The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.
Acetohydroxyacid synthase (AHAS) is the target for more than 50 commercial herbicides; first applied to crops in the 1980s. Since then, 197 site-of-action resistance isolates have been identified in weeds, with mutations at P197 and W574 the most prevalent. Consequently, AHAS is at risk of not being a useful target for crop protection. To develop new herbicides, a functional understanding to explain the effect these mutations have on activity is required. Here, we show that these mutations can have two effects (i) to reduce binding affinity of the herbicides and (ii) to abolish time-dependent accumulative inhibition, critical to the exceptional effectiveness of this class of herbicide. In the two mutants, conformational changes occur resulting in a loss of accumulative inhibition by most herbicides. However, bispyribac, a bulky herbicide is able to counteract the detrimental effects of these mutations, explaining why no site-of-action resistance has yet been reported for this herbicide.
Acetohydroxyacid synthase (AHAS,
EC 2.2.1.6), the first enzyme
in the branched chain amino acid biosynthesis pathway, is the target
for more than 50 commercially available herbicides, and is a promising
target for antimicrobial drug discovery. Herein, we have expressed
and purified AHAS from Candida auris, a newly identified
human invasive fungal pathogen. Thirteen AHAS inhibiting herbicides
have K
i values of <2 μM for this
enzyme, with the most potent having K
i values of <32 nM. Six of these compounds exhibited MIC50 values of <1 μM against C. auris (CBS10913
strain) grown in culture, with bensulfuron methyl (BSM) being fungicidal
and the most potent (MIC50 of 0.090 μM) in defined
minimal media. The MIC50 value increases to 0.90 μM
in media enriched by the addition of branched-chain amino acids at
the expected concentration in the blood serum. The sessile MIC50 for BSM is 0.6 μM. Thus, it is also an excellent inhibitor
of the growth of C. auris biofilms. BSM is nontoxic
in HEK-293 cells at concentrations >100 μM and thus possesses
a therapeutic index of >100. These data suggest that targeting
AHAS
is a viable strategy for treating C. auris infections.
Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.
Herbicides are commonly deployed as the front-line treatment to control infestations of weeds in native ecosystems and among crop plants in agriculture. However, the prevalence of herbicide resistance in many species is a major global challenge. The specificity and effectiveness of herbicides acting on diverse weed species are tightly linked to targeted proteins. The conservation and variance at these sites among different weed species remain largely unexplored. Using novel genome data in a genome-guided approach, 12 common herbicide-target genes and their coded proteins were identified from seven species of Weeds of National Significance in Australia: Alternanthera philoxeroides (alligator weed), Lycium ferocissimum (African boxthorn), Senecio madagascariensis (fireweed), Lantana camara (lantana), Parthenium hysterophorus (parthenium), Cryptostegia grandiflora (rubber vine), and Eichhornia crassipes (water hyacinth). Gene and protein sequences targeted by the acetolactate synthase (ALS) inhibitors and glyphosate were recovered. Compared to structurally resolved homologous proteins as reference, high sequence conservation was observed at the herbicide-target sites in the ALS (target for ALS inhibitors), and in 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (target for glyphosate). Although the sequences are largely conserved in the seven phylogenetically diverse species, mutations observed in the ALS proteins of fireweed and parthenium suggest resistance of these weeds to ALS-inhibiting and other herbicides. These protein sites remain as attractive targets for the development of novel inhibitors and herbicides. This notion is reinforced by the results from the phylogenetic analysis of the 12 proteins, which reveal a largely consistent vertical inheritance in their evolutionary histories. These results demonstrate the utility of high-throughput genome sequencing to rapidly identify and characterize gene targets by computational methods, bypassing the experimental characterization of individual genes. Data generated from this study provide a useful reference for future investigations in herbicide discovery and development.
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