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Exploring the Carbamazepine Interaction with Human Pregnane X Receptor and Effect on ABCC2 Using in Vitro and in Silico Approach

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Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

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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Abbreviations

ABC:

ATP-binding cassette

AEDs:

Antiepileptic drugs

CBZ:

Carbamazepine

CFDA:

Carboxyfluorescein diacetate

MD:

Molecular Dynamics

PXR:

Pregnane X receptor

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Acknowledgements

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.

Author information

Correspondence to Ritushree Kukreti.

Electronic supplementary material

Table S1

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Table S2

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Table S3

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Table S4

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Fig. S1

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)

Fig. S2

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)

Fig. S3

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)

Fig. S4

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)

Fig. S5

(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)

Fig. S6

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)

Fig. S7

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

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Key Words

  • ABCC2
  • carbamazepine
  • molecular dynamics simulation
  • pregnane xenobiotic receptor