# Expanding the chemical biological tools for the detection and regulation of human and bacterial proteases

> **NIH NIH R35** · SCRIPPS RESEARCH INSTITUTE, THE · 2020 · $213,000

## Abstract

PROJECT SUMMARY/ABSTRACT
 My laboratory is dedicated to the development, synthesis, and biochemical validation of small molecules and
peptides that quantitate and regulate human and bacterial proteases and the application of these tools with new
experimental methods to improve our understanding of proteases in human health and disease. Such protease-
directed chemical tools drive biological insights, and this is best evidenced by the seminal studies on serine
hydrolases by Balls and Jansen with diisopropylfluorophosphate (1952), as well as the use of tosyl phenylalanyl
chloromethyl ketone by Shaw and Schoellmann that established the reactive residues in chymotrypsin (1963).
Surprisingly, the types of chemistry used, and the proteases targeted have been fairly limited in scope in the
majority of protease-directed molecules available. For example, almost all small-molecule and peptide-based
probes target the non-prime-side region of the protease active site and the peptide probes typically consist of
natural amino acids. These constraints result in promiscuous molecules that prohibit their use in cells where one
needs to confidently assign function to individual proteases. Genetic approaches (e.g. CRISPR/Cas9) provide
exquisite control of proteins within cells; however, these methods cannot distinguish enzymatic contributions
from the potential roles involving protein:protein interactions. Thus, there is an urgent need for selective small
molecules and peptides that spatially and temporally detect and regulate individual proteases within cells.
 We have made significant advances in the chemistry used for protease-directed molecules, as well as in the
types of proteases studied. Our innovative use of unnatural amino acids and unique warheads resulted in the
first peptides with selectivity to individual human caspases. We have also made non-hydrolyzable peptides that
extend into the prime-side of the active site and have developed assays to discover procaspase activators and
inhibitors. We anticipate designing specific molecules for four human caspases within the next five years to
address major outstanding questions, including what are the cellular substrates for specific caspases and what
are the differences in substrates during apoptosis and T cell activation. We have expanded our probe and small
molecule designs to interrogate proteases in pathogenic and commensal bacteria in combination with structural
and biochemical methods. We plan to establish bacterial lipoprotein signal peptidases as targets for novel
antibiotics and identify how protease inhibition affects expression of surface-bound bacterial proteins. Proteases
secreted by gut commensal bacteria directly influence host homeostasis and represent an entire new set of
enzymes to be characterized. Our goal is to establish commonalities and differences among highly conserved
bacterial proteases and develop chemical probes to establish if aberrant proteolytic activity is a driver of disease;
...

## Key facts

- **NIH application ID:** 9930860
- **Project number:** 1R35GM136286-01
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** Dennis William Wolan
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $213,000
- **Award type:** 1
- **Project period:** 2020-05-01 → 2020-12-07

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9930860

## Citation

> US National Institutes of Health, RePORTER application 9930860, Expanding the chemical biological tools for the detection and regulation of human and bacterial proteases (1R35GM136286-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9930860. Licensed CC0.

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