Poster abstracts

Poster number 31 submitted by Jamison Law

Identification of triazolothiadiazoles as uncompetitive inhibitors of the Salmonella FraB deglycase, a drug target

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), Yuan Gao, Vicki H. Wysocki (Department of Chemistry and Biochemistry, Native Mass Spectrometry Guided Structural Biology Center, The Ohio State University), Kate Zakharova, Charles Bell (Department of Biological Chemistry and Pharmacology, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Non-typhoidal Salmonella is a prevalent cause of human foodborne illnesses worldwide, with no narrow-spectrum antibiotics or licensed vaccines available 1-4. During the host inflammatory response, Salmonella metabolizes fructose-asparagine as a nutrient using proteins encoded in the fra operon. Deletion of fraB leads to a build-up of its substrate (6-phosphofructose-aspartate) and intoxicates Salmonella 5,6. Because fra genes are absent in mammals and most members of the human gut microbiome, FraB inhibitors are expected to have limited off-target effects 7. We used an activity-based biochemical assay to screen 131,165 compounds and, identified (post-hit triage) 65 compounds that reduced FraB activity by 30-100% at 25 µM with 1 mM 6-phosphofructose-aspartate; nine of these hits shared a triazolothiadiazole pharmacophore. Guided by preliminary cell-based data, we further characterized three triazolothiadiazoles and found them to be uncompetitive inhibitors [IC50, 1 to 49 µM; Ki' (inhibitor constant), 2.2 to 42.5 µM]. Native mass spectrometry revealed that all three compounds were capable of binding FraB in the presence and, counterintuitively, absence of substrate. Understanding how these compounds achieve inhibition requires a knowledge of the structure and catalytic mechanism of FraB. Although we have a crystal structure of FraB (2Å), efforts to co-crystallize FraB with substrate ± inhibitor(s) have failed. Ongoing efforts seek to redress this issue using cryo-EM. To draw structure-function correlates, corroborating biochemical data are needed. The FraB homodimer consists of two active sites each composed of E214 and H230 drawn from each monomer, as supported by the loss of activity upon mutating either residue in both active sites. We used a novel (Sephadex-based) affinity chromatography method to isolate and characterize FraB heterodimers in which E214, H230, or both are mutated in only one of the active sites. Our findings inspire new questions regarding the FraB catalytic mechanism and will help forge an exciting path towards Salmonella-specific therapeutics.

References:
1. Kirk MD, Pires SM, Black R, et al. PLoS Med 12, e1001921
2. Sears KT, Galen JE, and Tennant SM. (2021) J Appl Microbiol 131, 2640-2658
3. Nasrin S, Fuche FJ, Sears KT, et al. (2021) Vaccines (Basel) 9
4. Higginson EE, Panda A, Toapanta FR, et al. (2021) Infect Immun 89, e0008721
5. Ali MM, Newsom DL, González JF, et al. (2014) PLoS Pathog 10, e1004209
6. Sabag-Daigle A, Blunk HM, Sengupta A, et al. (2016) Sci Rep 6, 28117
7. Sabag-Daigle A, Wu J, Borton MA, et al. (2018) Appl Environ Microbiol 84:e01957-17

Keywords: Salmonella, structure, therapeutic