*2.1. Interaction of CYP3A4 with Mibefradil*

Equilibrium titrations of the full-length CYP3A4 were conducted to determine and relate the binding affinity and dissociability of the investigated compounds to their association mode. Mibefradil is a type I ligand that causes a blue shift in the Soret band (Figure 2A). The spectral dissociation constant (Ks) derived from the titration plot (left inset in Figure 2A) was 3.3 μM, which is ~5-fold higher than the previously reported value [15], possibly due to differences in the CYP3A4 form. Since CYP3A4 precipitates during prolonged dialysis, the dissociation ability was assessed by titrating the ligand-bound protein with ritonavir, a high-affinity inhibitor that easily displaces other type I substrates, such as bromocryptine and midazolam (Figure S1). As seen in Figure 2B, ritonavir could fully replace mibefradil, but its binding affinity was 74-fold lower than for the ligand-free CYP3A4: Ks RIT of 1.4 μM vs. 0.019 μM [16], respectively. In contrast, only a 3.5-fold decrease in Ks RIT was observed for the bromocryptine- and midazolam-bound CYP3A4 (Table 1). Thus, mibefradil interacts with CYP3A4 stronger and/or is better protected and has a lower ability to dissociate.

**Table 1.** Parameters for the ligand binding to CYP3A4.


<sup>a</sup> Spectral dissociation constant for ligand-free CYP3A4. <sup>b</sup> Binding affinity of ritonavir for ligand-bound/free

CYP3A4. a,b Values represent an average of three measurements with the standard error. <sup>c</sup> S50; *<sup>n</sup>* is a Hill coefficient. <sup>d</sup> Dissociation constants for two binding sites.

**Figure 2.** Spectral and structural properties of the CYP3A4-mibefradil complex. (**A**,**B**) Spectral changes observed during equilibrium titrations of CYP3A4 with mibefradil and upon displacement of mibefradil with ritonavir, respectively. In panel (**A**), the spectrum of ligand-free CYP3A4 is in black. In panel (**B**), the spectrum of ritonavir-bound CYP3A4 is in brown. In both panels, the spectra of the CYP3A4-mibefradil complex and its ferrous and ferrous CO-bound forms are in red, green and blue, respectively. In the competitive displacement experiment (panel **B**), the concentration of mibefradil was 120 μM. The left and right insets are the difference spectra and titration plots with hyperbolic fittings, respectively. The derived Ks values are given in Table 1. (**C**) The active site of CYP3A4 bound to mibefradil (shown in orange sticks; PDB ID 6OO9). Green mesh is a polder omit electron density map contoured at 3σ level. Simulated annealing omit map for mibefradil is shown in Figure S2A. The labeled C35 atom of mibefradil is the closest to the heme iron (~3.8 Å away). (**D**) Interaction of mibefradil with surrounding residues (shown in green sticks and labeled). Red dotted lines are H-bonds. Cyan sphere is a water molecule. (**E**) A slice through the CYP3A4 molecule showing how well mibefradil (in space-filling representation) fits into the active site cavity. The visible helices are labeled.

### 2.1.1. Crystal Structure of the CYP3A4-Mibefradil Complex

Mibefradil inhibits microsomal and recombinant CYP3A4 with IC50 of 0.3–2 μM [17], but the chemical nature of its reactive metabolite(s) is still unknown. A previous attempt to elucidate the molecular basis for MBI led to a conclusion that the inactivation of CYP3A4 proceeds through heme destruction rather than covalent modification of the heme or apoprotein [15]. To identify the potential oxidation sites that could lead to bioactivation, CYP3A4 was co-crystallized with mibefradil. The crystal structure was determined to 2.25 Å resolution (Table S1) and contained one drug molecule in the active site (Figure 2C–E). The tetraline moiety is 3.3–3.6 Å above the heme plane, with the propyl C35 atom being the closest to the iron (~3.8 Å away; Figure 2C). The fluorophenyl portion is parallel to the I-helix and partially inserts into a hydrophobic pocket formed by F304, A305 and I301. The benzimidazole moiety provides additional hydrophobic and aromatic interactions with F57, F215 and M371 (Figure 2D). The methoxyacetate functionality, in turn, is H-bonded via the carbonyl oxygen to the R212 guanidine group. This polar interaction is part of the H-bonding network that links the F-F- -loop to the protein

core and likely contributes to the inhibitory potency, as the derivatives lacking the methoxyacetate group are weaker inhibitors of CYP3A4 than mibefradil [15,18]. The ligand binding mode is further stabilized by multiple van der Waals interactions.

As seen from Figures 2E and S3A, mibefradil binds compactly and fits into the catalytic cavity without triggering any notable conformational change. However, the compact binding mode may limit its motional freedom. This limitation and the fact that CYP3A4 remains in a resting conformation, tightly locked through the R212-mediated contacts, could explain the low dissociability of mibefradil.

#### 2.1.2. Possible Inhibitory Mechanism of Mibefradil

CYP3A4 metabolizes mibefradil via methoxyacetate and tertiary amine demethylation and hydroxylation of the benzimidazole ring (indicated in Figure 1) rather than the oxidation of the closest to the iron propyl group [10]. Thus, the re-entry or reorientation of mibefradil would be required to allow an access to the primary, non-inhibitory sites of metabolism. Whether the crystallographic binding mode could lead to products capable of escaping the active site is yet to be proven. Even so, based on our and earlier findings [10,15,18], it is plausible to suggest that the crystal structure represents an inhibitory complex, where mibefradil could decrease the CYP3A4 activity in two ways: (i) through formation of a slowly dissociable complex that would prevent other substrate molecules from reaching the catalytic center; and (ii) by producing an alkyl radical intermediate upon a hydrogen abstraction from the C35 atom, which could attack and destroy the heme. The formation of a highly reactive free radical would explain why the C35 oxidation product was not observed.
