Integrated in silico-in vitro strategy for addressing cytochrome P450 3A4 time-dependent inhibition.

Throughout the past decade, the expectations from the regulatory agencies for safety, drug-drug interactions (DDIs), pharmacokinetic, and disposition characterization of new chemical entities (NCEs) by pharmaceutical companies seeking registration have increased. DDIs are frequently assessed using in silico, in vitro, and in vivo methodologies. However, a key gap in this screening paradigm is a full structural understanding of time-dependent inhibition (TDI) on the cytochrome P450 systems, particularly P450 3A4.

Use of simple docking methods to screen a virtual library for heteroactivators of cytochrome P450 2C9.

Several laboratories have demonstrated that activation of drug metabolism by P450s may occur via a mechanism that resembles allosterism from an enzyme kinetic standpoint. Because the effector drug binding site may be located in the same P450 binding pocket where the drug substrate is located, the ability to find and characterize novel effectors (aka heteroactivators) will prove to be important in probing the mechanism of activation. We have used analogues of the prototypical CYP2C9 heteroactivator dapsone to validate a simple docking method that can be used to predict heteroactivators based on ligand binding location in a P450 crystal structure.

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9.

The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface.