Establishing that a molecule engages with a target protein (target engagement) <i>in situ</i> and <i>in vivo</i> is vital for increasing the probability of success for a drug discovery program. Target engagement along with observing desired pharmacological effect(s) upon compound treatment provides strong evidence for target validation and serves as a starting point for understanding compound mechanism of action. Chemical biology techniques such as bead-based affinity capture (AC), activity-based protein profiling (ABPP), affinity-based protein profiling (AfBPP) using gel-based immunoblots and fluorescence, or mass spectrometry have been traditionally used over the past two decades for target engagement studies. The toolbox for measuring target engagement has expanded with the addition of proteome-wide biophysical methods such as cellular thermal shift assay (CETSA®), stability of protein from rates of oxidation (SPROX), and limited proteolysis (LIP). These probe-free techniques provide target engagement information from detecting changes in protein physical properties when compound binding occurs. Coupling any chemical biology or proteome-wide biophysical technique with MS-based proteomics (chemoproteomics) enables off-targets to be identified to assess both target engagement and compound selectivity in the same experiment. In this presentation, we will show examples of how we use AC, ABPP, AfBPP, and CETSA® to establish target engagement and identify off-targets. We will also show how we are refining these methodologies to increase the diversity of biological systems investigated, number of compounds and conditions tested along with reducing the time to provide answers.

Methodologies and strategies for target engagement and off-target identification studies in drug discovery and development

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.