Mechanism of the decrease in catalytic activity of human cytochrome P450 2C9 polymorphic variants investigated by computational analysis

Abstract

Cytochrome P450 (CYP) is deeply involved in the metabolism of chemicals including pharmaceuticals. Therefore, polymorphisms of this enzyme have been widely studied to avoid unfavorable side effects of drugs in chemotherapy. In this work, we performed computational analysis of the mechanism of the decrease in enzymatic activity for three typical polymorphisms in CYP 2C9 species: *2, *3, and *5. Based on the equilibrated structure obtained by molecular dynamics simulation, the volume of the binding pocket and the fluctuation of amino residues responsible for substrate holding were compared between the wild type and the three variants. Further docking simulation was carried out to evaluate the appropriateness of the binding pocket to accommodate substrate chemicals. Every polymorphic variant was suggested to be inferior to the wild type in enzymatic ability from the structural viewpoint. F-G helices were obviously displaced outward in CYP2C9*2. Expansion of the binding pocket, especially the space near F' helix, was remarkable in CYP2C9*3. Disappearance of the hydrogen bond between K helix and β4 loop was observed in CYP2C9*5. The reduction of catalytic activity of those variants can be explained from the deformation of the binding pocket and the consequent change in binding mode of substrate chemicals. The computational approach is effective for predicting the enzymatic activity of polymorphic variants of CYP. This prediction will be helpful for advanced drug design because calculations forecast unexpected change in drug efficacy for individuals.

Prediction of the site of CYP3A4 metabolism of tolterodine by molecular dynamics simulation from multiple initial structures of the CYP3A4-tolterodine complex

“…The charges on the heme group and the tolterodine molecule were derived by quantum mechanical calculations carried out with Gaussian03 software [20] at the B3LYP/6-31G** level, and the charges were fitted to their respective atoms by means of the RESP method [21] in a manner similar to that described previously [22]. Each model of CYP3A4-tolterodine structure was inserted in a rectangular TIP3P water box [23] at a minimum distance of 18 Å from the box boundary.…”

“…MD simulations were performed with the PMEMD module of the Amber 12 software package in much the same way as described previously [22]. For CBZ, in an additional simulation after equilibration, we applied distance restraints to reflect the presence of a hydrogen bond between CBZ and CYP3A4.…”

High warfarin sensitivity in carriers of CYP2C9*35 is determined by the impaired interaction with P450 oxidoreductase

“…Alternatively, it has been suggested that this mutation may interfere with substrate binding via the expansion of the substrate-binding pocket and increased fluctuations of several important residues responsible for substrate holding, as observed in a previous molecular dynamics simulation study. 20 In conclusion, our data demonstrate that the Arg125Leu change in CYP2C9.35 strongly augments the modest inhibitory effect of the Arg144Cys mutation found in the carriers of CYP2C9*2 allelic variant. The in silico structural as well as the in vitro analyses suggest impaired functional CYP2C9-POR interaction as a major molecular mechanism behind such inhibitory effect.…”

“…[15][16][17][18][19] Thus, molecular dynamic simulations and subsequent substrate-docking simulations have been performed to clarify the mechanisms of enzymatic activity decrease in the different allelic variants of CYP2C9. 20 Amino-acid substitutions in these proteins were found to increase the fluctuation of the residues responsible for the substrate holding and alteration of the substrate-binding pocket. 20 Recently, we have described warfarin hypersensitivity in one Italian patient at the Centre of Haemostasis and Thrombosis of PTV (Policlinico Tor Vergata, Rome, Italy), who required only 2.25 mg warfarin per week to achieve therapeutic anticoagulation effect, a dose that is substantially below the normal prescription range.…”

“…20 Amino-acid substitutions in these proteins were found to increase the fluctuation of the residues responsible for the substrate holding and alteration of the substrate-binding pocket. 20 Recently, we have described warfarin hypersensitivity in one Italian patient at the Centre of Haemostasis and Thrombosis of PTV (Policlinico Tor Vergata, Rome, Italy), who required only 2.25 mg warfarin per week to achieve therapeutic anticoagulation effect, a dose that is substantially below the normal prescription range. 21 Sequencing of genes encoding enzymes involved in warfarin metabolism revealed a novel CYP2C9 allele designated as CYP2C9*35 (http://www.cypalleles.ki.se/).…”