Regioselectivity of CYP2B6 : homology modelling, molecular dynamics simulation, docking

Abstract

Human cytochrome P450 (CYP) 2B6 activates the anticancer prodrug cyclophosphamide (CPA) by 4-hydroxylation. In contrast, the same enzyme catalyzes N-deethylation of a structural isomer, the prodrug ifos-famide (IFA) thus causing severe adverse drug effects. To model the molecular interactions leading to a switch in regioselectivity, the structure of CYP2B6 was modelled based on the structure of rabbit CYP2C5. We mod-elled the lacking 22 residue loop in CYP2C5 between helix F and G (F-G loop) which is not resolved in the X-ray structure by molecular dynamics (MD) simulations using a simulated annealing protocol. The modelled conformation of the loop was validated by unconstrained MD simulations of the complete enzymes (CYP2C5 and CYP2B6) in water for 70 and 120 ps, respectively. The simulations were stable and led to a backbone r.m.s. deviation of 1.7 Å between the two CYPs. The shape of the substrate binding site of CYP2B6 was further analyzed. It consists of three well-defined hydro-phobic binding pockets adjacent to the catalytic heme. Size, shape and hydrophobicity of these pockets was compared to the shapes of the two structurally isomeric substrates. In their preferred orientation in the binding site both substrates fill all three binding pockets without repulsive interactions. The distance to the heme iron is short enough for 4-hydroxylation and N-deethylation to occur for CPA and IFA, respectively. However, if the substrates are docked in the non-preferred orientation (such that 4-hydroxylation and N-deethylation would occur for IFA and CPA, respectively), one pocket is left empty, and clashes were observed between the substrates.