# Screening for Cys-Reactive Ligands to Target PAX3-FOXO1

> **NIH NIH U54** · DUKE UNIVERSITY · 2022 · $369,418

## Abstract

Supplemental application U54 CA231630 PI: Angela Koehler, Co-PIs: Sara Buhrlage, Jarrod Marto
Abstract
Alveolar rhabdomyosarcoma (ARMS) is a lethal pediatric cancer driven by the oncogenic
transcription factor fusion protein PAX3-FOXO1 (P3F). While genetic data demonstrate P3F is a
potential therapeutic target, transcription factors are often considered challenging pharmalogical
targets. The goal of this U54 supplement is to expand drug identification strategies past our
current small-molecule microarrays (SMMs) and implement covalent ligand screening against
P3F protein Cys residues, thus increasing the pool of potential drug candidates. To achieve this
goal, team will collaborate to pursue three objectives. First, Dr. Koehler’s team will increase
production of purified full-length P3F as well as truncated forms of the protein which contain
Cys-793, a residue identified as a critical mediator of cofactor CBP. Second, Dr. Koehler’s team
will work with Drs. Buhrlage and Marto to optimize screening conditions for a full cys-reactive
library utilizing a pooling-approach to increase throughput. Individual hits will be validated
through LC-MS/MS to confirm biochemical results, such as covalent modifications and
stoichiometry. Lastly, covalent ligand screen hits will be subjected to secondary assays, such as
cellular thermal shift or reporter assays, outlined in the parent U45 for SMM drug discovery.
This collaboration between Dr. Koehler and Drs. Buhrlage and Marto will expand the U54
team’s drug discovery capabilities, and enable a more thorough and efficient assessment of
candidate drugs to target P3F. In the future, this technological approach could be extended to
other transcription factors for drug candidate identification.

## Key facts

- **NIH application ID:** 10611002
- **Project number:** 3U54CA231630-01A1S3
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** CHRISTOPHER M COUNTER
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $369,418
- **Award type:** 3
- **Project period:** 2022-08-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10611002

## Citation

> US National Institutes of Health, RePORTER application 10611002, Screening for Cys-Reactive Ligands to Target PAX3-FOXO1 (3U54CA231630-01A1S3). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10611002. Licensed CC0.

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