Related Experiment Video
Updated: Sep 15, 2025

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms
Published on: April 17, 2017
COOKIE-Pro: Covalent Inhibitor Binding Kinetics Profiling on the Proteome Scale.
Hanfeng Lin1,2,3, Bin Yang1,2, Lang Ding3,4
1The Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, Texas 77030, United States.
COOKIE-Pro is a new proteomics method to measure covalent inhibitor binding kinetics and selectivity across all proteins. This tool aids in developing more potent and selective covalent drugs.
Area of Science:
- Chemical Biology
- Proteomics
- Drug Discovery
Background:
- Covalent inhibitors are a promising therapeutic class.
- Limited methods exist for profiling their proteome-wide binding kinetics and selectivity.
Purpose of the Study:
- Introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), an unbiased method for quantifying covalent inhibitor binding kinetics.
- Enable proteome-wide assessment of inhibitor interactions with both on-target and off-target proteins.
Main Methods:
- Utilizes a two-step incubation process combined with mass spectrometry-based proteomics.
- Determines inactivation rate () and inhibition constant () values.
- Validated using BTK inhibitors spebrutinutin and ibrutinib.
Main Results:
- Accurately reproduced known kinetic parameters for BTK inhibitors.
- Identified expected and novel off-target proteins.
- Revealed spebrutinutin is over 10-fold more potent against TEC kinase than BTK.
- Demonstrated compatibility with streamlined cysteine activity-based protein profiling (SLC-ABPP) data.
Conclusions:
- COOKIE-Pro provides a comprehensive view of covalent inhibitor binding across the proteome.
- Represents a powerful tool for optimizing covalent drug potency and selectivity in preclinical development.
More Related Videos
13:57Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Published on: February 18, 2014
08:31Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions
Published on: December 1, 2020
Related Concept Videos
Protein-Drug Binding: Mechanism and Kinetics
Various forces drive these interactions, including hydrogen bonds, hydrophobic interactions, ionic bonds, electrostatic interactions, and van der Waals forces. These bonds enable drugs to bind to specific sites on proteins,...
Protein-protein Interfaces
Protein-Drug Binding: Determination Methods
Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound...
Protein Networks
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
The Equilibrium Binding Constant and Binding Strength
Nonlinear Pharmacokinetics: Bioavailability and Protein-Drug Binding
To quantify the extent of bioavailability, pharmacologists often use a parameter called .