Publications

• Structure-functional characterization of cytochrome P450 sterol 14alpha-demethylase (CYP51B) from Aspergillus fumigatus and molecular basis for the development of antifungal drugs.

  Hargrove T.Y., Wawrzak Z., Lamb D.C., Guengerich F.P., Lepesheva G.I.

  Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for ration ... >> More

  Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min(-1), respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections. << Less

  J. Biol. Chem. 290:23916-23934(2015) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• In vitro and in vivo efficacy of a novel and long-acting fungicidal azole, PC1244, on Aspergillus fumigatus infection.

  Colley T., Sehra G., Chowdhary A., Alanio A., Kelly S.L., Kizawa Y., Armstrong-James D., Fisher M.C., Warrilow A.G.S., Parker J.E., Kelly D.E., Kimura G., Nishimoto Y., Sunose M., Onions S., Crepin D., Lagasse F., Crittall M., Shannon J., McConville M., King-Underwood J., Naylor A., Bretagne S., Murray J., Ito K., Strong P., Rapeport G.

  The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against <i>Aspergillus fumigatus</i> were tested in a range of <i>in vitro</i> and <i>in vivo</i> studies. PC1244 demonstrated potent antifungal activities against clinical <i>A. fumigatus</i> isol ... >> More

  The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against <i>Aspergillus fumigatus</i> were tested in a range of <i>in vitro</i> and <i>in vivo</i> studies. PC1244 demonstrated potent antifungal activities against clinical <i>A. fumigatus</i> isolates (<i>n</i> = 96) with a MIC range of 0.016 to 0.25 μg/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 μg/ml. PC1244 was a strong tight-binding inhibitor of recombinant <i>A. fumigatus</i> CYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis in <i>A. fumigatus</i> with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 μg/ml), especially <i>Aspergillus terreus</i>, <i>Trichophyton rubrum</i>, <i>Candida albicans</i>, <i>Candida glabrata</i>, <i>Candida krusei</i>, <i>Cryptococcus gattii</i>, <i>Cryptococcus neoformans</i>, and <i>Rhizopus oryzae</i> PC1244 also proved to be quickly absorbed into both <i>A. fumigatus</i> hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 μg/ml) which indicated that it was 8-fold more potent than voriconazole. <i>In vivo</i>, once-daily intranasal administration of PC1244 (3.2 to 80 μg/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible <i>A. fumigatus</i> substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed <i>in vivo</i> effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of <i>A. fumigatus</i> infection in the lungs of humans. << Less

  Antimicrob. Agents Chemother. 62:0-0(2018) [PubMed] [EuropePMC]

• In Vitro biochemical study of CYP51-mediated azole resistance in Aspergillus fumigatus.

  Warrilow A.G., Parker J.E., Price C.L., Nes W.D., Kelly S.L., Kelly D.E.

  The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 recons ... >> More

  The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinant Aspergillus fumigatus CPR1 (AfCPR1), and Escherichia coli membrane suspensions containing recombinant A. fumigatus CYP51 proteins, allowing in vitro screening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed that A. fumigatus CYP51B (Af51B IC50, 0.50 μM) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC50, 17 μM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 μM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 μM triazole, which could confer azole resistance in A. fumigatus strains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance of A. fumigatus strains harboring G54W, L98H, and M220K Af51A point mutations. << Less

  Antimicrob. Agents Chemother. 59:7771-7778(2015) [PubMed] [EuropePMC]

• Ergosterol biosynthesis pathway in Aspergillus fumigatus.

  Alcazar-Fuoli L., Mellado E., Garcia-Effron G., Lopez J.F., Grimalt J.O., Cuenca-Estrella J.M., Rodriguez-Tudela J.L.

  The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (sus ... >> More

  The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (susceptible and resistant to azole drugs). Also, sterols were analyzed in several A. fumigatus mutant strains deficient in enzymatic steps of the ergosterol biosynthesis pathway such as 14-alpha sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A, Erg3B and Erg3C). All sterols identified from azole-resistant A. fumigatus strains were qualitatively and quantitatively similar to the susceptible strain (CM-237). However, sterol composition of mutants strains were different depending on the lacking enzyme. The analysis of the sterol composition in these mutant strains led to a better understanding of the ergosterol biosynthesis pathway in this important fungus. << Less

  Steroids 73:339-347(2008) [PubMed] [EuropePMC]