# Inhibitors of Intestinal Epithelial Cell Lysosomal-Mediated Necrosis

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $419,024

## Abstract

The intestinal epithelium plays a crucial role in barrier function. These cells are often injured and
progresses to death in inflammatory bowel disease (IBD)-like disorders. Epithelial cell loss typically occurs via
a mixture of cell death phenotypes such as apoptosis or necroptosis, but is dominated by lysosomal-mediated
(LM) necrosis when the insult is severe. Clinically, LM necrosis is manifest by large segments of the epithelial
lining sloughing into the intestinal lumen. Preservation of normal epithelial cell viability and function will help
ameliorate the complications of IBD-like disorders. Initially perceived as haphazard and irreversible, we show
that LM necrosis follows a molecular program and is reversible. We hypothesize that the development of a
high-throughput, high-content in vivo screen (HTS/HCS) for small molecule antidotes to LM necrosis will prime
the preclinical drug discovery pipeline for a form of intestinal epithelial cell death that currently lacks any means
of chemoprophylaxis. Unfortunately, the expense and complexity of mammalian systems make them
unsuitable for HTS/HCS formats. Moreover, these systems present with mixed cell death phenotypes, thereby
complicating the isolation of pure LM necrosis inhibitors. We identified a C. elegans strain null for an inhibitor of
lysosomal cysteine proteases. These animals are highly and exclusively susceptible to LM intestinal necrosis
when stressed by many of the noxious stimuli occurring in IBD-like disorders. This cell death pathway is
evolutionarily conserved, as fetal intestinal cell explant cultures from mice null for the homologous cysteine
protease inhibitor also preferentially undergo LM intestinal necrosis, even when exposed to apoptotic or
necroptotic stimuli. Our laboratory pioneered the use of mutant C. elegans strains to conduct live-animal,
HTS/HCS drug discovery campaigns (US Patent US8809617 B2). Compounds from these screens have
already proved effective in analogous mammalian models of disease. Using this robust technology, we
propose to use our mutant C. elegans strain to perfect an HTS/HCS assay for LM necrosis and conduct a
large-scale screen for small molecule inhibitors of intestinal epithelial LM necrosis. Promising hits have already
been identified in a small pilot screen using a library of FDA-approved drugs. Hits will be validated using a
number of secondary assays involving nematodes, mammalian cell lines and a mouse intestinal epithelial
injury protocol that models human necrotizing enterocolitis. The ability to conduct a live-animal, LM necrosis
phenotype-based screens for new therapeutics is unprecedented for episodes of IBD-like diseases marked by
extensive epithelial cell injury. This proposal was prepared in response to PA-16-374, Assay Development and
Screening to Discover Therapeutic or Imaging Agents for Diseases of interest to NIDDK.

## Key facts

- **NIH application ID:** 9895759
- **Project number:** 5R01DK114047-03
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Clifford James Luke
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $419,024
- **Award type:** 5
- **Project period:** 2018-04-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9895759

## Citation

> US National Institutes of Health, RePORTER application 9895759, Inhibitors of Intestinal Epithelial Cell Lysosomal-Mediated Necrosis (5R01DK114047-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9895759. Licensed CC0.

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