and compared with hyperforin (agonist of PXR) and ketoconazole (antagonist of PXR) through realtime PCR and western blot assay. Involvement of PXR was demonstrated through nuclear translocation and RNA interference and related effect of CBZ on ABCC2 through functional activity assay. Molecular docking and dynamic simulation approach was used to understand the interaction of CBZ with PXR.
CBZ and hyperforin increased the PXR and ABCC2 expression whereas reversed when present it in combination with ketoconazole. Experiments confirmed CBZ induced ABCC2 expression is PXR dependent. Molecular dynamic (MD) simulation and in vitro experiment indicated possibility of CBZ to be PXR agonist and PXR residue Gln285 to be important for CBZ-PXR interaction.
CBZ alters the functional activity of ABCC2 through PXR, which in turn can interfere with therapy. Mutational analysis of residues revealed the importance of Gln285 in ligand interaction.
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Pregnane X receptor
Wright J, Pickard N, Whitfield A, Hakin N. A population-based study of the prevalence, clinical characteristics and effect of ethnicity in epilepsy. Seizure. 2000;9(5):309–13.
Kwan P, Brodie MJ. Early identification of refractory epilepsy. N Engl J Med. 2000;342(5):314–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10660394
Loscher W, Potschka H. Drug resistance in brain diseases and the role of drug efflux transporters. Nat Rev Neurosci. 2005;6(8):591–602.
Löscher W, Potschka H. Role of drug efflux transporters in the brain for drug disposition and treatment of brain diseases. Prog Neurobiol. 2005;76:22–76. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16011870
Potschka H, Fedrowitz M, Löscher W. Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity. J Pharmacol Exp Ther. 2003;306(1):124–31.
Potschka H, Fedrowitz M, Löscher W. Brain access and anticonvulsant efficacy of carbamazepine, lamotrigine, and Felbamate in ABCC2/MRP2-deficient TR-rats. Epilepsia. 2003;44(12):1479–86.
Gilibili RR, Chatterjee S, Bagul P, Mosure KW, Murali BV, Mariappan TT, et al. Coproporphyrin-I: A fluorescent, endogenous optimal probe substrate for ABCC2 (MRP2) that is suitable for vesicle based MRP2 inhibition assay. Drug Metab Dispos. 2017; 8540:dmd.116.074740. Available from: http://dmd.aspetjournals.org/lookup/doi/10.1124/dmd.116.074740
Zamek-Gliszczynski MJ, Hoffmaster KA, Tweedie DJ, Giacomini KM, Hillgren KM. Highlights from the International Transporter Consortium second workshop. Clin Pharmacol Ther. 2012; 92(5):553–556. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23085880
Hillgren KM, Keppler D, Zur a a, Giacomini KM, Stieger B, Cass CE, et al. Emerging transporters of clinical importance: an update from the International Transporter Consortium. Clin Pharmacol Ther. 2013;94(1):52–63. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23588305
Nies AT, Keppler D. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch - Eur J Physiol. 2007;453:643–59.
Ye ZW, Camus S, Augustijns P, Annaert P. Interaction of eight HIV protease inhibitors with the canalicular efflux transporter ABCC2 (MRP2) in sandwich-cultured rat and human hepatocytes. Biopharm Drug Dispos. 2010;31(2–3):178–88.
Ufer M, von Stülpnagel C, Muhle H, Haenisch S, Remmler C, Majed A, et al. Impact of ABCC2 genotype on antiepileptic drug response in Caucasian patients with childhood epilepsy. Pharmacogenet Genomics. 2011;21(10):624–30.
Yi JH, Cho Y-J, Kim W-J, Lee MG, Lee JH. Genetic variations of ABCC2 Gene associated with adverse drug reactions to Valproic acid in Korean epileptic patients. Genomics Inform. 2013;11(4):254–62. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3897854&tool=pmcentrez&rendertype=abstract
Courtois A, Payen L, Guillouzo A, Fardel O. Up-regulation of multidrug resistance-associated protein 2 (MRP2) expression in rat hepatocytes by dexamethasone. FEBS Lett. 1999;459(3):381–5. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10526169
Fromm MF, Kauffmann HM, Fritz P, Burk O, Kroemer HK, Warzok RW, et al. The effect of rifampin treatment on intestinal expression of human MRP transporters. Am J Pathol. 2000;157(5):1575–80. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1885746&tool=pmcentrez&rendertype=abstract
Kim W-J, Lee JH, Yi J, Cho Y-J, Heo K, Lee SH, et al. A nonsynonymous variation in MRP2/ABCC2 is associated with neurological adverse drug reactions of carbamazepine in patients with epilepsy. Pharmacogenet Genomics. 2010;20:249–56. Available from: https://www.ncbi.nlm.nih.gov/books/NBK98215/
Oscarson M, Zanger UM, Rifki OF, Klein K, Eichelbaum M, Meyer UA. Transcriptional profiling of genes induced in the livers of patients treated with carbamazepine. Clin Pharmacol Ther. 2006;80(5):440–56.
Giessmann T, May K, Modess C, Wegner D, Hecker U, Zschiesche M, et al. Carbamazepine regulates intestinal P-glycoprotein and multidrug resistance protein MRP2 and influences disposition of talinolol in humans. Clin Pharmacol Ther. 2004;76(3):192–200.
Luo G, Cunningham M, Kim S, Burn TIM, Lin J, Sinz M, et al. Cyp3a4 induction by drugs : correlation between a Pregnane X receptor reporter Gene assay and Cyp3a4 expression in human hepatocytes. Dent Abstr. 2002;30(7):795–804.
Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, et al. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J Biol Chem. 2002;277(4):2908–15.
Jones SA, Moore LB, Shenk JL, Wisely GB, Hamilton GA, McKee DD, et al. The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol Endocrinol. 2000;14(1):27–39.
Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, Williams SP, et al. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science. 2001;292(5525):2329–2333. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11408620
Chen Y, Tang Y, Guo C, Wang J, Boral D, Nie D. Nuclear receptors in the multidrug resistance through the regulation of drug-metabolizing enzymes and drug transporters. Biochem Pharmacol. 2012;83(8):1112–26. England
Johannessen S. Therapeutic drug monitoring of antiepileptic drugs. In: Hempe G, Smith RM, editors. Handbook of analytical separation, vol. 5. Amsterdan: Elsevier; 2004. p. 221–53.
Albert D, Zündorf I, Dingermann T, Müller WE, Steinhilber D, Werz O. Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase. Biochem Pharmacol. 2002;64(12):1767–75.
Huang H, Wang H, Sinz M, Zoeckler M, Staudinger J, Redinbo MR, et al. Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole. Oncogene. 2007;26(2):258–68.
Di Salvo A, Dugois P, Tandeo D, Peltekian M, Kong Thoo Lin P. Synthesis, cytotoxicity and DNA binding of oxoazabenzo[de]anthracenes derivatives in colon cancer Caco-2 cells. Eur J Med Chem. 2013;69:754–61. France
Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 2001;29(9):e45.
Lebedeva IV, Pande P, Patton WF. Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters. PLoS One. 2011;6(7):e22429.
Maestro. version 9.2; Schrodinger, Inc.: New York. New York: Schrodinger, Inc.; 2011. Available from: http://www.schrodinger.com.
Schrodinger Suite 2011; Protein Preparation Wizard; Epik version 2.2, Schrodinger, LLC, New York, NY, 2011; Impact version 5.7, Schrodinger, LLC, New York, NY, 2011; Prime version 3.0, Schrodinger, LLC, New York, NY, 2011. Available from: http://www.schrodinger.com.
LigPrep. version 2.5; Schrodinger, Inc.: New York, NY. 2011. Available from: http://www.schrodinger.com.
Shelley JC, Cholleti A, Frye LL, Greenwood JR, Timlin MR, Uchimaya M. Epik: a software program for pKa prediction and protonation state generation for drug-like molecules. J Comput Aided Mol Des. 2007;21(12):681–91.
Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, et al. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem. 2004;47(7):1750–9.
Desmond Molecular Dynamics System (2011) Desmond Molecular Dynamics System, Version 3.0, D.E. Shaw Research, New York, NY, Maestro–Desmond Interoperability Tools, Version 3.0, Schrödinger, New York, NY. Available from: http://www.schrodinger.com.
Berendsen HJC, Postma JPM, van Gunsteren WF, Hermans J. Interaction models for water in relation to protein hydration. In: Intermolecular Forces. 1981. p. 331–42. Available from: http://link.springer.com/10.1007/978-94-015-7658-1_21.
Massova I, Kollman PA. Computational alanine scanning to probe protein-protein interactions: a novel approach to evaluate binding free energies. J Am Chem Soc. 1999;121(36):8133–43.
Moreira IS, Fernandes PA, Ramos MJ. Computational alanine scanning mutagenesis - an improved methodological approach. J Comput Chem. 2007;28(3):644–54.
Lyne PD, Lamb ML, Saeh JC. Accurate prediction of the relative potencies of members of a series of kinase inhibitors using molecular docking and MM-GBSA scoring. J Med Chem United States. 2006 Aug;49(16):4805–8.
Das D, Koh Y, Tojo Y, Ghosh AK, Mitsuya H. Prediction of potency of protease inhibitors using free energy simulations with polarizable quantum mechanics-based ligand charges and a hybrid water model. J Chem Inf Model. 2009;49(12):2851–62.
Koh Y, Das D, Leschenko S, Nakata H, Ogata-Aoki H, Amano M, et al. GRL-02031, a novel nonpeptidic protease inhibitor (PI) containing a stereochemically defined fused cyclopentanyltetrahydrofuran potent against multi-PI-resistant human immunodeficiency virus type 1 in vitro. Antimicrob Agents Chemother. 2009;53(3):997–1006.
Prime. 2014, Version 3.5 Schrodinger New York; 2014. Available from: http://www.schrodinger.com
Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit-Singh CJ, Willson TM, et al. St. John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci United States Am. 2000;97(13):7500–2. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC16574/pdf/pq007500.pdf
Li H, Dou W, Padikkala E, Mani S. Reverse yeast two-hybrid system to identify mammalian nuclear receptor residues that interact with ligands and/or antagonists. J Vis Exp. 2013;81:e51085.
Haenisch S, Laechelt S, Bruckmueller H, Werk A, Noack A, Bruhn O, et al. Down-regulation of ATP-binding cassette C2 protein expression in HepG2 cells after rifampicin treatment is mediated by microRNA-379. Mol Pharmacol. 2011;80(2):314–20. Available from. http://www.ncbi.nlm.nih.gov/pubmed/21540293
Alqahtani S, Mohamed LA, Kaddoumi A. Experimental models for predicting drug absorption and metabolism. Expert Opin Drug Metab Toxicol, Available from. 2013;9(10):1241–54. http://www.ncbi.nlm.nih.gov/pubmed/23687990
Korzekwa KR, Krishnamachary N, Shou M, Ogai A, Parise RA, Rettie AE, et al. Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites. Biochemistry. 1998;37(12):4137–47.
Kliewer SA, Goodwin B, Willson TM. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev. 2002;23(5):687–702.
Watkins RE, Maglich JM, Moore LB, Wisely GB, Noble SM, Davis-searles PR, et al. 2. 1 Å crystal structure of human PXR in complex with the St. John ’ s wort. Biochemistry. 2003;42:1430–8.
Li H, Redinbo MR, Venkatesh M, Ekins S, Chaudhry A, Bloch N, et al. Novel yeast-based strategy unveils antagonist binding regions on the nuclear xenobiotic receptor PXR. J Biol Chem. 2013;288(19):13655–68.
Chrencik JE, Orans J, Moore LB, Xue Y, Peng L, Collins JL, et al. Structural disorder in the complex of human pregnane X receptor and the macrolide antibiotic rifampicin. Mol Endocrinol. 2005;19(5):1125–34.
Watkins RE, Davis-Searles PR, Lambert MH, Redinbo MR. Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor. J Mol Biol. 2003;331(4):815–28.
Ostberg T, Bertilsson G, Jendeberg L, Berkenstam A, Uppenberg J. Identification of residues in the PXR ligand binding domain critical for species specific and constitutive activation. Eur J Biochem Germany. 2002 Oct;269(19):4896–904.
Ngan C-H, Beglov D, Rudnitskaya AN, Kozakov D, Waxman DJ, Vajda S. The structural basis of pregnane X receptor binding promiscuity. Biochemistry. 2009;48(48):11572–81. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2789303&tool=pmcentrez&rendertype=abstract
Nguyen TD, Markova S, Liu W, Gow JM, Baldwin RM, Habashian M, et al. Functional characterization of ABCC2 promoter polymorphisms and allele-specific expression. Pharm J. 2013;13(5):396–402. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3435480&tool=pmcentrez&rendertype=abstract
Fujita K, Nagashima F, Yamamoto W, Endo H, Sunakawa Y, Yamashita K, et al. Association of ATP-binding cassette, sub-family C, number 2 (ABCC2) genotype with pharmacokinetics of irinotecan in Japanese patients with metastatic colorectal cancer treated with irinotecan plus infusional 5-fluorouracil/leucovorin (FOLFIRI). Biol Pharm Bull, Available from. 2008;31(11):2137–42. http://www.ncbi.nlm.nih.gov/pubmed/18981587
Innocenti F, Kroetz DL, Schuetz E, Dolan ME, Ram Rez J, Relling M, et al. Comprehensive pharmacogenetic analysis of irinotecan neutropenia and pharmacokinetics. J Clin Oncol. 2009;27(16):2604–14.
de Jong FA, Scott-Horton TJ, Kroetz DL, McLeod HL, Friberg LE, Mathijssen RH, et al. Irinotecan-induced diarrhea: functional significance of the polymorphic ABCC2 transporter protein. Clin Pharmacol Ther United States. 2007;81(1):42–9.
Haenisch S, May K, Wegner D, Caliebe A, Cascorbi I, Siegmund W. Influence of genetic polymorphisms on intestinal expression and rifampicin-type induction of ABCC2 and on bioavailability of talinolol. Pharmacogenet Genomics. 2008;18(4):357–65. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18334920
Moriya Y, Nakamura T, Horinouchi M, Sakaeda T, Tamura T, Aoyama N, et al. Effects of polymorphisms of MDR1, MRP1, and MRP2 genes on their mRNA expression levels in duodenal enterocytes of healthy Japanese subjects. Biol Pharm Bull. 2002;25(10):1356–9. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12392094
Ufer M, Mosyagin I, Muhle H, Jacobsen T, Haenisch S, Häsler R, et al. Non-response to antiepileptic pharmacotherapy is associated with the ABCC2 -24C>T polymorphism in young and adult patients with epilepsy. Pharmacogenet Genomics. 2009;19(5):353–62.
Nishioka C, Sakaeda T, Nakamura T, Moriya Y, Okamura N, Tamura T, et al. Chemosensitivity in Japanese patients with colorectal adenocarcinomas. Rev Lit Arts Am. 2005;50(6):181–8.
Meyer Zu Schwabedissen HE, Jedlitschky G, Gratz M, Haenisch S, Linnemann K, Fusch C, et al. Variable expression of MRP2 (ABCC2) in human placenta: influence of gestational age and cellular differentiation. Drug Metab Dispos. 2005;33(7):896–904.
Krusekopf S, Roots I. St. John’s wort and its constituent hyperforin concordantly regulate expression of genes encoding enzymes involved in basic cellular pathways. Pharmacogenet Genomics. 2005;15(11):817–29.
Maglich JM, Stoltz CM, Goodwin B, Hawkins-Brown D, Moore JT, Kliewer SA. Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Mol Pharmacol. 2002;62(3):638–46. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12181440
Takeshita A, Taguchi M, Koibuchi N, Ozawa Y. Putative role of the orphan nuclear receptor SXR (steroid and xenobiotic receptor) in the mechanism of CYP3A4 inhibition by xenobiotics. J Biol Chem. 2002;277(36):32453–8.
Das BC, Madhukumar AV, Anguiano J, Kim S, Sinz M, Zvyaga TA, et al. Synthesis of novel ketoconazole derivatives as inhibitors of the human Pregnane X receptor (PXR; NR1I2; also termed SXR, PAR). Bioorg Med Chem Lett. 2008;18(14):3974–7.
Novotná A, Krasulová K, Bartoňková I, Korhoňová M, Bachleda P, Anzenbacher P, et al. Dual effects of ketoconazole cis-enantiomers on CYP3A4 in human hepatocytes and HepG2 cells. PLoS One. 2014;9(10):1–8.
Lee G, Piquette-miller M, De L. Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells. 2001;884:876–84.
Kalitsky-Szirtes J, Shayeganpour A, Brocks DR, Piquette-Miller M. Suppression of drug-metabolizing enzymes and efflux transporters in the intestine of endotoxin-treated rats. Drug Metab Dispos. 2004;32(1):20–7.
Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, et al. An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell. 1998;92(1):73–82.
Beutler AS, Li S, Nicol R, Walsh MJ. Carbamazepine is an inhibitor of histone deacetylases. Life Sci. 2005;76(26):3107–15.
Limbird L. Cell surface receptors: a short course on theory and. Methods. 2012:81–2.
Vanden Heuvel JP, Perdew GH, Mattes WB, editors. Vol. 14, Cellular and Molecular Toxicology. Gulf Professional Publishing; 2002. 646 p. Available from: https://www.elsevier.com/books/cellular-and-molecular-toxicology/vanden-heuvel/978-0-444-50868-3
Petrovic V, Teng S, Piquette-Miller M. Regulation of drug transporters during infection and inflammation. Mol Interv [Internet]. 2007;7(2):99–111. http://triggered.clockss.org/ServeContent?url=http%253A%252F%252Fmolinterv.aspetjournals.org%252Fcontent%252F7%252F2%252F99.full
Langmade SJ, Gale SE, Frolov A, Mohri I, Suzuki K, Mellon SH, et al. Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann-pick C disease. Proc Natl Acad Sci U S A 2006;103(37):13807–13812. http://www.scopus.com/inward/record.url?eid=2-s2.0-33748793667&partnerID=40&md5=51f9e02b9d4c1f28bef678af855e0af0%5Cnhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1564205/pdf/zpq13807.pdf.
Marchi N, Hallene KL, Kight KM, Cucullo L, Moddel G, Bingaman W, et al. Significance of MDR1 and multiple drug resistance in refractory human epileptic brain. BMC Med. 2004;2:37. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=524356&tool=pmcentrez&rendertype=abstract
We thank the Director, Dr. Rajesh Gokhale, Institute of Genomics and Integrative Biology (IGIB) and Dr. Shantanu Chowdhury for their motivation and unconditional support. We also acknowledge Dr. Rakesh Sharma, Dr. Neeru Saini and Dr. Lipi Thukral for valuable suggestions for this work. Financial support from Council of Scientific and Industrial Research (CSIR), Govt. of India is duly acknowledged. GKG and SK acknowledges DBT, Govt. of India, NK acknowledges CSIR (BSC0123) and CR acknowledge UGC, Govt. of India for providing fellowship.
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Cell viability Test. MTT assay was performed with A,D) carbamazepine, B,E) hyperforin and C, F) ketoconazole in HepG2 and Huh7 cells. Cells (10,000 cells/well-HepG2; 5000 cells/well-Huh7) were plated in 96-well plates for 24 h and subsequently treated with varying concentration of drugs for 72 h. Data represents the mean ± S.D. of five (n = 5) independent experiments. Statistical significance (***, p < 0.001, **, p < 0.01, *, p < 0.05) was determined using students t test. (DOCX 3627 kb)
Dose dependent effect of carbamazepine (CBZ), hyperforin (HYP) on mRNA level of PXR, ABCC2, ABCB1 (positive control) in HepG2 cells. Real-time RT –PCR analysis of total mRNA isolated from cells treated with (A) CBZ, (B) HYP for 24 h. The changes in level of PXR, ABCC2 and ABCB1 mRNA were normalized with Beta-Microgloublin. Statistical significance (***, p < 0.001, **, p < 0.01, *, p < 0.05) was determined using students t test by comparing CBZ or HYP with respect to VC (vehicle control, 0.1% DMSO). The data are the means ± S.D. of atleast (n = 5) independent real time PCR results. (DOCX 3627 kb)
Dose dependent effect of carbamazepine (CBZ) and hyperforin (HYP) and on protein expression of PXR and ABCC2 in HepG2 cells. Immunoblot analysis of whole cell lysates of HepG2 treated with (A) CBZ, (B) HYP (C) 2 μM HYP + 42 μM CBZ in combination were performed using monoclonal antibodies against human PXR and ABCC2 and Vinculin for 72 h. The bands of Vinculin were measured to normalise the results and expressed as normalized fold change over VC (vehicle control, 0.1% DMSO). Data shown are means ± S.D. from atleast (n = 4) independent experiments. Statistical significance (***, p < 0.001, **, p < 0.01, *, p < 0.05) was determined using Students t test compared with VC. (DOCX 3628 kb)
Competition ligand binding assay. Graph shows percentage displacement of SR12813 by carbamazepine (CBZ), hyperforin (HYP) and ketoconazole (KC) obtained by TR-FRET ligand binding assay. (DOCX 3628 kb)
(A) RMSD of Cα – PXR monitored during 50 ns MD simulation. (B) RMSF profile of PXR with three ligands. RMSD profile revealed that PXR protein complex showed stability during the entire run with minor fluctuation when bound with CBZ, hyperforin and ketoconazole around 3.5 Å (Fig.S5A). RMSF profile showed CBZ and hyperforin bound complexes fluctuated more when compared to PXR-ketoconazole complex. Active site residues Ser208, Ser247, Cys284, Gln285, Glu321, His327, His407 and Arg410 were having minimal fluctuations (Fig. S5B). (DOCX 3628 kb)
Matrices showing smallest distances between amino acid residue pairs. (A) Apo PXR, (B) PXR-CBZ, (C) PXR-hyperforin, (D) PXR-ketoconazole. Residue-residue distances were calculated to analyse the temporal changes of ligand binding residues. The distance matrix maps are represented in fig. S5 A, B, C, D. The distance between the residues 342–392 and 192–242 decreases upon ligand binding. This decrease was more pronounced on binding of ketoconazole as shown in fig. S5. The PXR-ketoconazole complex has more tightly packed residues between 192–242 (Helix 2, 3 and 4) and 342–392 (Helix 10, 11 and 12) as compared to the complex formed by CBZ and hyperforin. Additionally, closer packing of residues 342–392 and 392–440 was also observed. On the other hand, the residues 180–190 and 292–342 (Helix 8, 9 and 10) are closer in PXR-CBZ and PXR-hyperforin complexes than PXR–ketoconazole complex. Overall, these observations indicate conformational change of PXR in presence of CBZ and hyperforin is similar but different on ketoconazole binding. (DOCX 3628 kb)
Dose dependent effect of carbamazepine (CBZ) on mRNA level of ABCC2 in HepG2 cells. Real-time RT –PCR analysis of total mRNA isolated from cells treated with different doses of CBZ (21-168 μM) for 24 h. The changes in level of ABCC2 mRNA were normalized with Beta-Microgloublin. Statistical significance (***, p < 0.001, **, p < 0.01, *, p < 0.05) was determined using students t test by comparing CBZ with respect to VC (vehicle control, 0.1% DMSO). The data are the means ± S.D. of atleast (n = 5) independent real time PCR results. (DOCX 3628 kb)
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Grewal, G.K., Singh, K.D., Kanojia, N. et al. Exploring the Carbamazepine Interaction with Human Pregnane X Receptor and Effect on ABCC2 Using in Vitro and in Silico Approach. Pharm Res 34, 1444–1458 (2017). https://doi.org/10.1007/s11095-017-2161-z
- molecular dynamics simulation
- pregnane xenobiotic receptor