Synthesis and Structural Characterization of Ricin Inhibitors Targeting Ribosome Binding Using Fragment-Based Methods and Structure-Based Design

Abstract

Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved inhibitors against ricin. We used a new strategy to disrupt RTA–ribosome interactions by fragment screening using surface plasmon resonance. Here, using a structure-guided approach, we improved the affinity and inhibitory activity of small-molecular-weight lead compounds and obtained improved compounds with over an order of magnitude higher efficiency. Four advanced compounds were characterized by X-ray crystallography. They bind at the RTA–ribosome binding site as the original compound but in a distinctive manner. These inhibitors bind remotely from the catalytic site and cause local conformational changes with no alteration of the catalytic site geometry. Yet they inhibit depurination by ricin holotoxin and inhibit the cytotoxicity of ricin in mammalian cells. They are the first agents that protect against ricin holotoxin by acting directly on RTA.

Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins

“…With the recent discovery of the P-stalk binding site and demonstration that preventing P-stalk binding reduces the enzymatic activity of RTA, there is interest in developing small molecules against RTA that target P-stalk binding site ( 38 ). Those approaches have yielded compounds with high specificity that mimic interactions of Phe-10 within the P2C11 peptide and RTA as revealed by structural analysis, similar to the mimicry we observed for V9B2, V9E1, and V9F9.…”

Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction

“…However, targeting the ribosome-binding site of RIPs provides an attractive alternative with the potential for the development of the combination therapy. Recently, we have identified small-molecule inhibitors binding to the ribosome-binding site of RTA and established the CTD-binding pocket of RTA as a new drug target ( 40 , 41 ). Although the identified compounds bind remotely from the active site of RTA, they inhibit the SRL depurination activity mediated by the ricin holotoxin ( 40 , 41 ).…”

“…Recently, we have identified small-molecule inhibitors binding to the ribosome-binding site of RTA and established the CTD-binding pocket of RTA as a new drug target ( 40 , 41 ). Although the identified compounds bind remotely from the active site of RTA, they inhibit the SRL depurination activity mediated by the ricin holotoxin ( 40 , 41 ). Likewise, the ribosome-binding site of Stx2a is a convenient, yet unexplored, drug target.…”

Identification and Biological Evaluation of a Novel Small-Molecule Inhibitor of Ricin Toxin

“…The depurination assays were performed according to previously described methods [49,52]. SP2/0 cells were seeded in DMEM complete medium at a density of 2 × 10 5 cells/mL in 12-well plates with 500 µL per well and incubated overnight.…”

“…The q-PCR assays were performed on a Real-Time PCR System (BioRad CFX96, Hercules, CA, USA) using the Power SYBR Green Master Mix (Thermo Fisher Scientific, Seattle, WA, USA). Human 28S rRNA was used as the endogenous control, and the primer sequences for the q-PCR are listed in Table 2 [52]. RNA depurination levels were analyzed using the classic 2 −∆∆Ct method.…”