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

> **NIH NIH R35** · PURDUE UNIVERSITY · 2024 · $568,671

## Abstract

Project Summary/Abstract
Our research program involves the use of DNA-encoded chemical libraries (DELs) and DNA-linked enzyme
activity probes, which are new approaches for biomedical research that capitalize on the power of DNA analysis
techniques. Specifically, this work involves development of in vitro selection assays for both DELs and enzyme
activity probes. It is the in vitro selection that encodes transduces information (drug molecule activity or
biochemical activity of a sample) into DNA sequences to facilitate analysis. This work advances these techniques
into new areas, particularly for medicinal chemistry applications, to provide tools for biological discovery and
development of new therapeutics.
 We are using DELs in a directed, targeted way (on-DNA medicinal chemistry) to produce inhibitors to the
chromodomains in the CBX family and to several bromodomains. The homology of the chromodomains in the
eight chromobox (CBX) proteins and of the family of bromodomains (61 in humans) makes selective inhibition
challenging. Inhibitor probes generated will be used to the decipher roles of these proteins in transcriptional
regulation and in disease states. DELs are now routinely used for de novo discovery of compounds that bind to
a drug target to initiate a drug development campaign. The selection assay used for this discovery is a simple
affinity purification with a purified protein on a solid support. The requirement of a pure and active protein for this
assay severely limits the target scope of DELs, particularly for membrane bound protein targets, which constitute
a large portion of drug target space. We are developing selection assays to enable use of DELs to protein targets
both on and within live cells. These assays rely on bioluminescence resonance energy transfer (BRET), which
is a common modality for detection of interacting molecules in cells. We will apply these unique assays with
highly diverse (>109), commercially available DELs to challenging protein targets including Nrf2 (potential cancer
target) and adenylyl cyclase 1 (a potential target for pain). In addition, we are developing selection assays to
identify molecules that not only bind to a protein receptor but activate downstream signaling pathways. We are
applying this selection to the opioid family of GPCRs to identify novel agonists.
 We will advance to use DNA-linked enzyme activity probes for the proteomic profiling of tyrosine kinase
activities and for drug binding assays amenable to high throughput screening of traditional (off-DNA) compound
collections. We are implementing our DNA-based kinase activity profiling to further understand the mechanism
of drug resistance to tyrosine kinase inhibitors in cancer therapy. Also, the high sensitivity of the approach
(enabled by DNA amplification) will be used to assay kinase activities in single cells, which will provide a greater
understanding of cellular complexity within tumors.

## Key facts

- **NIH application ID:** 10765502
- **Project number:** 2R35GM128894-06
- **Recipient organization:** PURDUE UNIVERSITY
- **Principal Investigator:** Casey John Krusemark
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $568,671
- **Award type:** 2
- **Project period:** 2018-09-01 → 2028-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10765502

## Citation

> US National Institutes of Health, RePORTER application 10765502, Harnessing the In Vitro Selection for Activity-based Proteomics and Chemical Probe Development (2R35GM128894-06). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10765502. Licensed CC0.

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