AbstractShiga toxin (Stx) producing E. coli (STEC) and Shigella dysenteriae are foodborne pathogensthat can cause severe morbidity and mortality. STEC infections can progress to eitherhemorrhagic colitis (HC) or life-threatening hemolytic-uremic syndrome (HUS). HUS is the mostcommon cause of acute renal failure in US children. Presently there are no FDA approvedvaccines or therapeutics against STEC or Shigella infection. Moreover, the use of antibioticsexacerbates the disease. STEC and Shigella are classified as category B pathogens of nationalsecurity and public health risk. Shiga toxin has been a uniquely challenging drug target. Smallmolecule inhibitors of Shiga toxin enzymatic activity with high potency have not been identified.Interaction of A1 subunits with ribosomes has not been previously examined as a potential drugtarget. The goal of this proposal is to fill this gap by developing novel screens to identifyfragment and peptide inhibitors that disrupt activity of Stx2 by inhibiting its interaction with theribosome. We identified P stalk as the ribosome docking site of the A1 subunits of Stx1 (Stx1A1)and Stx2 (Stx2A1) and showed that an 11-mer peptide corresponding to the conserved last 11residues of P proteins binds to Stx2A1 and inhibits its activity. These studies established toxin-ribosome interactions as a new target for inhibitor discovery. We carried out a preliminaryfragment screen and identified fragments that bind to Stx2A1 with micromolar affinity. In aim 1we propose to develop Biacore-based primary screens to identify fragments, which bind toStx2A1 with higher affinity. We will validate the hits using activity assays and verify binding andselectivity of the inhibitors using ribosome binding and active site mutants. Medicinal chemistrywill be used to optimize the selected fragments into more potent leads based on theirexperimental X-ray crystal structure with Stx2. In aim 2 we will develop phage displayingmultiple copies of the P protein peptide to determine if multivalent display of this peptide motifwill disrupt the interaction of Stx2A1 with the ribosome. We will screen random P7 phagedisplay library to identify novel peptides that can bind to Stx2A1 more strongly than the native Pprotein peptide and inhibit its activity. In aim 3 we propose to solve the cryo-EM structure ofStx2 in complex with the ribosome to identify the binding sites of the P proteins to facilitateoptimization of the inhibitors. We expect that our unique assays to dissect toxin-ribosomeinteractions and toxin activity in combination with the medicinal chemistry and structural biologyexpertise will lead to the development of novel tool compounds and peptides, which can providebiochemical and mechanistic insight into STEC pathogenesis.