# Harnessing the In Vitro Selection for Activity-based Proteomics and Chemical Probe Development

> **NIH NIH R35** · PURDUE UNIVERSITY · 2020 · $376,148

## Abstract

Project Summary
Developments in genetic analysis technologies, particularly DNA sequencing, have been transformative to
biomedical research. In contrast to genomic information, the barrier to accessing proteomic information,
particularly enzyme activity, is dramatically higher. As aberrant enzyme activities are consistently observed in
disease, this information is critical for appropriate diagnosis and treatment. In addition, the ability to probe the
function of enzyme activities using chemical inhibitors is critical to our understanding of biology and disease.
DNA-encoded chemical libraries (DELs) have emerged as a new tool that enables medicinal chemists to
capitalize on the power of genetic techniques. The in vitro selection is the key process by which the function of
the synthetic molecules in a DEL are encoded with DNA sequence populations. Inspired by this transduction of
functional information of abiotic molecules, this project seeks to further exploit these capabilities into new areas.
Our research program centers on advancing DNA-encoded chemical approaches for the development of
chemical probes and for proteome-wide enzyme activity detection. This involves developing new approaches for
the synthesis and selection of DNA-encoded chemical libraries, as well as the implementation of a new, related
technology for DNA-based detection of enzyme activities using the in vitro selection of DNA-encoded proteomic
probes. For chemical probe development, we have chosen two target classes that involve protein:protein
interactions mediated through a short linear motifs: protein kinases and histone targeting domains. We will design
and build DELs that target the protein substrate site of protein kinases to develop both selective inhibitors and
non-natural substrates. Present use of ATP-competitive kinase inhibitors has been significantly limited by poor
selectivity. Similarly, the overlapping selectivities of peptide substrates has limited their use in activity detection.
Our targets for kinase protein substrate-competitive inhibitors and substrates are the Src family of tyrosine
kinases. Also, we will develop inhibitors to the trimethyllysine peptide binding site of chromodomains in the CBX
family of histone targeting proteins. The homology of the chromodomains in the eight chromobox (CBX) proteins
and the nature of their binding site has made the development of inhibitors difficult. Selective probes generated
here will be used to the decipher roles of CBX proteins in transcriptional regulation. With regards to activity
detection, we will specifically address two enzyme families: kinases and serine hydrolases. We will develop a
new approach to profile the activities of protein kinases by selection of DNA-linked substrates and DNA
sequencing. We will implement this approach in studies to characterize the changes in activity that occur in the
development of resistance to the kinase inhibitor lapatinib and also in the cellular process of epithelial-
mese...

## Key facts

- **NIH application ID:** 9996725
- **Project number:** 5R35GM128894-03
- **Recipient organization:** PURDUE UNIVERSITY
- **Principal Investigator:** Casey John Krusemark
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $376,148
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9996725

## Citation

> US National Institutes of Health, RePORTER application 9996725, Harnessing the In Vitro Selection for Activity-based Proteomics and Chemical Probe Development (5R35GM128894-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9996725. Licensed CC0.

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