Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents

Abstract

b High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidinesulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.

High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines

“…cerevisiae was performed to identify novel antifungal compounds [26, 27]. The ergot-related compound NGx04 (Fig 1A) was among the validated hits scoring an IC 50 < 20 μM.…”

Variation inα-acetolactate production within the hybrid lager yeast groupSaccharomyces pastorianusand affirmation of the central role of theILV6gene

“…This approach has been successfully applied to brewing yeast strains to increase stress tolerance (Blieck et al , ; James et al , ; Huuskonen et al , ; Yu et al , ; Ekberg et al , ) and alter the production of various flavour compounds (Strejc et al , ; Shen et al , ). Some success in altering diacetyl production has been achieved (Gjermansen et al , ), and the availability of a range of compounds that specifically inhibit the action of AHAS (Richie et al , ) suggests that this could be a potentially valuable approach for isolating natural variant strains with beneficial mutations in ILV2 or associated genes, particularly ILV6 .…”

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

“…This might be due to our strategy of inducing mutations rather than selecting spontaneously resistant clones. Multiple mutagenesis experiments in fungi and mammalian cells have revealed that there is a bias towards chromosomal aberrations and SNPs in pleiotropic drug-resistance genes if spontaneous mutants are selected and sequenced (Nyfeler et al, 2012;Richie et al, 2013;Sadlish et al, 2013;Shimada et al, 2013). The underlying mechanism might be that amino acid mutations in the essential, primary target can often be deleterious, and these cells are rapidly outcompeted.…”