biostudies-literatureKennedy CRFrancis Crick InstituteWellcome TrustBiotechnology and Biological Sciences Research CouncilEngineering and Physical Sciences Research Council153-168https://www.ebi.ac.uk/biostudies/studies/S-EPMC12584752biostudies-literatureUnknown7(1)As the global fight against antimicrobial resistance in bacteria becomes increasingly pressing, new tool compounds are needed to study and evaluate novel therapeutic targets. Here, cysteine-directed fragment-based drug discovery is coupled with high throughput chemistry direct-to-biology screening to target the catalytic cysteine of a family of bacterial effector proteins, the novel E3 ligases (NELs) from <i>Salmonella</i> and <i>Shigella</i>. These effector E3 ligases are attractive as potential drug targets because they are delivered into host cells during infection, have no human homologues and disrupt host immune response to infection. We successfully identify hit compounds against the SspH subfamily of NELs from <i>Salmonella</i> and show that these proteins are inhibited by compound RSC chemical biologyCovalent fragment screening to inhibit the E3 ligase activity of bacterial NEL enzymes SspH1 and SspH2.PMC12584752CC2075BB/T014547/1EP/V038028/1CC1063Dudley-Fraser JBush JHouse DMcPhie KAVuorinen AMcCarthy WJSkehel JMRittinger KKennedy CRMaslen SEsposito DPettinger JfalseCovalent fragment screening to inhibit the E3 ligase activity of bacterial NEL enzymes SspH1 and SspH2.As the global fight against antimicrobial resistance in bacteria becomes increasingly pressing, new tool compounds are needed to study and evaluate novel therapeutic targets. Here, cysteine-directed fragment-based drug discovery is coupled with high throughput chemistry direct-to-biology screening to target the catalytic cysteine of a family of bacterial effector proteins, the novel E3 ligases (NELs) from <i>Salmonella</i> and <i>Shigella</i>. These effector E3 ligases are attractive as potential drug targets because they are delivered into host cells during infection, have no human homologues and disrupt host immune response to infection. We successfully identify hit compounds against the SspH subfamily of NELs from <i>Salmonella</i> and show that these proteins are inhibited by compound 2026-01-01T00:00:00Z2026 Jan2026-06-12T04:56:37.072Z2026-06-12T03:07:44.801ZS-EPMC125847524119516710.1039/d5cb00177c