Claire M. Hull

The Clinical Candidate VT-1161 Is a Highly Potent Inhibitor of Candida albicans CYP51 but Fails To Bind the Human Enzyme

ABSTRACT The binding and cytochrome P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 (ERG11) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, characteristic of heme iron coordination. The binding affinity of VT-1161 for Candida albicans CYP51 was high (dissociation constant [Kd], ≤39 nM) and similar to that of the pharmaceutical azole antifungals (Kd, ≤50 nM). In stark contrast, VT-1161 at concentrations up to 86 μM did not perturb the spectrum of recombinant human CYP51, whereas all the pharmaceutical azoles bound to human CYP51. In reconstitution assays, VT-1161 inhibited Candida albicans CYP51 activity in a tight-binding fashion with a potency similar to that of the pharmaceutical azoles but failed to inhibit the human enzyme at the highest concentration tested (50 μM). In addition, VT-1161 (MIC = 0.002 μg ml−1) had a more pronounced fungal sterol disruption profile (increased levels of methylated sterols and decreased levels of ergosterol) than the known CYP51 inhibitor voriconazole (MIC = 0.004 μg ml−1). Furthermore, VT-1161 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. In summary, VT-1161 potently inhibited Candida albicans CYP51 and culture growth but did not inhibit human CYP51, demonstrating a >2,000-fold selectivity. This degree of potency and selectivity strongly supports the potential utility of VT-1161 in the treatment of Candida infections.

Facultative Sterol Uptake in an Ergosterol-Deficient Clinical Isolate of Candida glabrata Harboring a Missense Mutation in ERG11 and Exhibiting Cross-Resistance to Azoles and Amphotericin B

ABSTRACT We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 μg ml−1, respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium (glcYM). However, when grown on sterol-supplemented glcYM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ7-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented glcYM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using glcYM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using glcYM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 μg AMB ml−1, respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.

Two Clinical Isolates of Candida glabrata Exhibiting Reduced Sensitivity to Amphotericin B Both Harbor Mutations in ERG2

ABSTRACT Two novel isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B (MIC, 8 μg ml−1) were found to be ERG2 mutants, wherein Δ8-sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr121 in ERG2; the corresponding residue (Thr119) in Saccharomyces cerevisiae is essential for sterol Δ8-Δ7 isomerization. This constitutes the first report of C. glabrata harboring mutations in ERG2 and exhibiting reduced sensitivity to amphotericin B.

Clotrimazole as a potent agent for treating the oomycete fish pathogen Saprolegnia parasitica through inhibition of sterol 14α-demethylase (CYP51).

ABSTRACT A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks , 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd , ≤3 nM) and prothioconazole-desthio the lowest (K d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml−1). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml−1) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml−1). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml−1) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.

A case of unprovoked venous thromboembolism in a marathon athlete presenting atypical sequelae: What are the chances?

Marathon runners are exposed to multiple thrombogenic risk factors including dehydration and hemoconcentration, injury and inflammation, long‐distance travel between events, and contraceptive usage. However, despite awareness about thromboembolism and several case reports detailing life‐threatening hypercoagulopathies in athletes, the prevalence of venous thromboembolism in marathon runners remains uncharted. There is a lack of data and evidence‐based guidelines for these athletes and for healthcare providers, including general medical practitioners and sports physicians. We present an episode of unprovoked deep vein thrombosis (DVT) and pulmonary embolism (PE) in a female marathon athlete who presented with atypical sequelae over the course of 8 months, and identify some “easy‐to‐miss” warning signs and symptoms. Through dialogue with the patient regarding their personal questions and anxieties surrounding idiopathic DVT‐PE, we identify a clear need for more accessible information and comprehensive research concerning the detection, prevalence, and long‐term management of venous thromboembolism in athletes. We discuss the possibility that being an athlete might constitute a more significant risk factor for venous thromboembolism than is currently estimated by commonly used diagnostic protocols and conclude that there is quite possibly a need for more specific clinical guidelines for athletes in this area.

S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) Reduce In Vitro Inhibition by Fluconazole

ABSTRACT The effects of S279F and S279Y point mutations in Candida albicans CYP51 (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 higher Kd values observed.

Venous Thromboembolism and Marathon Athletes

V enous thromboembolism (VTE) is the collective term for deep vein thrombosis and pulmonary embolism, both of which constitute globally significant public health burdens. Because awareness of VTE is key to its prevention,1 efforts to disseminate advisory and educational information among medical professionals and the population at large, with specific resources for at-risk groups, remain crucial. The benefits of moderate and regular exercise for the general adult population are indisputable. However, for the marathon athlete who …

Mitigation of human-pathogenic fungi that exhibit resistance to medical agents: can clinical antifungal stewardship help?

Reducing indiscriminate antimicrobial usage to combat the expansion of multidrug-resistant human-pathogenic bacteria is fundamental to clinical antibiotic stewardship. In contrast to bacteria, fungal resistance traits are not understood to be propagated via mobile genetic elements, and it has been proposed that a global explosion of resistance to medical antifungals is therefore unlikely. Clinical antifungal stewardship has focused instead on reducing the drug toxicity and high costs associated with medical agents. Mitigating the problem of human-pathogenic fungi that exhibit resistance to antimicrobials is an emergent issue. This article addresses the extent to which clinical antifungal stewardship could influence the scale and epidemiology of resistance to medical antifungals both now and in the future. The importance of uncharted selection pressure exerted by agents outside the clinical setting (agricultural pesticides, industrial xenobiotics, biocides, pharmaceutical waste and others) on environmentally ubiquitous spore-forming molds that are lesserstudied but increasingly responsible for drug-refractory infections is considered.

Co-production of ethanol and squalene using a Saccharomyces cerevisiae ERG1 (squalene epoxidase) mutant and agro-industrial feedstock

BackgroundGenetically customised Saccharomyces cerevisiae that can produce ethanol and additional bio-based chemicals from sustainable agro-industrial feedstocks (for example, residual plant biomass) are of major interest to the biofuel industry. We investigated the microbial biorefinery concept of ethanol and squalene co-production using S. cerevisiae (strain YUG37-ERG1) wherein ERG1 (squalene epoxidase) transcription is under the control of a doxycycline-repressible tet07-CYC1 promoter. The production of ethanol and squalene by YUG37-ERG1 grown using agriculturally sourced grass juice supplemented with doxycycline was assessed.ResultsUse of the tet07-CYC1 promoter permitted regulation of ERG1 expression and squalene accumulation in YUG37-ERG1, allowing us to circumvent the lethal growth phenotype seen when ERG1 is disrupted completely. In experiments using grass juice feedstock supplemented with 0 to 50 μg doxycycline mL-1, YUG37-ERG1 fermented ethanol (22.5 [±0.5] mg mL-1) and accumulated the highest squalene content (7.89 ± 0.25 mg g-1 dry biomass) and yield (18.0 ± 4.18 mg squalene L-1) with supplements of 5.0 and 0.025 μg doxycycline mL-1, respectively. Grass juice was found to be rich in water-soluble carbohydrates (61.1 [±3.6] mg sugars mL-1) and provided excellent feedstock for growth and fermentation studies using YUG37-ERG1.ConclusionResidual plant biomass components from crop production and rotation systems represent possible substrates for microbial fermentation of biofuels and bio-based compounds. This study is the first to utilise S. cerevisiae for the co-production of ethanol and squalene from grass juice. Our findings underscore the value of the biorefinery approach and demonstrate the potential to integrate microbial bioprocess engineering with existing agriculture.

Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept

Microbial biotechnology and biotransformations promise to diversify the scope of the biorefinery approach for the production of high-value products and biofuels from industrial, rural and municipal waste feedstocks. In addition to bio-based chemicals and metabolites, microbial biomass itself constitutes an obvious but overlooked by-product of existing biofermentation systems which warrants fuller attention. The probiotic yeast Saccharomyces boulardii is used to treat gastrointestinal disorders and marketed as a human health supplement. Despite its relatedness to S. cerevisiae that is employed widely in biotechnology, food and biofuel industries, the alternative applications of S. boulardii are not well studied. Using a biorefinery approach, we compared the bioethanol and biomass yields attainable from agriculturally-sourced grass juice using probiotic S. boulardii (strain MYA-769) and a commercial S. cerevisiae brewing strain (Turbo yeast). Maximum product yields for MYA-769 (39.18 [±2.42] mg ethanol mL−1 and 4.96 [±0.15] g dry weight L−1) compared closely to those of Turbo (37.43 [±1.99] mg mL−1 and 4.78 [±0.10] g L−1, respectively). Co-production, marketing and/or on-site utilisation of probiotic yeast biomass as a direct-fed microbial to improve livestock health represents a novel and viable prospect for rural biorefineries. Given emergent evidence to suggest that dietary yeast supplementations might also mitigate ruminant enteric methane emissions, the administration of probiotic yeast biomass could also offer an economically feasible way of reducing atmospheric CH4.