Reduced catalytic activity of human CYP2C9 natural alleles for gliclazide: Molecular dynamics simulation and docking studies

• Published in: Biochimie 2011-06, Vol.93 (6), p.1028-1036
• Main Authors: Banu, Hussaina, Renuka, N., Vasanthakumar, Geetha
• Format: Article
• Language: English
• Subjects: Alleles; Aryl Hydrocarbon Hydroxylases - chemistry; Aryl Hydrocarbon Hydroxylases - genetics; Aryl Hydrocarbon Hydroxylases - metabolism; Binding Sites; Biocatalysis; Catalytic activity; Catalytic Domain; CYP2C92 and 3; Cytochrome P-450 CYP2C9; Docking simulation; Enzyme Stability; Genotype; Gliclazide; Gliclazide - chemistry; Gliclazide - metabolism; Heme - chemistry; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Hydroxylation; Hypoglycemic Agents - chemistry; Hypoglycemic Agents - metabolism; Molecular dynamics; Molecular Dynamics Simulation; Mutation; Natural alleles; Phenylalanine - chemistry; Protein Binding; Protein Structure, Tertiary
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2011.02.008

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. ► The size of the gliclazide access entry channel to the protein active site is significantly reduced in *3 and *2/*3 mutants. ► The distance between the substrate hydroxylation site and the heme is >5Å in *3 and *2/*3. ► The key residues, F100, F114 and F476, are absent in the interacting amino acid pocket of *3. ► Lack of stability of the active site residues R97 and R108 affects the stability of the heme and gliclazide in *2 and *3. ► Instabilities of heme and gliclazide at the active site affect their interaction, which in turn reduces the activity of mutants.