Ricin, one of the most toxic substances known and a type B biothreat agent, has the same mechanism of action as Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae, which are foodborne pathogens responsible for significant morbidity and mortality around the world. Despite years of research there are no effective small molecule inhibitors of ricin or Stxs and as of now only supportive care is available. Modulation of ribosome interactions by small molecules has not been previously explored as a strategy for inhibition. We showed that ricin toxin A subunit (RTA) and the A1 subunit of Stx2a (Stx2A1) bind to the C- terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop (SRL) at a distant site on the large subunit of the ribosome. We identified the critical residues at the P-stalk binding site of RTA and Stx2A1 and reported the first X-ray crystal structure of Stx2a with a P protein peptide and the first cryo-EM structure of Stx2a holotoxin with the purified P-stalk pentamer. Peptides mimicking the CTD of P proteins bind at a hydrophobic pocket on the opposite face of RTA/Stx2A1 relative to the active site and inhibit the activity of both toxins, identifying the P-stalk binding site as a new target for developing small molecules. We set up fragment-based lead discovery (FBLD) by surface plasmon resonance (SPR) and discovered two fragments that bind at the P-stalk binding site of RTA. Using structure-based design (SAR) we improved these inhibitors and obtained lead compounds that inhibit the activity and cytotoxicity of ricin and Stx2a holotoxin in mammalian cells. In this revised application, we propose to use a new fluorescence polarization assay to enable robust SAR evaluation and the design of second-generation lead compounds that inhibit the activity of ricin and Stx2a. We will test the hypothesis that the hydrophobic pocket at the P-stalk binding site of RTA and Stx2A1 can serve as a highly druggable site for developing small molecules that can be used to dissect the depurination mechanism of the ribosome inactivating proteins. In aim one, we will design new analogs based on trends in SAR revealed from our recent work, to improve binding interactions while improving physical properties. We will use docking and binding site analysis by SiteMap, WaterMap and FEP+ to optimize our leads. We will determine the affinities using a new fluorescence polarization assay, analyze potency in cell-free and cell-based assays and identify the binding modes by X-ray crystallography and NMR. A supplemental virtual screen against RTA will be used to identify new chemical scaffolds and new binding elements to combine with our current leads. In the second aim, we will use NMR analysis to investigate the mechanism of action of lead compounds to test the hypothesis that P-stalk binding indirectly influences the active site residues to allosterically regulate depurination activity at the catalytic site of RTA and Stx2A1. The product of our efforts will be chemical tools to elucidate P-stalk binding and activation mechanisms and may result in advanced lead compounds for the development of post-exposure therapeutics against ricin and STEC.

Ricin is one of the most potent toxins known and a type B biothreat agent and the related Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae remain enormous global health problems because at present, there is no known therapy against ricin or STEC. The proposed studies will develop novel small molecules that inhibit ricin and Shiga toxin 2 a by targeting their interactions with the ribosome. The findings are expected to be of great impact as they address the unmet need to develop novel ricin and STEC therapeutics by targeting toxin/ribosome interactions.