Antifungal Azoles: Structural Insights into Undesired Tight Binding to Cholesterol-Metabolizing CYP46A1

Abstract

Although there are currently three generations of antifungal azoles on the market, even the third-generation agents show undesirable interactions with human cytochrome P450 (P450) enzymes. CYP46A1 is a cholesterol-metabolizing P450 in the brain that tightly binds a number of structurally distinct azoles. Previously, we determined the crystal structures of CYP46A1 in complex with voriconazole and clotrimazole, and in the present work we cocrystallized the P450 with posaconazole at 2.5 Å resolution. This long antifungal drug coordinates the P450 heme iron with the nitrogen atom of its terminal azole ring and adopts a linear configuration occupying the whole length of the substrate access channel and extending beyond the protein surface. Numerous drug-protein interactions determine the submicromolar Kd of posaconazole for CYP46A1. We compared the crystal structure of posaconazole-bound CYP46A1 with those of the P450 in complex with other drugs, including the antifungal voriconazole and clotrimazole. We also analyzed the accommodation of posaconazole in the active site of the target enzymes, CYPs 51, from several pathogenic species. These and the solution studies with different marketed azoles, collectively, allowed us to identify the determinants of tight azole binding to CYP46A1 and generate an overall picture of azole binding to this important P450. The data obtained suggest that development of CYP51-specific antifungal agents will continue to be a challenge. Therefore, structural understanding of the azole binding not only to CYPs 51 from the pathogenic species but also to different human P450s is required to deal efficiently with this challenge.

Fig. 1.

Chemical structures of the three generations of systemic azoles developed for use in humans. The structures and drug names in black and gray are of the marketed and experimental azoles, respectively. The numbering of some of the heteroatoms in clotrimazole, posaconazole, and voriconazole is also shown.

Fig. 2.

Unbiased σ_A weighted 2|F_o|–|F_c| and |F_o|–|F_c| electron density for POS in the complex with CYP46A1 at 2.5 Å resolution. The 2|F_o|–|F_c| map (cyan) is contoured at 0.6 σ; the |F_o|–|F_c| map (green) is contoured at 3.0 σ. POS (PDB ligand X2N) carbon atoms are white, oxygen red, nitrogen blue, and fluorine purple; heme carbon atoms are orange, and iron purple.

Fig. 3.

Interactions of POS with CYP46A1. (A) Semitransparent representation of the CYP46A1 surface (in light blue) showing POS (carbon atoms are magenta) extending above the protein surface. (B) Amino acid residues of CYP46A1 within 4 Å from POS (except Leu229). The heme carbon atoms are red; the nitrogen, oxygen, fluorine, sulfur, and iron atoms are blue, red, black, yellow, and orange, respectively.

Fig. 4.

Binding of POS, VOR, and CLO to CYP46A1. (A) Superimposed views of POS-, CLO-, and VOR-bound CYP46A1 showing how the three structurally distinct azoles are accommodated in the enzyme active site. The azole carbon atoms are magenta (POS), wheat (CLO), and marine (VOR). Phe121 and the CYP46A1 secondary structural elements defining the Phe121 pocket are also shown. Their coloring is the same as that of the P450 carbon atoms in panel B. (B) Conformational flexibility of amino acid residues defining the Phe121 pocket (all drugs are omitted for clarity). The position of amino acid residues of the ligand-free CYP46A1 is shown for comparison. The CYP46A1 carbon atoms are colored light blue (POS-bound P450), cyan (CLO-bound P450), coral (VOR-bound P450), and yellow (ligand-free P450). The heme carbon atoms are red (POS-bound P450), salmon (CLO-bound P450), and brown (VOR-bound P450). Coloring of other atoms is the same as in Fig. 3.

Fig. 5.

POS conformations in different P450s. POS carbon atoms are magenta (in CYP46A1 cocomplex), cyan and orange (in TbCYP51 co-complexes), and brown (in TcCYP51 cocomplex). The heme carbon atoms are red (CYP46A1), purple (TcCYP51), and dark salmon and brown (the two TbCYPs 51). Coloring of other atoms is the same as in Fig. 3.

Fig. 6.

Binding of different ligands to CYP46A1. (A) Binding of POS (carbon atoms are magenta), VOR (carbon atoms are marine), and CLO (carbon atoms are wheat) as compared with that of tranylcypromine (carbon atoms are black), thioperamide (carbon atoms are lime), and cholesterol 3-sulfate (carbon atoms are gold). (B and C) Two views of POS binding as compared with that of bicalutamide (carbon atoms are green) and fluovoxamine (carbon atoms are brown). A and B also show Phe121 and the P450 secondary structural elements defining the Phe121-pocket. Panel C shows Trp368 and the CYP46A1 secondary structural elements defining the Trp368-pocket. The heme carbon atoms are red (POS-bound CYP46A1), brown (VOR-bound CYP46A1), salmon (CLO-bound CYP46A1), olive (tranylcypromine-bound CYP46A1), violet (thioperamide-bound CYP46A1), orange (cholesterol-sulfate-bound CYP46A1), light raspberry (bucalutamide-bound CYP46A1), and brown (fluvoxamine-bound CYP46A1). Coloring of other atoms is the same as in Fig. 3.