*2.2. Interaction of CYP3A4 with Azamulin*

Azamulin is also a medium affinity type I ligand (Ks of 1.7 μM; Figure 3A) which, unlike mibefradil, causes a complete high-spin shift in CYP3A4. The observed absorbance changes suggest that azamulin docks with the bulky pleuromutilin head on, as the heme ligation to the amino-triazolyl nitrogen would lead to a red shift in the Soret band (type II spectral perturbations). Despite the higher affinity for CYP3A4, azamulin was displaced by ritonavir more easily than mibefradil but less easily than other type I ligands (Figure 3B; Table 1). This implies that azamulin is a slowly dissociable ligand that forms fewer/weaker contacts and/or is less protected in the active site than mibefradil.

It should be noted also that Ks RIT serves as an estimate for the displaced ligand dissociation constant. The fact that the Ks RIT and Ks values derived for mibefradil and azamulin are very close (only 2-fold difference; Table 1) suggests that in the competitive displacement experiments the substrate dissociation is a limiting step, which is only weakly affected by ritonavir.
