Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4

Abstract

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F’ loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism

“…In our current work, TST is considered as a probe to investigate the heterotropic allostery in MDZ metabolism, which has the strongest allosteric effect on the MDZ metabolism as reported by Denisov et al Our results indicate that the presence of TST can promote the transition of the MDZ binding mode through long-range allosteric effects and can also increase the formation of 4-OH-MDZ. The work of Denisov et al 60 is a timely event that can provide experimental data to support our current study, and the important role of the F′−F loop reported by Denisov et al is also clarified by us that the movement of the F′−F loop can expand the CYP3A4 active site volume and open the "sandwich" structure providing favorable conditions for the transition of the MDZ binding mode. In this work, we propose two potential allosteric sites (Site 1 and Site 2) located in the periphery of CYP3A4, and we also discuss in detail that Site 2 seems to be more pronounced than Site 1 as it is modulated by three regulatory pathways.…”

“…The effects caused by TST binding can remotely promote the transition of the MDZ binding mode in the CYP3A4 active site via multiple allosteric communication pathways, which can increase the probability of pose C and facilitate the formation of 4-OH-MDZ. These findings could help us to understand the experimental fact 12,60 that TST can stimulate the formation of 4-OH-MDZ. According to the results of the residue interaction network (RIN), many key residues involved in the allosteric communication pathways are recognized; among them, R105 and F108 as well as heme are identified as key factors to trigger the transition of the MDZ binding mode.…”

“…Many studies 12,13,15,17,60,61 have shown that the CYP3A4mediated MDZ metabolism can be modulated by other drugs and generate two hydroxylated metabolites (1′-OH-MDZ and 4-OH-MDZ) in diverse proportions. TST is a steroid molecule that can greatly increase the formation of 4-OH-MDZ through allosteric effects.…”

“…12−16 Thus, CYP3A4mediated DDIs have received much attention in recent years. 14,15,17 During the preparation of our article, Denisov et al 60 reported a study on heterotropic DDIs mediated by CYP3A4. They monitored the effect of various drugs on MDZ metabolism by experimental means, suggesting TST and PGS could promote the formation of 4-OH-MDZ, and the effect of TST was particularly significant.…”

Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment

“…As mentioned above, membrane proteins can be reconstituted in a detergent-free lipid environment. Amphipathic MSP-based [ 80 , 81 , 82 ] and peptide-based [ 83 ] nanodiscs are commonly used systems for studying membrane proteins in the desired lipid environment [ 84 , 85 ]. However, the MSP protein or peptide in the nanodisc belt can interfere with the membrane protein characterization by many biophysical techniques, including circular dichroism (CD) and ultraviolet-visible (UV) absorption spectroscopy techniques.…”