Characterisation of Candida parapsilosis CYP51 as a Drug Target Using Saccharomyces cerevisiae as Host

The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole—enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.

Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

A theoretical analysis of single molecule protein sequencing via weak binding spectra

We propose and theoretically study an approach to massively parallel single molecule peptide sequencing, based on single molecule measurement of the kinetics of probe binding [1] to the N-termini of immobilized peptides. Unlike previous proposals, this method is robust to both weak and non-specific probe-target affinities, which we demonstrate by applying the method to a range of randomized affinity matrices consisting of relatively low-quality binders. This suggests a novel principle for proteomic measurement whereby highly non-optimized sets of low-affinity binders could be applicable for protein sequencing, thus shifting the burden of amino acid identification from biomolecular design to readout. Measurement of probe occupancy times, or of time-averaged fluorescence, should allow high-accuracy determination of N-terminal amino acid identity for realistic probe sets. The time-averaged fluorescence method scales well to extremely weak-binding probes. We argue that this method could lead to an approach with single amino acid resolution and the ability to distinguish many canonical and modified amino acids, even using highly non-optimized probe sets. This readout method should expand the design space for single molecule peptide sequencing by removing constraints on the properties of the fluorescent binding probes.Author summaryWe simplify the problem of single molecule protein sequencing by proposing and analyzing an approach that makes use of low-affinity, low-specificity binding reagents. This decouples the problem of protein sequencing from the problem of generating a high-quality library of binding reagents against each of the amino acids.

Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase

ABSTRACTProthioconazole is a new triazolinthione fungicide used in agriculture. We have usedCandida albicansCYP51 (CaCYP51) to investigate thein vitroactivity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment ofC. albicanscells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts fromC. albicansgrown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.

S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) ReduceIn VitroInhibition by Fluconazole

ABSTRACTThe effects of S279F and S279Y point mutations inCandida albicansCYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (Ks, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (Ks, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higherKdvalues observed.

Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B

ABSTRACT Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (Ks , 8.6 μM) and eburicol (Ks , 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol (Ks , 3.1 μM) and eburicol (Ks , 4.1 μM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the Kd (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 μM, respectively, in comparison with a Kd value of 4 μM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with Kd values ranging from 1 μM for itraconazole to 11.9 μM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.