**1. Introduction**

Human cytochrome P450 3A4 (CYP3A4) oxidizes over 50% of administered drugs [1], along with natural compounds, some of which can act as inhibitors of CYP3A4 [2]. The mechanism-based (suicide) inhibition (MBI) of CYP3A4 is the most common mechanism that could lead to clinically significant drug-drug interactions (DDIs), toxicity and therapeutic failures [3]. MBI is characterized by NADPH-, time- and concentration-dependent enzyme inactivation due to the formation and covalent attachment of a reactive metabolite(s) to the heme and/or apoprotein. The early elimination of the MBI/DDI potential in drug candidates is crucial [4] but highly challenging due to poor predictability of CYP3A4-ligand interactions. This results from high promiscuity [5] and conformational flexibility of CYP3A4 [6–8] and very limited structural information on the substrate association modes. To date, only three crystal structures of CYP3A4 with drug substrates bound in the active site have been reported [6,7,9] and none with suicide inactivators.

To fill this knowledge gap, this study investigated the binding manner of mibefradil, azamulin, bergamottin and 6- ,7- -dihydroxybergamottin (DHB) (Figure 1), which are known to act as potent mechanism-based inhibitors of CYP3A4. Mibefradil (or Posicor) is a benzimidazoyl-substituted tetraline, designed as a long-acting T-type calcium channel blocker for the treatment of chronic hypertension. Mibefradil interacts with multiple cytochrome P450 isoforms, but was withdrawn from the market mainly due to its high inhibitory potency for CYP3A4, leading to numerous life-threatening DDIs [10]. Azamulin is a semi-synthetic antibiotic and derivative of pleuromutilin, which failed stage I clinical trials due to poor bioavailability. Azamulin was recently identified as an effective and highly selective inhibitor of CYP3A4 and is currently recommended for use in reaction phenotyping studies instead of less specific ketoconazole [11,12]. Bergamottin and its derivative DHB, on the other

hand, are natural furanocoumarins found most abundantly in grapefruits. In addition to several health-promoting effects [13], both compounds mediate food-drug interactions primarily through inhibition of CYP3A4 [14]. Here, the results of spectral investigations are reported, as well as crystal structures of CYP3A4 bound to mibefradil, azamulin and DHB. Our findings help better understand the suicide substrate binding and inhibitory mechanism, and could improve the computational tools for modeling and prediction of the CYP3A4-ligand interactions, which is vital for designing safer and more effective drugs.

**Figure 1.** Chemical structures of the investigated compounds. Arrows indicate known sites of metabolism.
