CYP2C9: Typical substrates

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1 bing the structural basis for impaired drug metabolism associated with CYP2C9 poor metaboliser phenotype Flinders Medical Centre John. Miners PhD, DSc Dept of Clinical Pharmacology Flinders Medical Centre and Flinders University Adelaide, Australia CYP2C9: Typical substrates Antiasthmatics: montelukast, zafirlukast Anticoagulants: acenocoumarol, phenprocoumon, S-warfarin Anticonvulsants: phenytoin, valproic acid Antihypertensives: carvedilol, losartan Diuretic: torsemide Eicosanoids/fatty acids: arachidonic acid, linoleic acid NSAIDs: celecoxib, diclofenac, flurbiprofen, ibuprofen, meloxicam, naproxen.. ral hypoglycaemics: chlorpropamide, gliclazide, glipizide, tolbutamide. Statins: fluvastatin, rosuvastatin

2 CYP2C9: Atypical substrates PAH: dibenzo[a,h]anthracene Pesticides: disulfoton, methoxychlor Plant natural products: galangin, limonene, safrole N-demethylated drugs: amitriptyline, clozapine, fluoxetine. CYP2C9: Typical substrates Antiasthmatics: montelukast, zafirlukast Anticoagulants: acenocoumarol, phenprocoumon, S-warfarin Anticonvulsants: phenytoin, valproic acid Antihypertensives: carvedilol, losartan Diuretic: torsemide Eicosanoids/fatty acids: arachidonic acid, linoleic acid NSAIDs: celecoxib, diclofenac, flurbiprofen, ibuprofen, meloxicam, naproxen.. ral hypoglycaemics: chlorpropamide, gliclazide, glipizide, tolbutamide. Statins: fluvastatin, rosuvastatin

3 Frequency distribution of tolbutamide elimination rates Diabetes 28(1):41-51 (1978) Tolbutamide elimination in a poor metaboliser

4 Tolbutamide pharmacokinetics in a poor metaboliser Tolbutamide (CYP2C9) CL s = ml/min/kg ( ) t 1 / 2 = 39 hr (4.5 13) Theophylline (CYP1A2) CL S = ml/min/kg (0.670 ± 0.245) Debrisoquine (CYP2D6) MR = 0.68 Aust NZ J Med 15:348 (1985) Tolbutamide hydroxylation by CYP2C9 variants Biochem J 289:533 (1993)

5 Ile359Leu substitution is responsible for the CYP2C9 poor metaboliser phenotype The role of the CYP2C9-Leu 359 allelic variant in the tolbutamide polymorphism. Theresa Sullivan-Klose, Burhan Ghanayem, Douglas Bell, Zhi-Yi Zhang, Laurence Kaminsky, Gillian Shenfleld, John. Miners, Donald J. Birkett, Joyce A. Goldstein Tolbutamide undergoes hydroxylation in humans via a cytochrome P450-mediated pathway. The primary P450 isozyme responsible for metabolism is thought to be CYP2C9. Population studies have indicated the existence of slow metabolizers of tolbutamide (~1 in 500) suggesting a rare polymorphism associated with CYP2C9. Several allelic variants of CYP2C9 have been identified, however the effects of these allelic variations on metabolism in vivo is not established. In the present study, the coding regions, intron-exon junctions, and upstream region of CYP2C9 were amplified by PCR and sequenced in two slow metabolizers. ne individual was homozygous for Leu359 /Leu359 and the other individual was heterozygous for Arg/Cys144 and for Ile359/Leu359. No other genetic variations in CYP2C9 were detected in these individuals. PCR-RFLP showed that Arg144Tyr358Ile359Gly417 is the principal CYP2C9 allele. Frequencies of the rarer Leu359 and Cys144 alleles were 0.06 and 0.08, respectively, in a Caucasian-American population and and 0.01 respectively in African-Americans. The frequency of the Leu359 allele was in Chinese-Taiwanese, but the Cys144 allele was not detected in this population. Studies in a recombinant yeast expression system showed that the Leu359 variant had the highest K m and the lowest V max for hydroxylation of tolbutamide of all the CYP2C9 allelic variants. This allelic variant also had the highest K m for the 7-hydroxylation of S-warfarin. The present data suggest that the Leu359 allelic variant of CYP2C9 accounts for the occurrence of poor metabolizers of tolbutamide. Pharmacogenetics 6: (1996) Torsemide hydroxylation by CYP2C9 variants V (pmol/pmol P450/min) Torsemide (µm)

6 CYP2C9 Structure Recognition Sites From: Gotoh, J Biol Chem 267: 83 (1992) Crystal structure of CYP2C9 Ile359 is located 13.1Å from the heme Fe on helix-k

7 CYP2C9 Amino Acid Substitutions (position 359) C H 3 H H 3 C H Isoleucine (Ile) Leucine (Leu) CYP2C9 Amino Acid Substitutions (position 359) C H 3 H H 3 C H Isoleucine (Ile) Leucine (Leu) C H 3 H H H Valine (Val) Threonine (Thr)

8 CYP2C9 Amino Acid Substitutions (position 359) Small Aromatic Nucleophilic C H 3 H H H H Acidic Basic H H H H N H N H 2 H N H Hydrophobic Amide C H 3 S H H N H H 2 N H H Expression of CYP2C9 mutants

9 Spectral P450 content of CYP2C9 mutants Ile Leu Val Thr nmol CYP / mg protein CYP2C9 be Substrates H 3 C H 3 C S S CYP2C9 H N NADPH N Torsemide hylhydroxy torsemide H Cl H Cl CYP2C9 Cl NADPH Cl H Diclofenac 4'-Hydroxydiclofenac

10 K m values for torsemide hydroxylation by CYP2C9 mutants Ile Leu Val Thr K m (µm) V max values for torsemide hydroxylation by CYP2C9 mutants Ile Leu Val Thr V max (pmol /min/pmol CYP)

11 CL int values for torsemide hydroxylation by CYP2C9 mutants Ile Leu Val Thr CL int (µl/min.pmol CYP) K m values for diclofenac hydroxylation by CYP2C9 mutants Ile Leu Val Thr K m (µm)

12 V max values for diclofenac hydroxylation by CYP2C9 mutants Ile Leu Val Thr V max (pmol /min/pmol CYP) CL int values for diclofenac hydroxylation by CYP2C9 mutants Ile Leu Val Thr CL int (µl/min.pmol CYP)

13 Effect of the Ile359Leu substitution on CYP2C9 structure A B CYP2C9*1: edge-to-face π-stacking occurs between Tyr308 and Tyr358 (A). CYP2C9*3: 45º rotation in the Tyr308 sidechain perturbs π-stacking (B). CYP2C9*3: absence of β-branching creates a void volume for Tyr308 and Tyr358 to relax into (C). C Effect of the Ile359Leu substitution on CYP2C9 structure CYP2C9*1: Leu361 resides in SRS5 (K-L loop) 6.1Å from the Fe. CYP2C9*3: Leu361 is positioned 8.2Å from the heme Fe.

14 RCS overlay of 'typical' CYP2C9 structures RCS overlays with flurbiprofen. Sites of metabolism are shown in orange. From top left: naproxen, distance=0.71å; S- warfarin, distance=0.26å; tolbutamide, distance=1.57å; phenytoin, distance=0.75å. J Med Chem 51: (2008) RCS overlays with fluoxetine substrate amitriptyline clozapine tolterodine zopiclone distance Å * aligned yes yes yes yes * distance from site of fluoxetine N-demethylation to site of metabolism

15 Conclusions Position 359 is a critical site in CYP2C9 The presence of a β-branched amino acid at position 359 confers wild-type enzyme activity Substitution of leucine for isoleucine at position 359 causes conformational and spatial changes in helix-k, helix-i and SRS-5 CYP2C9 substrate selectivity may be predicted in silico Acknowledgments Donald Birkett Sandford Boye Benjamin Lewis David Elliot Matthew Sykes Grant support from the National Health and Medical Research Council of Australia