2. Academic Validation
  3. COOKIE-Pro: Covalent Inhibitor Binding Kinetics Profiling on the Proteome Scale

COOKIE-Pro: Covalent Inhibitor Binding Kinetics Profiling on the Proteome Scale

  • bioRxiv. 2025 Jun 22:2025.06.19.660637. doi: 10.1101/2025.06.19.660637.
Hanfeng Lin 1 2 3,  Bin Yang 1 2,  Lang Ding 3 4,  Yen-Yu Yang 5,  Matthew V Holt 6,  Sung Yun Jung 1,  Bing Zhang 6 7,  Meng C Wang 4,  Jin Wang 1 2 8
Affiliations

Affiliations

  • 1 The Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, Texas 77030, United States.
  • 2 Center for NextGen Therapeutics, Baylor College of Medicine, Houston, Texas 77030, United States.
  • 3 Graduate Program in Chemical, Physical & Structural Biology, Graduate School of Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, United States.
  • 4 Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 19700, United States.
  • 5 Thermo Fisher Scientific, San Jose, California 95134, United States.
  • 6 Lester and Sue Smith Breast Center and Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, Texas 77030, United States.
  • 7 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, United States.
  • 8 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, United States.
PMID: 40666940 DOI: 10.1101/2025.06.19.660637
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 k inact and K I values for covalent inhibitors against both on-target and off-target proteins. We validated COOKIE-Pro using the Btk inhibitors spebrutinib and ibrutinib, accurately reproducing known kinetic parameters and identifying both expected and novel off-targets. The method revealed that spebrutinib has over 10-fold higher potency for TEC kinase compared to its intended target Btk. We further demonstrate that COOKIE-Pro is compatible with streamlined cysteine activity-based protein profiling (SLC-ABPP) datasets, enabling efficient conversion of competition ratios to meaningful kinetic parameters. By providing a comprehensive view of covalent inhibitor binding across the proteome, COOKIE-Pro represents a powerful new tool for optimizing the potency and selectivity of covalent drugs during preclinical development.

Figures
Products