ABSTRACT
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. To address this, a number of high-throughput in vitro assays have been developed. This work describes an automated assay for TDI using two concentrations at two time points (2 + 2 assay). Data generated with this assay for over 2000 compounds from multiple therapeutic programs were used to generate in silico Bayesian classification models of P450 3A4-mediated TDI. These in silico models were validated using several external test sets and multiple random group testing (receiver operator curve value >0.847). We identified a number of substructures that were likely to elicit TDI, the majority containing indazole rings. These in vitro and in silico approaches have been implemented as a part of the Pfizer screening paradigm. The Bayesian models are available on the intranet to guide synthetic strategy, predict whether a NCE is likely to cause a TDI via P450 3A4, filter for in vitro testing, and identify substructures important for TDI as well as those that do not cause TDI. This represents an integrated in silico-in vitro strategy for addressing P450 3A4 TDI and improving the efficiency of screening.
Subject(s)
Cytochrome P-450 CYP3A Inhibitors , Cytochrome P-450 CYP3A/metabolism , Enzyme Inhibitors/pharmacology , Microsomes, Liver/metabolism , Enzyme Inhibitors/chemistry , Humans , Models, Biological , Time FactorsABSTRACT
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. As proof of concept for the described docking method, a protocol was developed to discover potential heteroactivators from a virtual chemical library through efficient sorting of >40,000 compounds. One of the top-scoring compounds identified was verified to be a CYP2C9 heteroactivator in vitro, and it possessed activity similar to dapsone.
Subject(s)
Aryl Hydrocarbon Hydroxylases/chemistry , Databases, Factual , Enzyme Activators/chemistry , Models, Molecular , Animals , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Cytochrome P-450 CYP2C9 , Dapsone/chemistry , Enzyme Inhibitors/chemistry , Flurbiprofen/chemistry , Humans , Molecular Structure , NADPH-Ferrihemoprotein Reductase/chemistry , Quantitative Structure-Activity Relationship , RatsABSTRACT
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. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.
