Heterologous expression and biochemical characterisation of the recombinant ß-carbonic anhydrase (MpaCA) from the warm-blooded vertebrate pathogen malassezia pachydermatis.

Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO2/HCO3-/pH-sensing in fungi is triggered by HCO3- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the ß-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.

Inhibitory Effects of Sulfonamide Derivatives on the ß-Carbonic Anhydrase (MpaCA) from Malassezia pachydermatis, a Commensal, Pathogenic Fungus Present in Domestic Animals.

Fungi are exposed to various environmental variables during their life cycle, including changes in CO2 concentration. CO2 has the potential to act as an activator of several cell signaling pathways. In fungi, the sensing of CO2 triggers cell differentiation and the biosynthesis of proteins involved in the metabolism and pathogenicity of these microorganisms. The molecular machineries involved in CO2 sensing constitute a promising target for the development of antifungals. Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial enzymes in the CO2 sensing systems of fungi, because they catalyze the reversible hydration of CO2 to proton and HCO3-. Bicarbonate in turn boots a cascade of reactions triggering fungal pathogenicity and metabolism. Accordingly, CAs affect microorganism proliferation and may represent a potential therapeutic target against fungal infection. Here, the inhibition of the unique ß-CA (MpaCA) encoded in the genome of Malassezia pachydermatis, a fungus with substantial relevance in veterinary and medical sciences, was investigated using a series of conventional CA inhibitors (CAIs), namely aromatic and heterocyclic sulfonamides. This study aimed to describe novel candidates that can kill this harmful fungus by inhibiting their CA, and thus lead to effective anti-dandruff and anti-seborrheic dermatitis agents. In this context, current antifungal compounds, such as the azoles and their derivatives, have been demonstrated to induce the selection of resistant fungal strains and lose therapeutic efficacy, which might be restored by the concomitant use of alternative compounds, such as the fungal CA inhibitors.

Skin Commensal Fungus Malassezia and Its Lipases.

Malassezia is the most abundant genus in the fungal microflora found on human skin, and it is associated with various skin diseases. Among the 18 different species of Malassezia that have been identified to date, M. restricta and M. globosa are the most predominant fungal species found on human skin. Several studies have suggested a possible link between Malassezia and skin disorders. However, our knowledge on the physiology and pathogenesis of Malassezia in human body is still limited. Malassezia is unable to synthesize fatty acids; hence, it uptakes external fatty acids as a nutrient source for survival, a characteristic compensated by the secretion of lipases and degradation of sebum to produce and uptake external fatty acids. Although it has been reported that the activity of secreted lipases may contribute to pathogenesis of Malassezia, majority of the data were indirect evidences; therefore, enzymes' role in the pathogenesis of Malassezia infections is still largely unknown. This review focuses on the recent advances on Malassezia in the context of an emerging interest for lipases and summarizes the existing knowledge on Malassezia, diseases associated with the fungus, and the role of the reported lipases in its physiology and pathogenesis.

Activation studies of the ß-carbonic anhydrases from Malassezia restricta with amines and amino acids.

The ß-carbonic anhydrase (CA, EC 4.2.1.1) from the genome of the opportunistic pathogen Malassezia restricta (MreCA), which was recently cloned and characterised, herein has been investigated for enzymatic activation by a panel of amines and amino acids. Of the 24 compounds tested in this study, the most effective MreCA activators were L-adrenaline (KA of 15 nM), 2-aminoethyl-piperazine/morpholine (KAs of 0.25-0.33 µM), histamine, L-4-amino-phenylalanine, D-Phe, L-/D-DOPA, and L-/D-Trp (KAs of 0.32 - 0.90 µM). The least effective activators were L-/D-Tyr, L-Asp, L-/D-Glu, and L-His, with activation constants ranging between 4.04 and 12.8 µM. As MreCA is involved in dandruff and seborrhoeic dermatitis, these results are of interest to identify modulators of the activity of enzymes involved in the metabolic processes of such fungi.

Molecular epidemiology, in vitro susceptibility and exoenzyme screening of Malassezia clinical isolates.

Introduction. Malassezia folliculitis (MF) and pityriasis versicolor (PV) are common dermatoses caused by Malassezia species. Their molecular epidemiology, drug susceptibility and exoenzymes are rarely reported in China.Aim. To investigate the molecular epidemiology, drug susceptibility and enzymatic profile of Malassezia clinical isolates.Methodology. Malassezia strains were recovered from MF and PV patients and healthy subjects (HS) and identified by sequencing analysis. The minimum inhibitory concentrations (MICs) of nine antifungals (posaconazole, voriconazole, itraconazole, fluconazole, ketoconazole, miconazole, bifonazole, terbinafine and caspofungin) and tacrolimus, the interactions between three antifungals (itraconazole, ketoconazole and terbinafine) and tacrolimus, and the extracellular enzyme profile were evaluated using broth and checkerboard microdilution and the Api-Zym system, respectively.Results. Among 392 Malassezia isolates from 729 subjects (289 MF, 218 PV and 222 HS), Malassezia furfur and Malassezia globosa accounted for 67.86 and 18.88 %, respectively. M. furfur was the major species in MF and PV patients and HS. Among 60M. furfur and 50M. globosa strains, the MICs for itraconazole, posaconazole, voriconazole and ketoconazole were <1 µg ml-1. M. furfur was more susceptible to itraconazole, terbinafine and bifonazole but tolerant to miconazole compared with M. globosa (P<0.05). Synergistic effects between terbinafine and itraconazole or between tacrolimus and itraconazole, ketoconazole or terbinafine occurred in 6, 7, 6 and 9 out of 37 strains, respectively. Phosphatases, lipases and proteases were mainly secreted in 51 isolates.Conclusions. Itraconazole, posaconazole, voriconazole and ketoconazole are theagents against which there is greatest susceptibility. Synergistic effects between terbinafine and itraconazole or tacrolimas and antifungals may be irrelevant to clinical application. Overproduction of lipases could enhance the skin inhabitation of M. furfur.

Cloning, Purification, and Characterization of a ß-Carbonic Anhydrase from Malassezia restricta, an Opportunistic Pathogen Involved in Dandruff and Seborrheic Dermatitis.

The cloning, purification, and initial characterization of the ß-carbonic anhydrase (CA, EC 4.2.1.1) from the genome of the opportunistic pathogen Malassezia restricta (MreCA), which a fungus involved in dandruff and seborrheic dermatitis (SD), is reported. MreCA is a protein consisting of 230 amino acid residues and shows high catalytic activity for the hydration of CO2 into bicarbonate and protons, with the following kinetic parameters: kcat of 1.06 × 106 s-1 and kcat/KM of 1.07 × 108 M-1 s-1. It is also sensitive to inhibition by the sulfonamide acetazolamide (KI of 50.7 nM). Phylogenetically, MreCA and other CAs from various Malassezia species seem to be on a different branch, distinct from that of other ß-CAs found in fungi, such as Candida spp., Saccharomyces cerevisiae, Aspergillus fumigatus, and Sordaria macrospora, with only Cryptococcus neoformans and Ustilago maydis enzymes clustering near MreCA. The further characterization of this enzyme and the identification of inhibitors that may interfere with its life cycle might constitute new strategies for fighting dandruff and SD.

Secreted Hydrolytic and Haemolytic Activities of Malassezia Clinical Strains.

Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms' lipase and phospholipase activities trigger inflammation in the host; thus, these lipase and phospholipase activities are widely recognised as some of the most important factors in Malassezia pathogenesis. In this study, we sought to investigate and examine the relationship between these secreted hydrolytic activities and haemolytic activity in newly isolated Malassezia clinical strains. This characterisation was expected to elucidate pathogenicity of this fungus. We isolated 35 clinical strains of Malassezia spp.; the most frequently isolated species were M. sympodialis and M. furfur. Next, we analysed the hydrolytic activities of all of these clinical isolates; all of these strains (except for one M. dermatis isolate) showed detectable lipase and phospholipase activities against 4-nitrophenyl palmitate and L-α-phosphatidylcholine, dipalmitoyl, respectively. Most of the M. globosa isolates showed higher lipase activities than isolates of other Malassezia species. In terms of phospholipase activity, no significant difference was observed among species of Malassezia, although one isolate of M. globosa showed considerably higher phospholipase activity than the others. All tested strains also exhibited haemolytic activity, both as determined using 5% (v/v) sheep blood agar (halo assay) and by quantitative assay. Although all tested strains showed detectable haemolytic activity, we did not observe an apparent correlation between the secreted lipase and phospholipase activities and haemolytic activity. We infer that the haemolytic activities of Malassezia spp. are mediated by non-enzymatic factor(s) that are present in the secreted samples.

Identification and Evaluation of Inhibitors of Lipase from Malassezia restricta using Virtual High-Throughput Screening and Molecular Dynamics Studies.

Recent studies revealed the role of lipase in the pathogenicity of Malassezia restricta in dandruff and seborrheic dermatitis (D/SD). The lipase from M. restricta (Mrlip1) is considered a potential target for dandruff therapy. In this work, we performed structure-based virtual screening in Zinc database to find the natural bioactive inhibitors of Mrlip1. We identified three compounds bearing superior affinity and specificity from the Traditional Chinese Medicine database (~60,000 compounds), and their binding patterns with Mrlip1 were analyzed in detail. Additionally, we performed three sets of 100 ns MD simulations of each complex in order to understand the interaction mechanism of Mrlip1 with known inhibitor RHC80267 and the newly identified compounds such as ZINC85530919, ZINC95914464 and ZINC85530320, respectively. These compounds bind to the active site cavity and cause conformational changes in Mrlip1. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) studies suggested that the average binding energy was stronger in the case of Mrlip1-ZINC85530919 and Mrlip1-ZINC95914464. The selected natural inhibitors might act as promising lead drugs against Mrlip1. Further, the present study will contribute to various steps involved in developing and creating potent drugs for several skin diseases including dandruff.

Benzenesulfonamides incorporating nitrogenous bases show effective inhibition of ß-carbonic anhydrases from the pathogenic fungi Cryptococcus neoformans, Candida glabrata and Malassezia globosa.

There is an urgent need for new chemotherapic agents to treat human fungal infections due to emerging and spreading globally resistance mechanisms. Among the new targets that have been recently investigated for the development of antifungal drugs there are the metallo-enzymes Carbonic Anhydrases (CAs, EC 4.2.1.1). The inhibition of the ß-CAs identified in many pathogenic fungi leads to an impairment of parasite growth and virulence, which in turn leads to a significant anti-infective effect. Based on antifungal nucleoside antibiotics, the inhibition of the ß-CAs from the resistance-showing fungi Candida glabrata (CgNce103), Cryptococcus neoformans (Can2) and Malasszia globosa (MgCA) with a series of benzenesulfonamides bearing nitrogenous bases, such as uracil and adenine, is here reported. Many such compounds display low nanomolar (<100 nM) inhibitory potency against Can2 and CgNce103, whereas the activity of MgCA is considerably less affected (inhibition constants in the range 138.8-5601.5 nM). The ß-CAs inhibitory data were compared with those against α-class human ubiquitous isoforms. Interesting selective inhibitory activities for the target fungal CAs over hCA I and II were reported, which make nitrogenous base benzenesulfonamides interesting tools and leads for further investigations in search of new antifungal with innovative mechanisms of action.

Understanding the Mechanism of Action of the Anti-Dandruff Agent Zinc Pyrithione against Malassezia restricta.

Dandruff is known to be associated with Malassezia restricta. Zinc pyrithione (ZPT) has been used as an ingredient in anti-dandruff treatments. The mechanism of ZPT has been investigated in several studies; however, a non-pathogenic model yeast, such as Saccharomyces cerevisiae was most often used. The aim of the present study was to understand how ZPT inhibits the growth of M. restricta. We analyzed the cellular metal content and transcriptome profile of ZPT-treated M. restricta cells and found that ZPT treatment dramatically increased cellular zinc levels, along with a small increase in cellular copper levels. Moreover, our transcriptome analysis showed that ZPT inhibits Fe-S cluster synthesis in M. restricta. We also observed that ZPT treatment significantly reduced the expression of lipases, whose activities contribute to the survival and virulence of M. restricta on human skin. Therefore, the results of our study suggest that at least three inhibitory mechanisms are associated with the action of ZPT against M. restricta: (i) an increase in cellular zinc levels, (ii) inhibition of mitochondrial function, and (iii) a decrease in lipase expression.

MGL_3741 gene contributes to pathogenicity of Malassezia globosa in pityriasis versicolor.

Dihydroxyacid dehydratase (DHAD) is a key enzyme in biosynthetic pathway of isoleucine and valine. This pathway is absent in human but exists in various organisms such as fungi. Using RNA-seq analysis in this study, we identified MGL_3741gene which encodes DHAD protein in Malassezia globosa (M. globosa). Furthermore, we found that mentioned gene is homologous to the Ustilago maydis, Saccharomyces cerevisiae, Aspergillus flavus, and Aspergillus fumigatus ILV3P. For understanding the probable role of this gene in pathogenicity of M. globosa, we applied Real-time PCR to investigate the differentially expressed of the MGL_3741 gene in healthy and pathogenic states. Our results indicate a significant difference between two mentioned stats. These results revealed that ILV3-like gene in M. globosa can be related to the pathogenicity of this yeast.

Malassezia pachydermatis from animals: Planktonic and biofilm antifungal susceptibility and its virulence arsenal.

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to>64 µg/mL for azole derivatives, 1 to >16 µg/mL for amphotericin B and 0.03 to 0.25 µg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.

Skin Commensal Malassezia globosa Secreted Protease Attenuates Staphylococcus aureus Biofilm Formation.

Skin provides the first defense against pathogenic micro-organisms and is also colonized by a diverse microbiota. Phylogenetic analysis of whole skin microbiome at different skin sites in health and disease has generated important insights on possible microbial involvement in modulating skin health. However, functional roles of the skin microbial community remain unclear. The most common sebaceous skin commensal yeasts are the basidiomycetes, Malassezia. Here, we characterized the dominant secreted Malassezia globosa protease in culture and subsequently named it Malassezia globosa Secreted Aspartyl Protease 1 (MgSAP1). We defined recombinant MgSAP1's substrate cleavage profile using an unbiased, mass-spectrometry-based technique. We show that this enzyme is physiologically relevant as mgsap1 expression was detected on at least one facial skin site of 17 healthy human volunteers. In addition, we demonstrated that this protease rapidly hydrolyzes Staphylococcus aureus protein A, an important S. aureus virulence factor involved in immune evasion and biofilm formation. We further observed that MgSAP1 has anti-biofilm properties against S. aureus. Taken together, our study defines a role for the skin fungus Malassezia in inter-kingdom interactions and suggests that this fungus and the enzymes it produces may be beneficial for skin health.

Malassezia globosa MgMDL2 lipase: Crystal structure and rational modification of substrate specificity.

Lipases play an important role in physiological metabolism and diseases, and also have multiple industrial applications. Rational modification of lipase specificity may increase the commercial utility of this group of enzymes, but is hindered by insufficient mechanistic understanding. Here, we report the 2.0 Å resolution crystal structure of a mono- and di-acylglycerols lipase from Malassezia globosa (MgMDL2). Interestingly, residues Phe278 and Glu282 were found to involve in substrate recognition because mutation on each residue led to convert MgMDL2 to a triacylglycerol (TAG) lipase. The Phe278Ala and Glu282Ala mutants also acquired ability to synthesize TAGs by esterification of glycerol and fatty acids. By in silicon analysis, steric hindrance of these residues seemed to be key factors for the altered substrate specificity. Our work may shed light on understanding the unique substrate selectivity mechanism of mono- and di-acylglycerols lipases, and provide a new insight for engineering biocatalysts with desired catalytic behaviors for biotechnological application.

Inhibition of Malassezia globosa carbonic anhydrase with phenols.

A panel of 22 phenols was investigated as inhibitors of the ß-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa (MgCA), a validated anti-dandruff drug target. The displayed inhibitory activities were compared to the ones previously reported against the off-target widely distributed human (h) isoforms hCA I and II. All tested phenols possessed a better efficacy in inhibiting MgCA than the clinically used sulfonamide acetazolamide, with KIs in the range of 2.5 and 65.0µM. A homology-built model of MgCA was also used for understanding the binding mode of phenols to the fungal enzyme. Indeed, a wide network of hydrogen bonds and hydrophobic interactions between the phenol and active site residues were evidenced. The OH moiety of the inhibitor was observed anchored to the zinc-coordinated water, also making hydrogen bonds with Ser48 and Asp49. The diverse substituents at the phenolic scaffold were observed to interact with different portions of the hydrophobic pocket according to their nature and position. Considering the effective MgCA inhibitory properties of phenols, beside to the rather low inhibition against the off-target hCA I and II, this class of compounds might be of considerable interest in the cosmetics field as potential anti-dandruff drugs.

Dithiocarbamates effectively inhibit the ß-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa.

A series of dithiocarbamates (DTCs) was investigated for the inhibition of the ß-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa, MgCA, a validated anti-dandruff drug target. These DTCs incorporate various scaffold, among which those of N,N-dimethylaminoethylenediamine, the aminoalcohols with 3-5 carbon atoms in their molecule, 3-amino-quinuclidine, piperidine, morpholine and piperazine derivatives, as well as phenethylamine and its 4-sulfamoylated derivative. Several DTCs resulted more effective in inhibiting MgCA compared to the standard sulfonamide drug acetazolamide (KI of 74µM), with KIs ranging between 383 and 6235nM. A computational approach, involving a homology modeling of the enzyme and docking inhibitors within its active site, helped us rationalize the results. This study may contribute to better understand the inhibition profile of MgCA, and offer new ideas for the design of modulators of activity which belong to less investigated chemical classes, thus potentially useful to combat dandruff and other fungal infections.

Whole genome sequencing analysis of the cutaneous pathogenic yeast Malassezia restricta and identification of the major lipase expressed on the scalp of patients with dandruff.

Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase-encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus-host interaction.

ß-Endorphin enhances the phospholipase activity of the dandruff causing fungi Malassezia globosa and Malassezia restricta.

ß-Endorphin is known to stimulate phospholipase production by Malassezia pachydermatis during canine dermatoses. The role of ß-endorphin in Malassezia infection in humans is not well studied. The present study compares the influence of ß-endorphin on Malassezia globosa and Malassezia restricta isolated from patients with seborrhoeic dermatitis/dandruff (SD/D) and healthy controls. Malassezia isolates (five each of the two species from patients and healthy controls) were grown on modified Dixon's agar with or without 100 nmol/L ß-endorphin. Phospholipase activity was quantified based on its ability to hydrolyze L-α-phosphatidylcholine dimyristoyl (phospholipid substrate). Free fatty acid was measured by a colorimetry method. In isolates from patients, the phospholipase activity significantly increased after exposure to ß-endorphin (M. globosa, P = .04; M. restricta, P = .001), which did not occur in isolates from healthy controls. Moreover, after ß-endorphin exposure the patient isolates had significantly higher (P = .0004) phospholipase activity compared to the healthy control isolates. The results suggest that isolates of M. globosa and M. restricta from patients may differ from those of healthy humans.

Simplified Enzymatic Upgrading of High-Acid Rice Bran Oil Using Ethanol as a Novel Acyl Acceptor.

One of the major challenges in the upgrading of high-acid rice bran oil (RBO) is to efficiently reduce the amount of free fatty acids. Here we report a novel method for upgrading high-acid RBO using ethanol as a novel acyl acceptor in combination with a highly selective lipase from Malassezia globosa (SMG1-F278N). This process enabled an unprecedented deacidification efficiency of up to 99.80% in a short time (6 h); the immobilized SMG1-F278N used in deacidification exhibited excellent operational stability and could be used for at least 10 consecutive batches without detectable loss in activity. Scale-up was performed under optimized conditions to verify the applicability of this process, and low-acid (0.08%) RBO with a high level of γ-oryzanol (27.8 g/kg) and γ-oryzanol accumulation fold (1.5) was obtained after molecular distillation at lower temperature (120 °C). Overall, we report a simplified and efficient procedure for the production of edible RBO from high-acid RBO.

Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa.

Malassezia globosa cytochromes P450 CYP51 and CYP5218 are sterol 14α-demethylase (the target of azole antifungals) and a putative fatty acid metabolism protein (and a potential azole drug target), respectively. Lanosterol, eburicol and obtusifoliol bound to CYP51 with Kd values of 32, 23 and 28 µM, respectively, catalyzing sterol 14α-demethylation with respective turnover numbers of 1.7 min(-1), 5.6 min(-1) and 3.4 min(-1). CYP5218 bound a range of fatty acids with linoleic acid binding strongest (Kd 36 µM), although no metabolism could be detected in reconstitution assays or role in growth on lipids. Clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole and ketaminazole bound tightly to CYP51 (Kd ≤ 2 to 11 nM). In contrast, fluconazole did not bind to CYP5218, voriconazole and ketaminazole bound weakly (Kd ~107 and ~12 µM), whereas ketoconazole, clotrimazole and itraconazole bound strongest to CYP5218 (Kd ~1.6, 0.5 and 0.4 µM) indicating CYP5218 to be only a secondary target of azole antifungals. IC50 determinations confirmed M. globosa CYP51 was strongly inhibited by azole antifungals (0.15 to 0.35 µM). MIC100 studies showed itraconazole should be considered as an alternative to ketoconazole given the potency and safety profiles and the CYP51 assay system can be used in structure-activity studies in drug development.