High‐throughput screening assays to identify inhibitors of the enterohemorrhagic E. coli NleB1 virulence factor

Enterohemorrhagic Escherichia coli (EHEC) are especially significant bacterial pathogens because they can cause hemorrhagic colitis and a type of renal failure (hemolytic uremic syndrome) for which therapy is limited. The molecular mechanisms by which EHEC and the closely related enteropathogenic E. coli (EPEC), as well as Salmonella enterica serovar Typhimurium, a common cause of human gastroenteritis, inhibit host innate immune responses to promote bacterial colonization are under intense investigation. Understanding these host‐pathogen dynamics in molecular detail is likely to impact positively our ability to develop pharmacological approaches to treat infections. We have characterized the NleB virulence protein because of its proven importance to human E. coli infections, as well as to the virulence of Citrobacter rodentium , a pathogen of mice used for in vivo studies of E. coli . NleB is one of many effector proteins that are injected into infected intestinal epithelial cells by the pathogen's type III secretion system. The NleB effectors are translocated glycosyltransferase enzymes that covalently modify host proteins with N‐ acetylglucosamine (GlcNAc) to subvert host pro‐inflammatory signaling pathways. These enzymes, as well as several S. Typhimurium NleB orthologs named SseK/1/2/3, glycosylate arginines of host protein substrates ( N ‐GlcNAcylation). We hypothesize that inhibiting NleB activity may be efficacious in treating bacterial infections. Small molecules that bind NleB may also facilitate structural analyses of these enzymes. Here we developed and utilized a high‐throughput screening (HTS) assay to identify NleB inhibitors. We first developed in vitro assays based upon the Promega UDP‐Glo™ Glycosyltransferase Assay to quantify EHEC NleB1 self‐glycosylation. We then determined the K cat and K m for this reaction and adapted the assay to a 384‐well plate format amenable to HTS. We screened a library of 5,160 compounds from the Diversity Set synthesized by The Chemical Methodologies and Library Development Center from the University of Kansas. These compounds utilize new principles of scaffold design that maximize drug‐like characteristics and pharmacological activity. The average Z′ score across all assay plates was 0.88 + 0.048. We identified 52 compounds that inhibited NleB1 activity greater than 3 standard deviations as compared to the plate median inhibition from all compounds. After performing dose‐response assays, we selected 18 compounds with IC50s in the low μM range for resynthesis and purification. These compounds were retested against all NleB/SseK orthologs as well as against the human O‐GlcNAc transferase enzyme. We propose that the NleB inhibitors we have identified may have utility both as anti‐virulence compounds and as research tools for understanding the NleB enzyme mechanism. Support or Funding Information NIAID AI099002, NIAID AI127973, NIGMS GM113117 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .