# Technology for evaluating drug-binding responses to small-molecule perturbation

> **NIH NIH R35** · UNIVERSITY OF WASHINGTON · 2024 · $388,750

## Abstract

PROJECT SUMMARY | ABSTRACT
Proteins act as the effector molecules of cells – carrying out most of the structural, regulatory, and enzymatic
functions. Proteins themselves are often regulated through direct interaction with ligands, including metals,
lipids, other proteins, and drugs. These protein-ligand interactions are fundamental to diverse biological
processes. Yet, technologies to explore these interactions are limited in terms of both throughput and their
ability to scale. The limits of these technologies are in part highlighted be the fact that for nearly 30 years,
proteomics and genomics technologies research have been unable to fully characterize the functions of the
20,000 protein coding genes in human cells.
To address this, we propose to build a cornerstone technology suite for high-throughput, proteome-wide
protein-ligand interaction profiling. In this work we will demonstrate the development and implementation in a
focused way to highlight the potential of this technology to bring robust quantitative approaches to study ligand
binding at scale. Our technological innovations center on using high-throughput methods to detect protein-
ligand interactions across the entire proteome in a single analysis. To do this, we will measure the change in
thermal stability of proteins induced by binding to a ligand. We measure this thermal stability as a relative
difference in protein abundance using sample multiplexing based on tandem mass tags (TMT). Sample
multiplexing enables quantitation of up to 18 samples’ proteomes simultaneously. Sample multiplexing with
TMT increases sample throughput, reduces missing values across samples, and enables complex
experimental designs – e.g., time courses, dose dependency, and knockout-rescue experiments.
Over the course of the proposed work, we will build new proteomics technologies to harness the benefits of
proteome-wide thermal stability assays and TMT quantitation to characterize protein-ligand interactions. The
combination of (1) intelligent mass spectrometric data acquisition, (2) proteome thermal stability profiling, and
(3) sample multiplexing will enable us to decipher the complex interplay between proteins and ligands across
the proteome. With an eye towards translational research, we will focus at first on small-molecule drugs as
ligands as we can acquire diverse libraries with known primary protein targets. These data and methods will be
used to reveal the functional and secondary effects of ligand perturbation of the proteome by leveraging
matched whole proteome and gene expression profiles to determine to what extent specific drug-protein-
engagement drives cellular responses.

## Key facts

- **NIH application ID:** 10931378
- **Project number:** 5R35GM150919-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Devin Karl Schweppe
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $388,750
- **Award type:** 5
- **Project period:** 2023-09-25 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10931378

## Citation

> US National Institutes of Health, RePORTER application 10931378, Technology for evaluating drug-binding responses to small-molecule perturbation (5R35GM150919-02). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10931378. Licensed CC0.

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