In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

Abstract

bThe 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 (IC 50 ) showed that A. fumigatus CYP51B (Af51B IC 50 , 0.50 M) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC 50 , 17 M) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC 50 s of 0.16 to 0.38 M). 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 M 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.

The Tetrazole VT-1161 Is a Potent Inhibitor of Trichophyton rubrum through Its Inhibition of T. rubrum CYP51

“…Both the T. rubrum and M. graminicola CYP51 enzymes can turn over eburicol but not lanosterol (23; this study). Aspergillus fumigatus CYP51 isoenzymes A and B turn over both eburicol and lanosterol, albeit with a 4-to 7-fold preference for eburicol in terms of measured velocity using purified proteins (29) or more than an 18-fold preference for eburicol using membrane fractions. Candida albicans, Cryptococcus neoformans, and Malassezia globosa CYP51 enzymes, on the other hand, all readily turn over both eburicol and lanosterol (29)(30)(31).…”

“…Aspergillus fumigatus CYP51 isoenzymes A and B turn over both eburicol and lanosterol, albeit with a 4-to 7-fold preference for eburicol in terms of measured velocity using purified proteins (29) or more than an 18-fold preference for eburicol using membrane fractions. Candida albicans, Cryptococcus neoformans, and Malassezia globosa CYP51 enzymes, on the other hand, all readily turn over both eburicol and lanosterol (29)(30)(31). Analysis of the amino acid sequences of the six SRSs between the seven fungal CYP51 enzymes did not identify any residue changes that could be directly linked to the change in substrate specificity observed in the T. rubrum and M. graminicola CYP51 enzymes.…”

“…Additional CYP51 enzymes that exhibit narrow substrate specificities include obtusifoliol-specific Trypanosoma brucei CYP51 and plant CYP51 enzymes, such as Sorghum bicolor CYP51 (36), while the Aspergillus fumigatus CYP51A and CYP51B isoenzymes have a strong preference for eburicol (29). The Trub51 K m for eburicol of 2 M was comparable to the substrate K m values previously obtained for CYP51 enzymes from C. albicans and Saccharomyces cerevisiae (32, 37, 38) but was 5-to 30-fold lower than those determined for CYP51 enzymes from Leishmania infantum, Homo sapiens, Mycosphaerella graminicola, and Malassezia globosa (23,31,36,39).…”

In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection

“…A CYP51 reconstitution assay system was used to determine 50% inhibitory concentrations (IC 50 s) (37). Test agent was added to a mixture of 0.5 μM CYP51, 1 μM A.…”

Eine strukturelle Evaluierung medizinalchemischer Strategien gegen Wirkstoffresistenzen

“…Tatsächlich bewirkt die TR34/ Leu98His-Mutation eine Verengung der Liganden-Zugangskanäle sowie eine Verdrängung von Ty r121 und Tyr107, von denen bekannt ist, dass sie fürd ie Triazol-Bindung wichtig sind. [396] Überraschenderweise sind nur exogene Liganden von dieser Mutation betroffen, während die biologische Aktivitätd es Enzyms unverändert bleibt. [395] Kürzlich wurde aufgrund von 3D-Strukturmodellen vorgeschlagen, dass die Leu98His-Mutation auch die Stabilitätd es Proteins verringern kçnnte und dies zur Azolresistenz beiträgt.…”