Language

English

Publication Date

9-30-2025

Journal

Nature Communications

DOI

10.1038/s41467-025-63491-2

PMID

41027871

PMCID

PMC12484723

PubMedCentral® Posted Date

9-30-2025

PubMedCentral® Full Text Version

Post-print

Abstract

Covalent inhibitors are an emerging class of therapeutics, but methods to comprehensively profile their binding kinetics and selectivity across the proteome have been limited. Here we introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), an unbiased method for quantifying irreversible covalent inhibitor binding kinetics on a proteome-wide scale. COOKIE-Pro uses a two-step incubation process with mass spectrometry-based proteomics to determine kinact and KI values for covalent inhibitors against both on-target and off-target proteins. We validated COOKIE-Pro using BTK inhibitors spebrutinib and ibrutinib, accurately reproducing known kinetic parameters and identifying both expected and unreported off-targets. The method revealed that spebrutinib has over 10-fold higher potency for TEC kinase compared to its intended target BTK. To demonstrate the method’s utility for high-throughput screening, we applied a streamlined two-point strategy to a library of 16 covalent fragments. This approach successfully generated thousands of kinetic profiles, enabling the quantitative decoupling of intrinsic chemical reactivity from binding affinity at scale and validating the method’s broad applicability. By providing a comprehensive view of covalent inhibitor binding across the proteome, COOKIE-Pro represents a powerful tool for optimizing the potency and selectivity of covalent drugs during preclinical development.

Keywords

Kinetics, Proteome, Protein Kinase Inhibitors, Humans, Proteomics, Pyrimidines, Piperidines, Protein Binding, Adenine, Pyrazoles, Agammaglobulinaemia Tyrosine Kinase, High-Throughput Screening Assays, Mass Spectrometry, Target identification, Proteomic analysis, Proteomics

Published Open-Access

yes

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