Allosteric activation of cytochrome P450 3A4 by efavirenz facilitates midazolam binding

Abstract

1. The purpose of this study is to investigate the heteroactivation mechanism of CYP3A4 by efavirenz, which enhances metabolism of midazolam in vivo, in terms of its binding to CYP3A4 with in vitro spectroscopic methods. 2. Efavirenz exhibited a type II spectral change with binding to CYP3A4 indicating a possible inhibitor. Although dissociation constant (K ) was approximated as 520 μM, efavirenz enhanced binding affinity of midazolam as a co-existing drug with an estimated iK value of 5.6 µM which is comparable to a clinical concentration. 3. Efavirenz stimulated the formation of 1'-hydroxymidazolam, and the product formation rate (V ) concentration-dependently increased without changing the K. Besides, an efavirenz analogue, [6-chloro-1,4-dihydro-4-(1-pentynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one] (efavirenz impurity) slightly facilitated the binding affinity of midazolam in a concentration-dependent manner. These results propose that efavirenz affects midazolam-binding via binding to the peripheral site which is apart from the active site of CYP3A4. 4. A molecular dynamics simulation also suggested the bound-efavirenz was repositioned to effector-binding site. As a consequence, our spectroscopic studies clarified the heteroactivation of CYP3A4 caused by efavirenz with a proper affinity to the peripheral site, and we concluded the method can be a useful tool for characterising the potential for drug-drug interactions.

A Covalently Attached Progesterone Molecule Outcompetes the Binding of Free Progesterone at an Allosteric Site of Cytochrome P450 3A4

“…[10][11][12] This hypothesis was greatly reinforced when the first CYP3A4 crystal structure was published by William et al in 2004, revealing an unexpected peripheral binding site for PRG, proposed to be an allosteric pocket (Figure 1). 13 Since then, many publications [14][15][16][17][18][19][20][21][22][23][24][25] have reported results that are consistent with this PRG binding site being an allosteric site, yet its exact location is still questioned as the identified PRG binding pocket may result from a crystallographic artifact. Several steroids are known to act as allosteric effectors of CYP3A4, often in a substrate-dependent manner.…”

Efavirenz Metabolism: Influence of Polymorphic CYP2B6 Variants and Stereochemistry

“…Cooperativity among CYP2B6 substrates is relatively uncommon, having been described for CYP2B6.1 and 7-hydroxy-4-trifluoromethylcoumarin (n 5 1.4) (Ekins et al, 1997), testosterone (n 5 1.3) (Ekins et al, 1998), methadone (Totah et al, 2007), and S-efavirenz (n 5 1.5) (Ward et al, 2003) but not several other substrates (Ekins et al, 1998;Liu et al, 2016), and also for CYP2B6.4 and 7-ethoxycoumarin (n 5 1.7) (Ariyoshi et al, 2001). Interestingly, heteroactivation by efavirenz was recently reported, with enhanced midazolam hydroxylation by CYP3A4 via interaction at an allosteric site (Ichikawa et al, 2018).…”

Combining Small-Molecule Bioconjugation and Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) to Expose Allostery: the Case of Human Cytochrome P450 3A4

“…Important insights into the location of the CYP3A4 allosteric site came from a crystal structure in which the substrate progesterone was found to bind at a site distal to the heme, near the F′ helix (Figure S23A). Although it has been speculated that this peripheral binding pocket may stem from a crystallographic artifact, many biochemical and biophysical studies support the hypothesis that the F′ helix is involved in effector binding and allosteric activation. ,,,− How ligand binding in this region may lead to CYP3A4 catalytic enhancement remains unknown. Structural changes leading to allosteric activation can be dynamic and transient, making them difficult to fully capture with X-ray crystallography.…”