7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching, suggesting binding at multiple protein sites

Abstract

Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern-Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (Kd 1.7-2.5 μM and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (Kd 1.7 μM) than the quenching of protein fluorescence at 335 and 355 nm (Kd 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (Kd 0.048 μM and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.

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.…”

Combining small-molecule bioconjugation and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to expose allostery: the case of human cytochrome P450 3A4

“…25 Although it has been speculated that this peripheral binding site 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. 14,22,23,26,[29][30][31][32][33][34][35] How a binding event in this region may lead to CYP3A4 catalytic enhancement remains unknown. Structural changes leading to allosteric activation are often dynamic, and can be transient, making them difficult to fully capture with X-ray crystallography.…”

Time-dependent enzyme inactivation: Numerical analyses of in vitro data and prediction of drug-drug interactions

“…CYPs are known to exhibit multiple substrate binding kinetics, leading to non-MM kinetics such as biphasic, sigmoidal, or substrate inhibition (Atkins, 2005;Korzekwa, et al, 1998;Marsch, et al, 2018). There has been significant development in terms of mechanistic understanding and inclusion of atypical kinetics in in vitro-in vivo extrapolation (IVIVE) of reversible inhibition (Davydov & Halpert, 2008;Galetin, et al, 2003;Houston & Kenworthy, 2000;Kenworthy, et al, 2001;Yang, et al, 2012).…”