Small Molecule Inhibitors Targeting the Interaction of Ricin Toxin A Subunit with Ribosomes

Li, Xiaoping; Harijan, R.K.; Kahn, Jennifer Nielsen; Schramm, Vern L.; Tumer, Nilgun E.

doi:10.1021/acsinfecdis.0c00127

Cited by 14 publications

(29 citation statements)

References 65 publications

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“…Recent results identified small molecules that bind to the ribosome-binding site of RTA and inhibit its activity, establishing ribosome-binding site of RTA as a new target for therapeutic intervention (49). The ribosome-binding site of Stxs has not been previously explored as a target for therapeutic development.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins

Rudolph

Davis

Tumer

et al. 2020

Journal of Biological Chemistry

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The principal virulence factor of human-pathogenic enterohemorrhagic Escherichia coli is Shiga toxin (Stx). Shiga toxin 2a (Stx2a) is the subtype most commonly associated with severe disease outcomes such as hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 subunit (Stx2A1) binds to the conserved elongation factor binding C-terminal domain (CTD) of ribosomal P stalk proteins to inhibit translation. Stx2a holotoxin also binds to the CTD of P stalk proteins because the ribosome binding site is exposed. We show here that Stx2a binds to an 11mer peptide (P11) mimicking the CTD of P stalk proteins with low micromolar affinity. We cocrystallized Stx2a with P11 and defined their interactions by X-ray crystallography. We found that the last six residues of P11 inserted into a shallow pocket on Stx2A1 and interacted with Arg172, Arg176 and Arg179, which were previously shown to be critical for binding of Stx2A1 to the ribosome. Stx2a formed a distinct P11 binding mode within a different surface pocket relative to ricin toxin A subunit and trichosanthin, suggesting different ribosome recognition mechanisms for each ribosome inactivating protein (RIP). The binding mode of Stx2a to P11 is also conserved among the different Stx subtypes. Furthermore, the P stalk protein CTD is flexible and adopts distinct orientations and interaction modes depending on the structural differences between the RIPs. Structural characterization of the Stx2a-ribosome complex is important for understanding the role of the stalk in toxin recruitment to the sarcin/ricin loop and may provide a new target for inhibitor discovery.

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Section: Discussionmentioning

confidence: 99%

Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins

Rudolph

Davis

Tumer

et al. 2020

Journal of Biological Chemistry

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“…40 Leu9 makes several hydrophobic interactions with Ile251, while the aromatic side chain of Phe10 forms an offsetting π-stack with Tyr183 and Phe240. 43 The aromatic ring of CC10501 similarly establishes π-stacking interactions with Tyr183 and Phe240 and comparable hydrophobic associations with Ile251. 43 The carboxylate moiety on CC10501 and the carboxylate of C-terminal Asp11 form similar hydrogen bond interactions with the guanidine group of Arg234 and Arg235 just outside the hydrophobic pocket (Figure 2).…”

Section: ■ Resultsmentioning

confidence: 98%

“…43 The aromatic ring of CC10501 similarly establishes π-stacking interactions with Tyr183 and Phe240 and comparable hydrophobic associations with Ile251. 43 The carboxylate moiety on CC10501 and the carboxylate of C-terminal Asp11 form similar hydrogen bond interactions with the guanidine group of Arg234 and Arg235 just outside the hydrophobic pocket (Figure 2). 43 CC10501 binds RTA with a dissociation constant (K D ) of 270 μM and inhibits its ribosome depurination activity with an IC 50 of 181 μM, supporting the biological function for this interaction.…”

Section: ■ Resultsmentioning

confidence: 98%

“…Compound Design for Fragment Optimization. The crystal structure of RTA in complex with CC10501 (PDB ID: 6URX) (Figure 2) 43 revealed that the inhibitor binds in the same deep hydrophobic pocket of RTA as Leu9 and Phe10 of the P11 peptide (SDDDMGFGLFD) (5GU4). 40 Leu9 makes several hydrophobic interactions with Ile251, while the aromatic side chain of Phe10 forms an offsetting π-stack with Tyr183 and Phe240.…”

Section: ■ Resultsmentioning

confidence: 99%

“…We used a surface plasmon resonance (SPR) based fragment screen and discovered two small molecule fragments, CC10501 and CC70601, which bind the hydrophobic pocket at the P stalk binding site away from the active site and inhibit the activity of RTA. 43 Here, we used a structure-based design exploring the CC10501 chemical scaffold as the starting point for optimization and constructed a focused library. Structure activity relationships (SARs) of the compounds in the library were examined by measuring their binding affinity and inhibitory activity on eukaryotic ribosomes.…”

Section: ■ Introductionmentioning

confidence: 99%

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

Harijan

Cao

et al. 2021

J. Med. Chem.

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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.

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1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit

Bhattacharya,

Dahmane,

Goger

et al. 2024

Biomol NMR Assign

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Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA’s depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

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Small Molecule Inhibitors Targeting the Interaction of Ricin Toxin A Subunit with Ribosomes

Cited by 14 publications

References 65 publications

Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins

Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins

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

1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit

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