This proposal brings together investigators from Rutgers University (Tumer, Kimball, Augery), Albert Einstein College of Medicine (Schramm, Almo and Cameron) and Wadsworth Center (Mantis) with expertise in biochemistry, structural biology, medicinal chemistry, and toxicology to identify inhibitors that target ribosome interactions and catalytic activity of ricin. Currently, there is no proven, safe treatment for ricin intoxication or infection by related Shiga toxin producing Shigella or E. coli. Two ricin vaccines in clinical trials do not elicit robust toxin neutralizing activity. The goal of this proposal is to fill this gap by by identifying peptides and small molecule fragments that bind to key pockets on ricin A chain (RTA) and inhibit its activity. During the previous funding period we identified the host target of ricin as the conserved C-terminal 11-mer (P11) of the ribosomal P-protein stalk. We showed that the ribosome binding surface of RTA is on the opposite face of the active site cleft and proposed a model where binding to the ribosomal stalk stimulates ribosome depurination by reorienting the active site of RTA towards the SRL. These studies established a new paradigm for the mechanism of depurination and identified toxin/ribosome interactions as a new target for inhibitor discovery. We recently discovered a new hydrophobic pocket anchored by an essential arginine critical for ribosome interactions of RTA. Our overall hypothesis is that we can inhibit the catalytic activity and the toxicity of ricin by interfering with ribosome interactions of RTA. We will identify the key interacting residues at the ribosome binding surface of RTA as a starting point in inhibitor discovery. Using peptide arrays of the ribosomal target we will identify peptide analogs that bind to the ribosome binding surface and block the ribosome interactions of RTA. We will use fragment based lead discovery (FBLD) with surface plasmon resonance (SPR) to identify fragments that can bind to the ribosome binding pocket, the active site cleft or previously unidentified pockets and inhibit the depurination activity and toxicity of RTA. Ribosome binding and active site mutants will be used to determine the binding site selectivity and X-ray crystal structure analysis will be used to elucidate the binding mode of the peptides and the fragments. The fragment hits will be optimized and evaluated in cell-based assays and in a mouse model of ricin intoxication. The promising leads will be used as a starting point to build a scaffold. Our innovative approach, rigorous methodology and deep insight into the structure function analysis of RTA will provide new knowledge about the basic mechanism for molecular recognition of the stalk and will help identify inhibitors that can be used as new leads for future therapeutic design.

Public Health Relevance

Ricin intoxication causes serious and sometimes fatal effects. There is no proven safe treatment for ricin intoxication or infection by related Shiga toxin producing E. coli. We propose to characterize the ribosome interactions of ricin to identify inhibitors that inhibit ribosome binding, catalytic activity and toxicity of ricin.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI072425-14
Application #
10090551
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Baqar, Shahida
Project Start
2007-03-15
Project End
2022-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
14
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Rutgers University
Department
Other Basic Sciences
Type
Earth Sciences/Resources
DUNS #
001912864
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Li, Xiao-Ping; Kahn, Jennifer N; Tumer, Nilgun E (2018) Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding. Toxins (Basel) 10:
Zhou, Yijun; Li, Xiao-Ping; Kahn, Jennifer N et al. (2018) Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins. Toxins (Basel) 10:
Li, Xiao-Ping; Tumer, Nilgun E (2017) Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins. Toxins (Basel) 9:
Zhou, Yijun; Li, Xiao-Ping; Chen, Brian Y et al. (2017) Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk. Sci Rep 7:42912
Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N et al. (2016) The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1. Infect Immun 84:149-61
Basu, Debaleena; Kahn, Jennifer N; Li, Xiao-Ping et al. (2016) Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome. Infect Immun 84:3290-3301
Jetzt, Amanda E; Li, Xiao-Ping; Tumer, Nilgun E et al. (2016) Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome. Toxicol Appl Pharmacol 310:120-128
Basu, Debaleena; Tumer, Nilgun E (2015) Do the A subunits contribute to the differences in the toxicity of Shiga toxin 1 and Shiga toxin 2? Toxins (Basel) 7:1467-85
Grela, Przemys?aw; Li, Xiao-Ping; Tch├│rzewski, Marek et al. (2014) Functional divergence between the two P1-P2 stalk dimers on the ribosome in their interaction with ricin A chain. Biochem J 460:59-67
Yan, Qing; Li, Xiao-Ping; Tumer, Nilgun E (2014) Wild type RTA and less toxic variants have distinct requirements for Png1 for their depurination activity and toxicity in Saccharomyces cerevisiae. PLoS One 9:e113719

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