Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs

Abstract

Background:The fungus Aspergillus fumigatus causes human diseases that are treated with CYP51 azole inhibitors. Results: We report crystal structures of A. fumigatus CYP51B complexes with two inhibitors, voriconazole and VNI. Conclusion:The structures reveal fungus-specific features not observed in CYP51 enzymes from other biological kingdoms. Significance: This molecular insight facilitates rational design of novel antifungals without inhibition of human enzymes.

Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design

“…Trypanosoma cruzi, Aspergillus fumigatus, and full-length human CYP51 and T. brucei and rat NADPH-cytochrome P450 reductase were expressed in Escherichia coli and purified as described previously (49,50). The full-length proteins were used for functional studies (ligand binding, enzymatic activity, and inhibition).…”

“…P450 concentrations were determined from the Soret band intensity using  417 117 mM 1 cm 1 for the low-spin ferric form of the protein or  450-490 91 mM 1 cm 1 for the reduced carbon monoxide difference spectra; the spin state of the P450 samples was estimated from the absolute absorbance spectra (53). Substrate binding was monitored as a "type I" spectral response (blue shift in the Soret band maximum from 417 to 393 nm) (54) reflecting low-to-high spin transition of the ferric P450 heme iron as a result of displacement of the heme-coordinated water molecule (50). Various aliquots of sterols (dissolved in 45% HPCD, w/v) (49) were added to the sample cuvette (1 cm optical path length), and the same volume of HPCD was added to the reference cuvette.…”

“…Michaelis-Menten parameters were calculated using GraphPad Prism 6, with the reaction rates (nanomoles of product formed per nanomole of P450 per minute) being plotted against total substrate concentration. The inhibitory potencies of azoles on T. cruzi, A. fumigatus, and human CYP51 activity were compared on the basis of decreases in substrate conversion in 60 min reactions (50,51,57,58). Fig.…”

“…The low-to-high spin transitions (saturation with lanosterol and obtusifoliol) were 18 and 36%, respectively. Titration curves are presented in the insets; the corresponding calculated binding parameters are given in Table 2 (50,60)], the human enzyme binds all of the five natural CYP51 substrates (Fig. 1A) and catalyzes their 14-demethylation quite efficiently (Fig.…”

Clinical Candidate VT-1161's Antiparasitic Effect In Vitro , Activity in a Murine Model of Chagas Disease, and Structural Characterization in Complex with the Target Enzyme CYP51 from Trypanosoma cruzi

“…The enzyme was purified by affinity chromatography on Ni 2ϩ -nitrilotriacetic acid (NTA) agarose (Qiagen), followed by cation-exchange chromatography on a carboxymethyl (CM) Sepharose Fast Flow cation exchanger (GE Healthcare) (27). The samples of CYP51 enzymes from Aspergillus fumigatus and Candida albicans were expressed and purified as previously described (28,29). The CYP51 electron donor partner NADPH-cytochrome P450 reductase (CPR) was from T. brucei and rat for protozoan and fungal orthologs, respectively (29)(30)(31).…”