Poster abstracts
Poster number 22 submitted by Jamison Law
Identifying inhibitors of the FraB deglycase as therapeutics for Salmonella-mediated gastroenteritis
Jamison Law (Department of Chemistry and Biochemistry, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Non-typhoidal Salmonella enterica (Salmonella) annually causes significant morbidity/mortality (154 million/121,000 worldwide), healthcare-related costs ($12 billion, USA), and meat spoilage (13 million pounds recalled in 2018, USA) 1-3. No vaccines are available and extant antibiotics are disfavored because they exacerbate the infection 4-6. Few drug targets exist for Salmonella due to its versatility in exploiting redundancy/overlap in metabolic pathways 7-8. However, fructose-asparagine (F-Asn), an Amadori compound, was recently discovered as a nutrient utilized by Salmonella due to the activity of five proteins encoded in the fraRBDAE operon 9. 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 10. The absence of FraB in mammals and most members of the human microbiota makes FraB an attractive anti-Salmonella target 11. Therefore, to identify potential inhibitors of FraB, we sought to leverage the ~500,000-compound library from the ICCB-Longwood Screening Facility at Harvard. To this end, cell-based (Ahmer, OSU) and biochemical assays were developed to enable high-throughput screening of compounds capable of inhibiting Salmonella growth and FraB activity, respectively. Of 200,000 compounds that we tested, ~150 were confirmed as bona fide inhibitors of FraB with 18 hits overlapping between both assays. Ongoing biochemical studies with these compounds seek to characterize their Ki values and mode of inhibition. Further medicinal chemistry-based optimization of these hits, however, requires a high-resolution structure of FraB (± substrate/inhibitor). To overcome poor outcomes in our longstanding crystallization efforts, we recently used the Surface Entropy Reduction approach to gain insights into mutations that would promote crystal formation. One such mutant, FraB K275A-E276A, yielded robust crystals that diffracted to 2.0 Å and a structure! This key advance bolsters co-crystallization prospects and helps forge an exciting path towards Salmonella-specific therapeutics.
References:
1. Sabag-Daigle et al. Sci Rep. 2016;6:28117.
2. Kirk MD et al. PLoS Med. 2015;12(12):e1001921.
3. Scharff. J Food Prot. 2012;75(1):123-131.
4. Summary of Recall Cases in Calendar Year 2018. In: United States Department of Agriculture Food Safety and Inspection Services; 2019.
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. Appl Environ Microbiol. 2018;84(5).
Keywords: Salmonella, crystallography, therapeutic