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Functional and structural characterisation of common cytochrome P450 2D6 allelic variants—roles of Pro34 and Thr107 in catalysis and inhibition

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Abstract

One major source of inter-individual variability in drug pharmacokinetics is genetic polymorphism of the cytochrome P450 (CYP) genes. This study aimed to elucidate the enzyme kinetic and molecular basis for altered activity in three major alleles of CYP2D6, namely CYP2D6*2, CYP2D6*10 and CYP2D6*17. The E. coli-expressed allelic variants were examined using substrate (venlafaxine and 3-cyano-7-ethoxycoumarin[CEC]) and inhibitor (quinidine, fluoxetine, paroxetine, terbinafine) probes in enzyme assays as well as molecular docking. The kinetics data indicated that R296C and S486T mutations in CYP2D6*2 have caused enhanced ligand binding (enhanced intrinsic clearance for venlafaxine and reduced IC50 for quinidine, paroxetine and terbinafine), suggesting morphological changes within the active site cavity that favoured ligand docking and binding. Mutations in CYP2D6*10 and CYP2D6*17 tended to cause deleterious effect on catalysis, with reduced clearance for venlafaxine and CEC. Molecular docking indicated that P34S and T107I, the unique mutations in the alleles, have negatively impacted activity by affecting ligand access and binding due to alteration of the substrate access channel and active site morphology. IC50 values however were quite variable for quinidine, fluoxetine and terbinafine, and a general decrease in IC50 was observed for paroxetine, suggesting ligand-specific altered susceptibility to inhibition in the alleles. This study indicates that CYP2D6 allele selectivity for ligands was not solely governed by changes in the active site architecture induced by the mutations, but that the intrinsic properties of the substrates and inhibitors also played vital role.

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Funding

This work was supported by the Monash University Malaysia (Monash Seed Grant under the Bioactive Compounds Research Strength), the Ministry of Science, Technology & Innovation (grant no. 02-02-10-SF0077), as well as the Ministry of Higher Education (grant no. FRGS/1/2014/SKK03/MUSM/02/1).

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Authors and Affiliations

  1. School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor, Malaysia

    Amelia Nathania Dong & Nafees Ahemad

  2. Department of Biomedical Science, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia

    Yan Pan

  3. Jeffrey Cheach School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor, Malaysia

    Uma Devi Palanisamy

  4. School of Pharmacy, International Medical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000, Kuala Lumpur, Malaysia

    Beow Chin Yiap & Chin Eng Ong

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  1. Amelia Nathania Dong
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  2. Nafees Ahemad
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  3. Yan Pan
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  4. Uma Devi Palanisamy
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Contributions

CEO, YP and UDP designed and conceptualised the research. AND, NA and BCY conducted and analysed the experiments. AND, NA and CEO drafted the manuscript and revised it for critically important intellectual content. All authors approved the final version of the manuscript.

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Correspondence to Chin Eng Ong.

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Dong, A.N., Ahemad, N., Pan, Y. et al. Functional and structural characterisation of common cytochrome P450 2D6 allelic variants—roles of Pro34 and Thr107 in catalysis and inhibition. Naunyn-Schmiedeberg's Arch Pharmacol 392, 1015–1029 (2019). https://doi.org/10.1007/s00210-019-01651-0

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