Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay

Sabag-Daigle, Anice; Boulanger, Erin F; Thirugnanasambantham, Pankajavalli; Law, Jamison D; Bogard, Alex J; Behrman, E. J.; Gopalan, Venkat; Ahmer, Brian M. M.

doi:10.1128/spectrum.04606-22

Cited by 3 publications

(3 citation statements)

References 37 publications

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“…Cellular metabolism, though essential to life, inescapably results in production of intermediate metabolites, some of which have toxic effects if permitted to accumulate. Leveraging this toxicity may lead to development of new antimicrobials and anticancer drugs [9,10]. The potential of this approach has been demonstrated in a series of elegant studies of fructose-asparagine (F-Asn) metabolism in Salmonella enterica Typhimurium.…”

Section: Introductionmentioning

confidence: 99%

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Lethal metabolism of Candida albicans respiratory mutants

Kane,

Burke,

Diaz

et al. 2024

PLoS ONE

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The destructive impact of fungi in agriculture and animal and human health, coincident with increases in antifungal resistance, underscores the need for new and alternative drug targets to counteract these trends. Cellular metabolism relies on many intermediates with intrinsic toxicity and promiscuous enzymatic activity generates others. Fuller knowledge of these toxic entities and their generation may offer opportunities of antifungal development. From this perspective our observation of media-conditional lethal metabolism in respiratory mutants of the opportunistic fungal pathogen Candida albicans was of interest. C. albicans mutants defective in NADH:ubiquinone oxidoreductase (Complex I of the electron transport chain) exhibit normal growth in synthetic complete medium. In YPD medium, however, the mutants grow normally until early stationary phase whereupon a dramatic loss of viability occurs. Upwards of 90% of cells die over the subsequent four to six hours with a loss of membrane integrity. The extent of cell death was proportional to the amount of BactoPeptone, and to a lesser extent, the amount of yeast extract. YPD medium conditioned by growth of the mutant was toxic to wild-type cells indicating mutant metabolism established a toxic milieu in the media. Conditioned media contained a volatile component that contributed to toxicity, but only in the presence of a component of BactoPeptone. Fractionation experiments revealed purine nucleosides or bases as the synergistic component. GC-mass spectrometry analysis revealed acetal (1,1-diethoxyethane) as the active volatile. This previously unreported and lethal synergistic interaction of acetal and purines suggests a hitherto unrecognized toxic metabolism potentially exploitable in the search for antifungal targets.

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Section: Introductionmentioning

confidence: 99%

“…In the absence of fraB, 6-phosphofructose-aspartate accumulates and attenuates growth and virulence [11,13]. Several small molecule inhibitors of the fraB glycanase were recently identified as potential narrow spectrum antimicrobials [9]. This approach was recently expanded to consider other toxic sugar-phosphate intermediates [14,15].…”

Section: Introductionmentioning

confidence: 99%

Lethal metabolism of Candida albicans respiratory mutants

Kane,

Burke,

Diaz

et al. 2024

PLoS ONE

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“…Although FrlB is the focus of this study, FraB, a related deglycase, has recently been identified as a novel drug target since disrupting the FraB‐dependent metabolism of F‐Asn by Salmonella results in the accumulation of a toxic sugar‐phosphate metabolite (Sabag‐Daigle et al, 2016). To this end, we have invested much effort to design a narrow‐spectrum therapeutic to inhibit Salmonella FraB (Sabag‐Daigle et al, 2023; Thirugnanasambantham et al, 2022). Our interest in FraB inspired our studies of the catalytic mechanism of Salmonella FrlB.…”

Section: Introductionmentioning

confidence: 99%

Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine

et al. 2023

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Amadori rearrangement products are stable sugar-amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose-lysine (F-Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F-Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6-phosphofructose-lysine (6-P-F-Lys). FrlB, a deglycase, then converts 6-P-F-Lys to L-lysine and glucose-6-phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8-Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6-P-F-Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine-6-phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB-6-P-F-Lys on GlmS-fructose-6-phosphate structures revealed parallels in their active-site arrangement and guided our selection of seven putative active-site residues in FrlB for site-directed mutagenesis. Activity assays with eight recombinant single-substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface-induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x-ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure-function and mechanistic studies of enzymes.

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Serendipitous Discovery of a Competitive Inhibitor of FraB, a Salmonella Deglycase and Drug Target

et al. 2022

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Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 μM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

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Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay

Abstract: Nontyphoidal salmonellosis is a serious threat in the United States and globally. We have recently identified an enzyme, FraB, that when mutated renders Salmonella growth defective in vitro and unfit in mouse models of gastroenteritis.

Cited by 3 publications

References 37 publications

Lethal metabolism of Candida albicans respiratory mutants

Lethal metabolism of Candida albicans respiratory mutants

Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine

Serendipitous Discovery of a Competitive Inhibitor of FraB, a Salmonella Deglycase and Drug Target

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