Crystal Structure of CYP3A4 Bound to Azamulin

The crystal structure of the CYP3A4-azamulin complex was determined to 2.52 Å resolution (Table S1) and contains one drug molecule bound to the active site in an extended conformation (Figure 3C). As the spectral data predicted, the pleuromutilin functionality is placed near the heme. The complex could be productive, because two carbon atoms of the hexane ring, C08 and C09, are close enough for oxidation: 4.6 Å and 4.3 Å from the iron, respectively. The thioacetyl linker is stretched above the I-helix and, as a result, the amino-triazolyl end-group points upward rather than toward the substrate channel (Figure 3C,D). This conformation is stabilized by multiple van der Waals contacts and two hydrogen bonds, formed between the hydroxyl group of pleuromutilin's eight-membered ring and S119 side chain, and between the amino-triazolyl nitrogen and E308 carboxyl (Figure 3E). Importantly, due to steric clashing with the amino-triazolyl moiety, the F-F- -loop (residues 210–217) becomes disordered, leaving the end-portion of azamulin partially solvent exposed (Figure S4). This suboptimal binding mode could explain why azamulin is displaced by ritonavir more easily than mibefradil.

**Figure 3.** Spectral and structural properties of the CYP3A4-azamulin complex. (**A**,**B**) Spectral changes observed during equilibrium titrations of CYP3A4 with azamulin and upon displacement of azamulin 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-azamulin 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 azamulin was 60 μM. The left and right insets are the difference spectra and titration plots with hyperbolic fittings, respectively. The derived Ks values are listed in Table 1. (**C**) The binding mode of azamulin (in cyan; PDB ID 6OOA). Green mesh is a polder omit electron density map contoured at 3σ level. Simulated annealing omit map for azamulin is shown in Figure S2B. The closest to the iron C08 and C09 atoms are indicated. (**D**) A slice through the CYP3A4 molecule showing that azamulin (in space-filling representation) extends over the I-helix rather than along the substrate channel. The visible helices are labeled. (**E**) Interaction of azamulin with surrounding residues (shown in beige sticks and labeled). The H-bonds are depicted as red dotted lines.

Azamulin was identified as a potent and highly selective competitive and mechanism-based inhibitor of microsomal and recombinant CYP3A4 (IC50 of 0.03–0.24 μM) [11]. The pleuromutilin group is thought to undergo metabolic activation [11], but the reactive intermediates are yet to be identified. The crystal structure corroborates the notion that the pleuromutilin is required for MBI, because this functionality is the closest to the heme, mediates the majority of protein-ligand interactions and orients suitably for oxidation. It needs to be tested though whether the C08/9 oxidation products could be reactive or they would have to dissociate and rebind in a distinct orientation to enable bioactivation at other sites. In any case, considering the bulkiness, high hydrophobicity and low dissociability of azamulin, it is plausible to suggest that this compound could effectively inhibit CYP3A4 by crowding/blocking the active site as well.

#### *2.3. Interaction of CYP3A4 with Bergamottin and DHB*

Bergamottin, but not DHB, causes a partial high-spin shift in CYP3A4 (Figure 4A,B). An increase in DHB concentration leads to a small decrease rather than a shift in the Soret band, meaning that DHB could approach and alter the heme environment without changing the coordination state. Another notable difference was in the shape of the titration curves: sigmoidal for bergamottin and hyperbolic for DHB (right insets in Figure 4A,B). The S50 and *n* values (substrate concentration at half-saturation and the Hill coefficient, respectively) derived from the sigmoidal plot suggest that bergamottin binds to CYP3A4 cooperatively and with affinity comparable to those of mibefradil and azamulin (Table 1). The DHB titration curve, in turn, was best fit to a two-site binding hyperbolic equation, indicating that (i) two DHB molecules can simultaneously bind to CYP3A4, and (ii) the occupation of the high affinity site (Ks of 0.22 μM) leads to very small changes in the Soret band (~17% of the total absorbance decrease). Another notable feature is the incomplete reduction of bergamottin- and DHB-bound CYP3A4 with sodium dithionite, as evident from the red-shifted absorbance maxima of their ferrous forms: 413 and 416 nm, respectively, versus 410 nm for mibefradil- and azamulin-bound CYP3A4 (compare green spectra in Figures 2A, 3A and 4A,B). Thus, both furanocoumarins seem to limit an access of the reductant to the heme iron. Since the reaction of CYP3A4 with sodium dithionite was carried out under aerobic conditions and was the slowest for the DHB-bound form, the lower than expected 450 nm absorption of the ferrous DHB/CO-bound species (blue spectrum in Figure 4B) could be the consequence of both the incomplete reduction and oxidative damage of the heme. Despite the markedly distinct binding manner, both bergamottin and DHB could be easily displaced by ritonavir (Figure S5), whose binding affinity was only mildly affected (<2-fold decrease; Table 1).

**Figure 4.** Interaction of CYP3A4 with bergamottin and DHB. (**A**,**B**) Spectral changes observed during equilibrium titration of CYP3A4 with bergamottin and 6- ,7- -dihydroxybergamottin (DHB), respectively. The spectra of ligand-free CYP3A4 are in black. The spectra of the CYP3A4-substrate complexes and their ferrous and ferrous CO-bound forms are in red, green and blue, respectively. The left and right insets are the difference spectra and titration plots with sigmoidal and hyperbolic fittings, respectively. The derived Ks values are listed in Table 1. (**C**) The binding mode of DHB (in magenta; PDB ID 6OOB). Green mesh is a polder omit electron density map contoured at 3σ level. Simulated annealing omit map for DHB is shown in Figure S2C. Cyan sphere is the heme-bound water molecule. (**D**) A slice through the CYP3A4 molecule showing how DHB (in space-filling representation) orients in the active site cavity. The visible helices are labeled. (**E**) Interaction of DHB with surrounding residues (shown in cyan sticks and labeled). Red dotted lines are H-bonds. Cyan sphere is a water molecule. The furan double bond, a possible bioactivation site, is indicated by an arrow. The C22 atom of psoralen's phenyl ring, the closest to the heme iron, is labeled.
