Identification of inhibitors of the Salmonella FraB deglycase, a drug target.

Abstract

Nontyphoidal Salmonella is one of the most prevalent causes of human foodborne illnesses worldwide, with no narrow-spectrum antibiotics or vaccines available. Here, we seek to address this gap. 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 6-phosphofructose-aspartate, the substrate of FraB, and intoxicates Salmonella. 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 and offer prospects as potential therapeutics. To identify such inhibitors, we conducted a high-throughput screening of small-molecule libraries using a FraB activity-based biochemical assay. We screened 131,165 compounds and identified 126 hits that could be obtained commercially for further characterization. When tested at 25 μm inhibitor in the presence of 1 mm 6-phosphofructose-aspartate, FraB activity was reduced ~ 30-100% by 65 compounds. Guided by preliminary cell-based data, we further characterized six compounds (one triazolidine, two thiadiazolidines, and three triazolothiadiazoles) and found them to exhibit IC₅₀ values from ~ 3 to 100 μm and K_I (inhibitor constant) values from ~ 1 to 29 μm. Native mass spectrometry revealed that all three triazolothiadiazoles were capable of binding FraB; we also obtained evidence that one of the triazolothiadiazoles binds FraB even in the presence of substrate. The recurrence of multiple pharmacophores bolsters prospects for farming more hits from compound libraries and for designing therapeutics against nontyphoidal Salmonella.