Probe Substrate Dependencies in CYP3A4 Allosteric Inhibition: A Novel Molecular Mechanism Involving F–F′ Loop Perturbations

The biochemical basis for substrate dependences in apparent
inhibition
constant values (Ki) remains unknown.
Our study aims to elucidate plausible structural determinants underpinning
these observations. In vitro steady-state inhibition assays conducted
using human recombinant CYP3A4 enzyme and testosterone substrate revealed
that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib
and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9
μM, respectively. However, when rivaroxaban was adopted as the
probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into
this phenomenon, erdafitinib and pemigatinib were docked to allosteric
sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations
of apo- and holo-CYP3A4 were conducted to investigate the structural
changes induced. Comparative structural analyses of representative
MD frames extracted by hierarchical clustering revealed that the allosteric
inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially
modulate its active site characteristics. In contrast, we discovered
that allosteric binding of the FGFR inhibitors reduces the structural
flexibility of the F–F′ loop region, an important gating
mechanism to regulate access of the substrate to the catalytic heme.
We surmised that the increased rigidity of the F-F′ loop engenders
a more constrained entrance to the CYP3A4 active site, which in turn
impedes access to the larger rivaroxaban molecule to a greater extent
than testosterone and culminates in more potent inhibition of its
CYP3A4-mediated metabolism. Our findings suggest a potential mechanism
to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of
CYP3A4.