Abstract
Ricin toxin A subunit (RTA) removes an adenine from the universally conserved sarcin/ricin loop (SRL) on eukaryotic ribosomes, thereby inhibiting protein synthesis. No high affinity and selective small molecule therapeutic antidotes have been reported against ricin toxicity. RTA binds to the ribosomal P stalk to access the SRL. The interaction anchors RTA to the P protein C-termini at a well-defined hydrophobic pocket, which is on the opposite face relative to the active site. The RTA ribosome binding site has not been previously targeted by small molecule inhibitors. We used fragment screening with surface plasmon resonance to identify small molecular weight lead compounds that bind RTA and defined their interactions by crystallography. We identified five fragments, which bound RTA with mid-micromolar affinity. Three chemically distinct binding fragments were cocrystallized with RTA, and crystal structures were solved. Two fragments bound at the P stalk binding site, and the third bound to helix D, a motif distinct from the P stalk binding site. All fragments bound RTA remote from the catalytic site and caused little change in catalytic site geometry. Two fragments uniquely bound at the hydrophobic pocket with affinity sufficient to inhibit the catalytic activity on eukaryotic ribosomes in the low micromolar range. The binding mode of these inhibitors mimicked the interaction of the P stalk peptide, establishing that small molecule inhibitors can inhibit RTA binding to the ribosome with the potential for therapeutic intervention.