title: Reduced catalytic activity of human CYP2C9 natural alleles for gliclazide: molecular dynamics simulation and docking studies.
abstract: Amongst sulfonylureas, gliclazide is one of the mostly prescribed drugs to diabetic patients and is metabolized extensively by P450 CYP2C9. Among 24-CYP2C9 alleles, the *2/*2 and *3/*3 genotypes showed significantly lower gliclazide clearances with reductions of 25 and 57%, respectively. However, the reason for the change in drug-metabolizing activity induced by these natural alleles is unknown. In the present study, we used molecular dynamics simulation and autodocking studies to provide models for gliclazide-bound complexes of CYP2C9*2, *3 and *2/*3 mutants, which give insight into CYP2C9-gliclazide interactions and explain the reduced enzymatic activity seen in these variants. Our data shows that the size of the substrate-access entry site is significantly reduced in mutants, which limits the access of gliclazide to heme and the active site. The distance from the substrate oxidation site and heme is >5Å in *3 and *2/*3. Therefore, the addition of an active oxygen molecule by heme-Fe is hindered. The absence of F100, F114 and F476 in the interacting amino acid pocket in *3 reduces catalytic efficiency toward gliclazide. In *1, gliclazide is stabilized by the formation of two hydrogen bonds with R108 while it is absent in mutants. Further in *3 and *2/*3, the key heme-stabilizing residue, R97 stabilization is greatly reduced. Therefore, the decreased catalytic activity of these variants can be explained from the reduced access of the gliclazide to heme, and the interaction between heme and substrate is affected due to their instability in the active site.
authors.affiliation: Department of Biotechnology, Manipal University, Dubai, United Arab Emirates.
authors.forename: Hussaina
authors.initials: H
authors.lastname: Banu
authors.forename: N
authors.initials: N
authors.lastname: Renuka
authors.forename: Geetha
authors.initials: G
authors.lastname: Vasanthakumar
mesh_terms: D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D065729:Cytochrome P-450 CYP2C9; D004795:Enzyme Stability; D005838:Genotype; D005907:Gliclazide / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D006900:Hydroxylation; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D007004:Hypoglycemic Agents; D006418:Heme; D010649:Phenylalanine; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D005907:Gliclazide