In a new study published in Nature Communications titled, “COOKIE-Pro: Covalent Inhibitor Binding Kinetics 2 Profiling on the Proteome Scale,” researchers at Baylor College of Medicine have developed a new data analysis method that provides a comprehensive, unbiased view of how covalent inhibitors interact with proteins throughout the cell. The technique aims to accelerate the design of more effective and safer therapeutics by precisely measuring both the binding strength and reaction speed of these drugs against thousands of potential targets simultaneously.
“Covalent inhibitors, which include well-known drugs like aspirin and the cancer therapeutic ibrutinib, are highly effective because they form a strong, permanent bond with their target protein,” said Jin Wang, PhD, director of the Center for NextGen Therapeutics, professor of biochemistry and molecular pharmacology at Baylor, and corresponding author of the study. “However, this strength can be a double-edged sword; these drugs can also bind to unintended off-target proteins, potentially leading to unwanted side effects.”
Optimizing these drugs requires a delicate balance between how strongly they bind to a target and how quickly they form the permanent bond. Limitations in methods to measure these two parameters across the entire proteome have traditionally bottlenecked drug development.
“The challenge was getting a clear, complete picture,” said Hanfeng Lin, the study’s first author and a graduate student in the Wang lab. “We knew we needed to measure both affinity and reactivity, but doing it for one protein takes time, let alone thousands. COOKIE-Pro gives us a comprehensive map in which we can see for the first time, not just if a drug binds off-target, but how well and how fast, which is critical information for drug designers.”
To achieve high throughput affinity and reactivity assessment, the study introduced a specially designed “chaser” probe that latches onto protein-binding sites left unoccupied by the drug. Mass spectrometry then measured chase probe binding to deduce the degree of occupation of each protein by the drug. This approach allows researchers to calculate both the binding affinity and the inactivation rate for thousands of proteins at once.
The team validated the method using two well-studied drugs, spebrutinib and ibrutinib. Results showed that spebrutinib, a highly selective enzymatic inhibitor, is over 10 times more potent against an off-target protein, TEC kinase, than the intended target, Bruton’s tyrosine kinase (BTK). For the less-selective drug ibrutinib, COOKIE-Pro identified known off-targets and generated profiles that aligned with previously published values to validate the method’s accuracy.
When applying this high-throughput approach to a library of 16 covalent inhibitor fragments, the team generated thousands of profiles to illustrate the method’s capability to efficiently guide the earliest stages of drug discovery.
“The ultimate goal is rational drug design,” said Wang. “A drug might appear potent because it binds quickly, but if that is simply because it has a ‘hot’ reactive group, it might cause side effects by binding everywhere. COOKIE-Pro allows us to separate that intrinsic reactivity from true binding affinity. We can now help chemists prioritize compounds that are potent because they bind specifically to the right target, not just because they are broadly reactive. This is a crucial step toward creating the next generation of highly selective and safer covalent medicines.”
