Researchers have developed a novel method to measure how covalent drugs interact with proteins across the proteome.
In the study, the researchers showed that spebrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, was more than 10 times more potent for TEC kinase than for BTK. The method also reproduced benchmark kinetic parameters for ibrutinib and identified its known off-targets, including BLK and TEC.
The approach, called COOKIE-Pro, used mass spectrometry to quantify the binding kinetics of covalent inhibitors. It calculated kinact, the inactivation rate, and the apparent affinity constant KI for both intended and unintended targets. This allowed direct comparison of drug selectivity across thousands of proteins.
Validation experiments were conducted in Ramos B-cell lymphoma cells. For spebrutinib, the researchers measured covalent binding across concentrations and time points, using a probe to capture unbound proteins. Among more than 400 proteins identified, BTK and TEC showed strong dose-dependent binding. TEC reached high occupancy at lower doses and shorter times compared with BTK, confirmed by immunoblotting. For ibrutinib, tests at 12.5 to 200 nM identified BTK as the main target but also quantified off-target effects on BLK and TEC. Lead study author Hanfeng Lin, a PhD candidate at Baylor College of Medicine, and colleagues explained: “Maintaining the intrinsic reactivity of the warhead is crucial for ensuring the effectiveness of the probe."
The researchers also applied a streamlined two-point COOKIE-Pro protocol for high-throughput screening. Using this approach on 16 covalent inhibitor fragments, they generated over 100,000 potential kinetic curves, allowing them to separate chemical reactivity from binding affinityto identify compounds with specific interactions.
The researchers reported limitations. The two-point strategy used only two concentrations to estimate kinetic parameters, making the analysis prone to error. Small variations could lead to inaccurate results, and biological replicates were required to increase confidence. Protein-level enrichment improved the detection of key targets, but the method could miss low-abundance proteins or misinterpret peptide-level binding.
The findings showed that COOKIE-Pro could reproduce known drug kinetics while revealing new information on off-target activity and relative potency. Its application to fragment screening suggested potential for use in early-stage drug discovery, with future studies likely to extend the method to other covalent inhibitors and cell systems.
The study was funded by the National Institutes of Health, the Cancer Prevention and Research Institute of Texas, and a Michael E. DeBakey, MD, Professorship in Pharmacology. One researcher reported co-founding companies and consulting activities unrelated to the study.
Source: Nature Communications
