Research

22 pages, 5452 KiB

Open AccessArticle

Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate

by Danyon O. Graham, Rajni K. Wilson, Yasmeen N. Ruma, Mikhail V. Keniya, Joel D. A. Tyndall and Brian C. Monk

J. Fungi 2021, 7(11), 897; https://doi.org/10.3390/jof7110897 - 24 Oct 2021

Cited by 3 | Viewed by 2061

Abstract

Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to [...] Read more.

Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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17 pages, 5327 KiB

Open AccessArticle

Raman Characterization of Fungal DHN and DOPA Melanin Biosynthesis Pathways

by Benjamin D. Strycker, Zehua Han, Aysan Bahari, Tuyetnhu Pham, Xiaorong Lin, Brian D. Shaw, Alexei V. Sokolov and Marlan O. Scully

J. Fungi 2021, 7(10), 841; https://doi.org/10.3390/jof7100841 - 07 Oct 2021

Cited by 11 | Viewed by 2627

Abstract

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we [...] Read more.

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we characterize the Raman spectra of wild-type Aspergillus fumigatus and Cryptococcus neoformans and their melanin biosynthetic mutants and provide a rough “map” of the DHN (A. fumigatus) and DOPA (C. neoformans) melanin biosynthetic pathways. We compare this map to the Raman spectral data of Aspergillus nidulans wild-type and melanin biosynthetic mutants obtained from a previous study. We find that the fully polymerized A. nidulans melanin cannot be classified according to the DOPA pathway; nor can it be solely classified according to the DHN pathway, consistent with mutational analysis and chemical inhibition studies. Our approach points the way forward for an increased understanding of, and methodology for, investigating fungal melanins. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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9 pages, 429 KiB

Open AccessArticle

In Vitro Activity of Novel Antifungal Olorofim against Filamentous Fungi and Comparison to Eight Other Antifungal Agents

by Ourania Georgacopoulos, Natalie S. Nunnally, Eric M. Ransom, Derek Law, Mike Birch, Shawn R. Lockhart and Elizabeth L. Berkow

J. Fungi 2021, 7(5), 378; https://doi.org/10.3390/jof7050378 - 12 May 2021

Cited by 19 | Viewed by 3220

Abstract

Olorofim is a novel antifungal drug that belongs to the orotomide drug class which inhibits fungal dihydroorotate dehydrogenase (DHODH), thus halting pyrimidine biosynthesis and ultimately DNA synthesis, cell growth and division. It is being developed at a time when many invasive fungal infections [...] Read more.

Olorofim is a novel antifungal drug that belongs to the orotomide drug class which inhibits fungal dihydroorotate dehydrogenase (DHODH), thus halting pyrimidine biosynthesis and ultimately DNA synthesis, cell growth and division. It is being developed at a time when many invasive fungal infections exhibit antifungal resistance or have limited treatment options. The goal of this study was to evaluate the in vitro effectiveness of olorofim against a large collection of recently isolated, clinically relevant American mold isolates. In vitro antifungal activity was determined for 246 azole-susceptible Aspergillus fumigatus isolates, five A. fumigatus with TR₃₄/L98H-mediated resistance, 19 Rhizopus species isolates, 21 Fusarium species isolates, and one isolate each of six other species of molds. Olorofim minimum inhibitory concentrations (MICs) were compared to antifungal susceptibility testing profiles for amphotericin B, anidulafungin, caspofungin, isavuconazole, itraconazole, micafungin, posaconazole, and voriconazole. Olorofim MICs were significantly lower than those of the echinocandin and azole drug classes and amphotericin B. A. fumigatus wild type and resistant isolates shared the same MIC50 = 0.008 μg/mL. In non-Aspergillus susceptible isolates (MIC ≤ 2 μg/mL), the geometric mean (GM) MIC to olorofim was 0.54 μg/mL with a range of 0.015–2 μg/mL. Olorofim had no antifungal activity (MIC ≥ 2 μg/mL) against 10% of the collection (31 in 297), including some isolates from Rhizopus spp. and Fusarium spp. Olorofim showed promising activity against A. fumigatus and other molds regardless of acquired azole resistance. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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0 pages, 5813 KiB

Open AccessArticle

Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins

by Cristina Jiménez-Ortigosa, Jennifer Jiang, Muyuan Chen, Xuyuan Kuang, Kelley R. Healey, Paul Castellano, Nikpreet Boparai, Steven J. Ludtke, David S. Perlin and Wei Dai

J. Fungi 2021, 7(2), 120; https://doi.org/10.3390/jof7020120 - 06 Feb 2021

Cited by 15 | Viewed by 5629 | Correction

Abstract

Fungal plasma membrane proteins have long been recognized as targets for the development of antifungal agents. Despite recent progress in experimental approaches and computational structural predictions, our knowledge of the structural dynamics and spatial distribution of these membrane proteins in the context of [...] Read more.

Fungal plasma membrane proteins have long been recognized as targets for the development of antifungal agents. Despite recent progress in experimental approaches and computational structural predictions, our knowledge of the structural dynamics and spatial distribution of these membrane proteins in the context of their native lipid environment remains limited. By applying cryo-electron tomography (cryoET) and subtomogram analysis, we aim to characterize the structural characteristics and spatial distribution of membrane proteins present in Candida glabrata plasma membranes. This study has resulted in the identification of the membrane-embedded structure of the fungal H⁺-ATPase, Pma1. Tomograms of the plasma membrane revealed that Pma1 complexes are heterogeneously distributed as hexamers that cluster into distinct membrane microdomains. This study characterizes fungal membrane proteins in the native cellular landscape and highlights the unique potential of cryoET to advance our understanding of cellular biology and biological systems. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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Review

Jump to: Research, Other

16 pages, 1562 KiB

Open AccessReview

Directed Mutational Strategies Reveal Drug Binding and Transport by the MDR Transporters of Candida albicans

by Atanu Banerjee, Jorgaq Pata, Suman Sharma, Brian C. Monk, Pierre Falson and Rajendra Prasad

J. Fungi 2021, 7(2), 68; https://doi.org/10.3390/jof7020068 - 20 Jan 2021

Cited by 11 | Viewed by 2321

Abstract

Multidrug resistance (MDR) transporters belonging to either the ATP-Binding Cassette (ABC) or Major Facilitator Superfamily (MFS) groups are major determinants of clinical drug resistance in fungi. The overproduction of these proteins enables the extrusion of incoming drugs at rates that prevent lethal effects. [...] Read more.

Multidrug resistance (MDR) transporters belonging to either the ATP-Binding Cassette (ABC) or Major Facilitator Superfamily (MFS) groups are major determinants of clinical drug resistance in fungi. The overproduction of these proteins enables the extrusion of incoming drugs at rates that prevent lethal effects. The promiscuity of these proteins is intriguing because they export a wide range of structurally unrelated molecules. Research in the last two decades has used multiple approaches to dissect the molecular basis of the polyspecificity of multidrug transporters. With large numbers of drug transporters potentially involved in clinical drug resistance in pathogenic yeasts, this review focuses on the drug transporters of the important pathogen Candida albicans. This organism harbors many such proteins, several of which have been shown to actively export antifungal drugs. Of these, the ABC protein CaCdr1 and the MFS protein CaMdr1 are the two most prominent and have thus been subjected to intense site-directed mutagenesis and suppressor genetics-based analysis. Numerous results point to a common theme underlying the strategy of promiscuity adopted by both CaCdr1 and CaMdr1. This review summarizes the body of research that has provided insight into how multidrug transporters function and deliver their remarkable polyspecificity. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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35 pages, 2021 KiB

Open AccessReview

Roles for Structural Biology in the Discovery of Drugs and Agrochemicals Targeting Sterol 14α-Demethylases

by Brian C. Monk and Mikhail V. Keniya

J. Fungi 2021, 7(2), 67; https://doi.org/10.3390/jof7020067 - 20 Jan 2021

Cited by 11 | Viewed by 2925 | Correction

Abstract

Antifungal drugs and antifungal agrochemicals have significant limitations. These include several unintended consequences of their use including the growing importance of intrinsic and acquired resistance. These problems underpin an increasingly urgent need to improve the existing classes of antifungals and to discover novel [...] Read more.

Antifungal drugs and antifungal agrochemicals have significant limitations. These include several unintended consequences of their use including the growing importance of intrinsic and acquired resistance. These problems underpin an increasingly urgent need to improve the existing classes of antifungals and to discover novel antifungals. Structural insights into drug targets and their complexes with both substrates and inhibitory ligands increase opportunity for the discovery of more effective antifungals. Implementation of this promise, which requires multiple skill sets, is beginning to yield candidates from discovery programs that could more quickly find their place in the clinic. This review will describe how structural biology is providing information for the improvement and discovery of inhibitors targeting the essential fungal enzyme sterol 14α-demethylase. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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13 pages, 2782 KiB

Open AccessReview

Strategies to Better Target Fungal Squalene Monooxygenase

by Alia A. Sagatova

J. Fungi 2021, 7(1), 49; https://doi.org/10.3390/jof7010049 - 13 Jan 2021

Cited by 13 | Viewed by 3973

Abstract

Fungal pathogens present a challenge in medicine and agriculture. They also harm ecosystems and threaten biodiversity. The allylamine class of antimycotics targets the enzyme squalene monooxygenase. This enzyme occupies a key position in the sterol biosynthesis pathway in eukaryotes, catalyzing the rate-limiting reaction [...] Read more.

Fungal pathogens present a challenge in medicine and agriculture. They also harm ecosystems and threaten biodiversity. The allylamine class of antimycotics targets the enzyme squalene monooxygenase. This enzyme occupies a key position in the sterol biosynthesis pathway in eukaryotes, catalyzing the rate-limiting reaction by introducing an oxygen atom to the squalene substrate converting it to 2,3-oxidosqualene. Currently, terbinafine—the most widely used allylamine—is mostly used for treating superficial fungal infections. The ability to better target this enzyme will have significant implications for human health in the treatment of fungal infections. The human orthologue can also be targeted for cholesterol-lowering therapeutics and in cancer therapies. This review will focus on the structural basis for improving the current therapeutics for fungal squalene monooxygenase. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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22 pages, 1412 KiB

Open AccessReview

Sterol 14α-Demethylase Ligand-Binding Pocket-Mediated Acquired and Intrinsic Azole Resistance in Fungal Pathogens

by Katharina Rosam, Brian C. Monk and Michaela Lackner

J. Fungi 2021, 7(1), 1; https://doi.org/10.3390/jof7010001 - 22 Dec 2020

Cited by 27 | Viewed by 4182

Abstract

The fungal cytochrome P450 enzyme sterol 14α-demethylase (SDM) is a key enzyme in the ergosterol biosynthesis pathway. The binding of azoles to the active site of SDM results in a depletion of ergosterol, the accumulation of toxic intermediates and growth inhibition. The prevalence [...] Read more.

The fungal cytochrome P450 enzyme sterol 14α-demethylase (SDM) is a key enzyme in the ergosterol biosynthesis pathway. The binding of azoles to the active site of SDM results in a depletion of ergosterol, the accumulation of toxic intermediates and growth inhibition. The prevalence of azole-resistant strains and fungi is increasing in both agriculture and medicine. This can lead to major yield loss during food production and therapeutic failure in medical settings. Diverse mechanisms are responsible for azole resistance. They include amino acid (AA) substitutions in SDM and overexpression of SDM and/or efflux pumps. This review considers AA affecting the ligand-binding pocket of SDMs with a primary focus on substitutions that affect interactions between the active site and the substrate and inhibitory ligands. Some of these interactions are particularly important for the binding of short-tailed azoles (e.g., voriconazole). We highlight the occurrence throughout the fungal kingdom of some key AA substitutions. Elucidation of the role of these AAs and their substitutions may assist drug design in overcoming some common forms of innate and acquired azole resistance. Full article

(This article belongs to the Special Issue The Application of Structural Biology in Antifungal Drug Discovery)

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