Poster abstracts

Poster number 60 submitted by Jamison Law

Identifying inhibitors of the FraB deglycase to design therapeutics for Salmonella-mediated gastroenteritis

Jamison Law (Department of Chemistry and Biochemistry, The Ohio State University), Pankajavalli Thirugnanasambantham (Department of Chemistry and Biochemistry, The Ohio State University), Sravya Kovvali (Department of Microbiology, The Ohio State University), Kate Zakharova, Charles Bell (Department of Biological Chemistry and Pharmacology, The Ohio State University), Anice Sabag-Daigle, Brian Ahmer (Department of Microbial Infection and Immunity, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Non-typhoidal Salmonella enterica (Salmonella) causes significant morbidity, mortality, and economic costs worldwide 1,2. No vaccines are available and existing antibiotics are disfavored 3-7. Drug targets in Salmonella are scarce due to the pathogen’s versatility in exploiting redundancy/overlap in metabolic pathways 8,9. An Achilles’ heel became evident with the discovery that fructose-asparagine (F-Asn) is a nutrient utilized by Salmonella. F-Asn catabolism requires five proteins encoded in the fraRBDAE operon 10. Surprisingly, ΔfraB mutants could grow in glucose but not in glucose + F-Asn. This growth inhibition is due to the toxic buildup of 6-phosphofructose-aspartate, the substrate of FraB 11. The absence of FraB in mammals and most members of the human microbiota makes FraB an attractive anti-Salmonella target 12. We utilized a biochemical assay in high-throughput fashion to screen 131,165 compounds; this effort yielded ~30 hits that inhibit FraB with Ki’ values from ~0.2 to 60 µM. Five of these compounds share a triazole-thiadiazole pharmacophore and three from this subset (excitingly!) inhibited the growth of ΔtolC Salmonella, a mutant whose efflux capability is dampened. Although FraB has proven recalcitrant to substrate/inhibitor co-crystallization efforts, we have obtained structures of FraB alone (2 Å) with its C-terminal tail in two different conformations. Computational analyses revealed that these two poses differ with respect to their tunnel topography/volume and ligand docking and the distance between putative catalytic residues (general base E214 and general acid H230) that narrowed from ~18 to 5 Å. These data shed light on the conformational changes obligatory for catalysis. Because each of the two active sites in the FraB homodimer consists of E214 and H230 drawn from each monomer, we are using a novel affinity chromatography method to isolate FraB heterodimers in which either the general acid or general base is mutated in one of the monomers. Our findings inspire new questions regarding the FraB catalytic mechanism and will help forge an exciting path towards Salmonella-specific therapeutics.

References:
1. Collaborators GDD. Lancet Infect Dis. 11 2018;18(11):1211-1228.
2. Scharff. J Food Prot. 2012;75(1):123-131.
3. Strugnell RA, Scott TA, Wang N, et al. Curr Opin Microbiol. Feb 2014;17:99-105.
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5. Gopinath et al. Proc Natl Acad Sci USA. 2014;111(44):15780-15785.
6. Diard et al. Curr Biol. 2014;24(17):2000-2005.
7. Wiström et al. Ann Intern Med. 1992;117(3):202-208.
8. Becker et al. Nature. 2006;440(7082):303-307.
9. Steeb B et al. PLoS Pathog. 2013;9(4):e1003301.
10. Ali et al. PLoS Pathog. 2014;10(6):e1004209.
11. Sabag-Daigle et al. Sci Rep. 2016;6:28117.
12. Sabag-Daigle et al. Appl Environ Microbiol. 2018;84(5).

Keywords: Salmonella, crystallography, therapeutic