# Assay development and screening for inhibitors targeting Shiga toxin 2

> **NIH NIH R01** · RUTGERS, THE STATE UNIV OF N.J. · 2022 · $511,743

## Abstract

Abstract
Shiga toxin (Stx) producing E. coli (STEC) and Shigella dysenteriae are foodborne pathogens
that can cause severe morbidity and mortality. STEC infections can progress to either
hemorrhagic colitis (HC) or life-threatening hemolytic-uremic syndrome (HUS). HUS is the most
common cause of acute renal failure in US children. Presently there are no FDA approved
vaccines or therapeutics against STEC or Shigella infection. Moreover, the use of antibiotics
exacerbates the disease. STEC and Shigella are classified as category B pathogens of national
security and public health risk. Shiga toxin has been a uniquely challenging drug target. Small
molecule inhibitors of Shiga toxin enzymatic activity with high potency have not been identified.
Interaction of A1 subunits with ribosomes has not been previously examined as a potential drug
target. The goal of this proposal is to fill this gap by developing novel screens to identify
fragment and peptide inhibitors that disrupt activity of Stx2 by inhibiting its interaction with the
ribosome. We identified P stalk as the ribosome docking site of the A1 subunits of Stx1 (Stx1A1)
and Stx2 (Stx2A1) and showed that an 11-mer peptide corresponding to the conserved last 11
residues of P proteins binds to Stx2A1 and inhibits its activity. These studies established toxin-
ribosome interactions as a new target for inhibitor discovery. We carried out a preliminary
fragment screen and identified fragments that bind to Stx2A1 with micromolar affinity. In aim 1
we propose to develop Biacore-based primary screens to identify fragments, which bind to
Stx2A1 with higher affinity. We will validate the hits using activity assays and verify binding and
selectivity of the inhibitors using ribosome binding and active site mutants. Medicinal chemistry
will be used to optimize the selected fragments into more potent leads based on their
experimental X-ray crystal structure with Stx2. In aim 2 we will develop phage displaying
multiple copies of the P protein peptide to determine if multivalent display of this peptide motif
will disrupt the interaction of Stx2A1 with the ribosome. We will screen random P7 phage
display library to identify novel peptides that can bind to Stx2A1 more strongly than the native P
protein peptide and inhibit its activity. In aim 3 we propose to solve the cryo-EM structure of
Stx2 in complex with the ribosome to identify the binding sites of the P proteins to facilitate
optimization of the inhibitors. We expect that our unique assays to dissect toxin-ribosome
interactions and toxin activity in combination with the medicinal chemistry and structural biology
expertise will lead to the development of novel tool compounds and peptides, which can provide
biochemical and mechanistic insight into STEC pathogenesis.

## Key facts

- **NIH application ID:** 10322373
- **Project number:** 5R01AI141635-04
- **Recipient organization:** RUTGERS, THE STATE UNIV OF N.J.
- **Principal Investigator:** Xiao-Ping Li
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $511,743
- **Award type:** 5
- **Project period:** 2019-01-25 → 2024-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10322373

## Citation

> US National Institutes of Health, RePORTER application 10322373, Assay development and screening for inhibitors targeting Shiga toxin 2 (5R01AI141635-04). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10322373. Licensed CC0.

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