On the human CYP2C9*13 variant activity reduction: a molecular dynamics simulation and docking study
Introduction
Cytochrome P450s (CYPs) are the most important superfamily of biotransformation enzymes that are involved in oxidative metabolism of a wide variety of endogenous and exogenous compounds [1], [2], [3]. The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism and plays a key role in the drugs pharmacological and toxicological effects [4]. CYP2C9 is the most abundant 2C subfamily isozyme in human liver which is responsible for the metabolic clearance of a wide variety of the therapeutic agents, estimated up to 16% of drugs in current clinical used [5].
The human CYP2C9 gene is highly polymorphic. At least 24 CYP2C9 alleles have been identified to date and most of them show reduced activity (http://www.imm.ki.se/CYPalleles/cyp2c9.htm). Some narrow therapeutic index drugs, of which very small changes in the dosage level could cause toxic results, may lead more dangerous to the individuals who carries mutant CYP2C9 allele. Numerous studies have been performed in vitro and in vivo to evaluate the influence of CYP2C9 genotypes on metabolic activity and drug disposition [5], [6], [7], [8], [9], [10], [11]. How these different alleles influence enzymatic activity has attracted much interest.
A CYP2C9 allele designated CYP2C9*13 has been identified in a Chinese poor metabolizer of lornoxicam [12]. This allele involves a T269C transversion in exon 2 that leads to a Leu90Pro substitution in the encoded protein, with an allele frequency of about 1.02% in the Chinese population [12] and 0.6% in the Korea population [13]. Two classical substrates, lornoxicam and diclofenac, were chosen to measure the catalytic activity of CYP2C9 [7], [8], [14]. Results of the kinetic experiments showed that CYP2C9*13 had lower intrinsic clearance than CYP2C9*1 due to the increase in Michaelis–Menten constant (Km) and decrease in maximal reaction velocity (Vmax) and the metabolic impact of CYP2C9*13 depends on the substrate being metabolized (Table 1). The mutant CYP2C9 allele which has lower activity is associated with increased risk of adverse drug events. Therefore, it is of significance to find out the reasons for the decreasing activity of CYP2C9*13 for clinic therapy.
CYP2C9 is the first human P450 isozyme that the X-ray crystal structures both unliganded and in complex with warfarin has been determined by Williams et al. [15] (PDB code 1OG2/1OG5). Then, Wester et al. [16] determined the structure of CYP2C9 complexed with flurbiprofen (PDB code 1R9O). The X-ray crystal structures can help to understand the structure–function relationship of CYP2C9, but the relationship between structure and lower catalytic activity of its natural allelic variant CYP2C9*13 are still unclear.
Before CYP2C9 crystal structure was determined, many research groups [17], [18], [19], [20] had used the method of homology modeling based on the X-ray crystal structure of CYP2C5 etc. to construct the three-dimensional (3D) model of CYP2C9. In this study, 3D structures of substrate-free CYP2C9*1 and *13 were constructed based on the X-ray crystal structure of wild-type CYP2C9 complexes with flurbiprofen (PDB code: 1R9O). They were then used to characterize the explicit enzyme complexes with two structurally and chemically diverse substrates, lornoxicam and diclofenac, in order to identify the most important residues in CYP2C9*13 for binding the two substrates.
Section snippets
Methods
All molecular modeling studies were performed on SGI O3800 workstation using Gaussian 03 [21] and the InsightII software package developed by Accelrys [22]. The consistent-valence force field (CVFF) was used for energy minimization and molecular dynamics (MD) simulations.
3D structures of CYP2C9*1 and *13
Williams et al. [15] and Wester et al. [16] obtained the X-ray structure of CYP2C9 successively. In this work, 1R9O, the result of Wester et al., has been used as the initial structure in further simulations, for its better resolution and less residues has been substituted in the crystal structure. The trans/cis configuration of the bond between Asp89 and Pro90 in CYP2C9*13 has not been determined in experiments. In this work, besides the substrate-free 3D model of CYP2C9*1, two 3D models of
Conclusion
CYP2C9 polymorphisms are associated with increasing the risk of adverse drug events and arouse an extensive research. A mutant allele, CYP2C9*13, which has a Leu90Pro amino acid substitution has been identified by some of the present authors [12]. Consequent kinetic study in vitro indicates that this variant of CYP2C9 has less active in eliminating diclofenac and lornoxicam by the increase of Km and the decrease of Vmax depending on the substrate. For investigating the mechanism of the
Acknowledgments
This work was supported by the National Science Foundation of China (20333050, 20073014, 30472062), Doctor Foundation by the Ministry of Education, Foundation for University Key Teacher by the Ministry of Education, Key Subject of Science and Technology by the Ministry of Education of China, and Key Subject of Science and Technology by Jilin Province.
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