Affinity Map: Global Protein-Ligand Binding Affinity Profiling Using Photocatalytic Labeling

Background & Unmet Need

• Measuring binding affinities between ligands and proteins is crucial for understanding the potency, selectivity, and specific effects of molecules in complex biological systems
• Existing methods for measuring binding affinity are typically accomplished in vitro, requiring purified proteins and prior knowledge of the interaction, limiting use in complex samples like live cells or serum
• Proximity labeling technologies can identify novel interactions in vivo (e.g., protein-protein, protein-DNA, protein-RNA) but lack quantitative binding affinity measurements
• Unmet Need: Robust, proteome-wide, and quantitative measurement of binding affinities in complex biological samples such as live cells, cell lysates, and organ extracts

Technology Overview

• The Technology: Affinity Map is a novel platform that enables robust measurement of binding affinities between ligands and proteins across the proteome using photocatalytic labeling and high-throughput mass spectrometry
• A “hot” ligand conjugated to a photocatalyst and a “cold” unmodified ligand are used to covalently label proteins, enabling competitive binding assays that yield quantitative affinity measurements
• Affinity Map is applicable to major classes of ligands: small molecules, linear and cyclic peptides, proteins
• PoC Data: Affinity Map measured Kd values for the protein interactions of dasatinib, a tyrosine inhibitor, and JQ1, a selective bromodomain binding compound, with high accuracy and selectivity, even identifying previously unknown interactions
• Affinity Map was used to measure Kd values for the macrocyclic cRBDfk peptide and human IgG on live cell surfaces, finding close concurrence with reported literature values

Technology Applications

• Global small molecule-protein, peptide-protein, or protein-protein binding affinity determination
• Can be leveraged in complex biological systems such as cell lysates, protein extracts, and live cells
• Use in early drug discovery to screen novel chemical matter or profile interactions of small molecule candidates, including binding affinity, target engagement, and proteome-wide off-target binding affinities

Technology Advantages

• Measures binding affinities across all protein classes, including membrane proteins and non-kinase targets
• Maintains a low false detection negative rate, outperforming methods like CETSA and limited proteolysis
• Provides quantitative binding affinities using competitive kinetics, offering insights beyond qualitative fold enrichment data from other platforms