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Updated: Jan 16, 2026
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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Published on: February 18, 2014
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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, TX, USA.
Nature Communications
|September 30, 2025
Summary
COOKIE-Pro is a new proteomics method to measure how covalent inhibitors bind to proteins. This tool helps optimize drug potency and selectivity for better covalent therapeutics development.
Area of Science:
- Biochemistry
- Chemical Biology
- Proteomics
Background:
- Covalent inhibitors are a promising therapeutic class.
- Existing methods for profiling covalent inhibitor binding kinetics and selectivity are limited.
- Comprehensive proteome-wide analysis is needed to understand off-target effects.
Purpose of the Study:
- Introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), a novel method for quantifying covalent inhibitor binding kinetics.
- Enable unbiased, proteome-wide assessment of inhibitor interactions.
- Facilitate optimization of covalent drug potency and selectivity.
Main Methods:
- Utilizes a two-step incubation process combined with mass spectrometry-based proteomics.
- Determines kinact and KI values for covalent inhibitors.
- Applies a streamlined two-point strategy for high-throughput screening.
Main Results:
- COOKIE-Pro accurately quantifies covalent inhibitor binding kinetics on a proteome-wide scale.
- Validated using BTK inhibitors, reproducing known parameters and identifying off-targets.
- Revealed spebrutinib is more potent against TEC kinase than BTK.
- Generated thousands of kinetic profiles from covalent fragment screening.
Conclusions:
- COOKIE-Pro is a powerful tool for preclinical covalent drug development.
- Enables quantitative decoupling of reactivity and affinity at scale.
- Provides a comprehensive view of covalent inhibitor binding, aiding in optimizing drug properties.
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