Structural analyses of Candida albicans sterol 14α-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis

Abstract

With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14α-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: ()-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against and , pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.

Crystal Structure of the New Investigational Drug Candidate VT-1598 in Complex with Aspergillus fumigatus Sterol 14α-Demethylase Provides Insights into Its Broad-Spectrum Antifungal Activity

“…3c), with the fifth axial coordination bond (on the proximal surface of the heme plane) being formed with the thiolate ion of Cys463. Interestingly, considering that the relatively lower electronegativity of the tetrazole ring N-4 atom would predict a greater distance between atoms, the length of the Fe-VT-1598 coordination bond is 2.1 Å, which is more typical for coordination bonds between the CYP51 iron and 1,2,4-triazoles (13,17,22). The difluorinated ␤-phenyl ring of VT-1598 protrudes deeper into the CYP51 substrate binding cavity, while the long arm lies within the substrate access channel, with its benzonitrile portion being seen above the surface of the channel entry (Fig.…”

“…VT-1598 completely blocked the initial rate of the A. fumigatus CYP51B reaction at a 1:1 molar ratio of inhibitor/enzyme (data not shown). Because we previously observed such a behavior for many inhibitors of other CYP51 orthologs (20,21), in the present study we used a long-term reaction (1 h) and a 2-fold molar excess of the inhibitor over the enzyme, as these conditions allow screening out of the inhibitors that can be replaced in the CYP51 active site by the substrate over time and thus reveal the most potent compounds (18,22). Figure 2 shows that VT-1598 had the strongest inhibitory effect on the activity of A. fumigatus CYP51B: 100% inhibition of substrate conversion at a P450/inhibitor/eburicol molar ratio of 1:2:50.…”

“…Since His377 in C. albicans corresponds to His374 in A. fumigatus CYP51B (Fig. 5a and b) and VT-1161 is one of the strongest C. albicans CYP51 inhibitors identified (22), it is, at first glance, unclear why VT-1161 produces a weaker effect on the A. fumigatus ortholog (Fig. 2).…”

“…The crucial role of H bonding in strengthening the CYP51/inhibitor complexes was first discovered for the VNI scaffold inhibitors of protozoan CYP51 (21,30). A strong H bond (2.8 Å) between the trifluoroethoxyphenyl oxygen of VT-1161 and His377 was found in its complex with Candida albicans CYP51 (22). Since His377 in C. albicans corresponds to His374 in A. fumigatus CYP51B (Fig.…”

Acceleration of murine hepatocyte proliferation by imazalil through the activation of nuclear receptor PXR

“…Imazalil inhibits the sterol biosynthesis of fungi through inhibiting 14α-demethylase (Hargrove et al, 2017). Imazalil is usually left in citrus, pears and apples (Kodama et al, 2003;Donnarumma et al, 2005) and its ADI is set at 0.025 mg/kg body weight/day in Japan.…”