Abstract
Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.
Highlights
Shiga toxin (Stx) produced by the bacteria Shigella dysenteriae and Shiga-like toxins (Stx1 and Stx2) produced by certain strains of Escherichia coli are potent ribosome-inactivating proteins (RIPs) [1]
According to site-directed mutagenesis and X-ray diffraction studies along with a transition-state analysis of the depurination caused by ricin subunit A (RTA) [9,10,11,12,13], the catalytic mechanism of depurination by RTA begins with sandwiching of the adenine ring of the substrate ribosomal RNA (rRNA) between Tyr80 and Tyr123 of RTA via pi-pi interactions [9] at the Michaelis-Menten state [14]
Informed by these seminal findings and the aforementioned challenge of obtaining proteinNpolynucleotide-interaction inhibitors, we decided to use a doorstop approach to identify smallmolecule inhibitors of RTA and Stx2. This new approach aims to identify small molecules that work as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting the active conformation thereby blocking the function of the protein rather than work as contenders in the competition for binding to the RIP
Summary
Shiga toxin (Stx) produced by the bacteria Shigella dysenteriae and Shiga-like toxins (Stx and Stx2) produced by certain strains of Escherichia coli are potent ribosome-inactivating proteins (RIPs) [1]. According to site-directed mutagenesis and X-ray diffraction studies along with a transition-state analysis of the depurination caused by ricin subunit A (RTA) [9,10,11,12,13], the catalytic mechanism of depurination by RTA begins with sandwiching of the adenine ring of the substrate rRNA between Tyr and Tyr123 of RTA via pi-pi interactions [9] at the Michaelis-Menten state [14] These interactions enable the protonation of the adenine ring at N3 [9] by the cationic Arg180 of RTA that forms a hydrogen bond to the anionic Glu177 of RTA at the transition state [10]. A water molecule activated by the neutral Arg180 subsequently attacks the ribocation to form the ribose product and resume the cationic Arg180 [10,11,12,13]
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