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Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry

The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can b...

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Autores principales: Liebl, Maximilian, Huber, Ludwig, Elsaman, Hesham, Merschak, Petra, Wagener, Johannes, Gsaller, Fabio, Müller, Christoph
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10381423/
https://www.ncbi.nlm.nih.gov/pubmed/37504756
http://dx.doi.org/10.3390/jof9070768
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author Liebl, Maximilian
Huber, Ludwig
Elsaman, Hesham
Merschak, Petra
Wagener, Johannes
Gsaller, Fabio
Müller, Christoph
author_facet Liebl, Maximilian
Huber, Ludwig
Elsaman, Hesham
Merschak, Petra
Wagener, Johannes
Gsaller, Fabio
Müller, Christoph
author_sort Liebl, Maximilian
collection PubMed
description The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can be used to identify compounds interacting with the enzymes of isoprenoid biosynthesis, an important part of the ergosterol biosynthesis pathway. The method was validated according to the EMEA guideline on bioanalytical method validation. Aspergillus fumigatus hyphae and Saccharomyces cerevisiae cells were lysed mechanically in an aqueous buffer optimized for the enzymatic deconjugation of isoprenoid pyrophosphates. The residual alcohols were extracted, silylated and analyzed by GC-MS. The obtained isoprenoid pattern provides an indication of the inhibited enzyme, due to the accumulation of specific substrates. By analyzing terbinafine-treated A. fumigatus and mutant strains containing tunable gene copies of erg9 or erg1, respectively, the method was verified. Downregulation of erg9 resulted in a high accumulation of intracellular farnesol as well as elevated levels of geranylgeraniol and isoprenol. The decreased expression of erg1 as well as terbinafine treatment led to an increased squalene content. Additional analysis of growth medium revealed high farnesyl pyrophosphate levels extruded during erg9 downregulation.
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spelling pubmed-103814232023-07-29 Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry Liebl, Maximilian Huber, Ludwig Elsaman, Hesham Merschak, Petra Wagener, Johannes Gsaller, Fabio Müller, Christoph J Fungi (Basel) Article The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can be used to identify compounds interacting with the enzymes of isoprenoid biosynthesis, an important part of the ergosterol biosynthesis pathway. The method was validated according to the EMEA guideline on bioanalytical method validation. Aspergillus fumigatus hyphae and Saccharomyces cerevisiae cells were lysed mechanically in an aqueous buffer optimized for the enzymatic deconjugation of isoprenoid pyrophosphates. The residual alcohols were extracted, silylated and analyzed by GC-MS. The obtained isoprenoid pattern provides an indication of the inhibited enzyme, due to the accumulation of specific substrates. By analyzing terbinafine-treated A. fumigatus and mutant strains containing tunable gene copies of erg9 or erg1, respectively, the method was verified. Downregulation of erg9 resulted in a high accumulation of intracellular farnesol as well as elevated levels of geranylgeraniol and isoprenol. The decreased expression of erg1 as well as terbinafine treatment led to an increased squalene content. Additional analysis of growth medium revealed high farnesyl pyrophosphate levels extruded during erg9 downregulation. MDPI 2023-07-20 /pmc/articles/PMC10381423/ /pubmed/37504756 http://dx.doi.org/10.3390/jof9070768 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liebl, Maximilian
Huber, Ludwig
Elsaman, Hesham
Merschak, Petra
Wagener, Johannes
Gsaller, Fabio
Müller, Christoph
Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry
title Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry
title_full Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry
title_fullStr Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry
title_full_unstemmed Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry
title_short Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry
title_sort quantifying isoprenoids in the ergosterol biosynthesis by gas chromatography–mass spectrometry
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10381423/
https://www.ncbi.nlm.nih.gov/pubmed/37504756
http://dx.doi.org/10.3390/jof9070768
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