Researchers at Baylor College of Medicine have introduced a new analytical method called COOKIE-Pro (Covalent Occupancy Kinetic Enrichment via Proteomics) to study how covalent inhibitors interact with proteins inside cells. The details of this technique are published in Nature Communications.
COOKIE-Pro is designed to measure both the binding strength and reaction speed of covalent drugs against thousands of proteins at once. This approach could help scientists develop more effective and safer medications by giving them detailed information about drug interactions throughout the cell.
“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 Dr. Jin Wang, director of the Center for NextGen Therapeutics and Michael E. DeBakey, M.D. Endowed Professor in Pharmacology at Baylor. “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.”
The process developed by the team involves breaking down cells in solution and adding the covalent drug so it can bind to its targets. A specially designed probe is then added to attach to any remaining unoccupied sites on proteins. Using mass spectrometry, researchers determine how much of this probe binds, allowing them to calculate both how strongly and how quickly each protein was bound by the drug.
“The challenge was getting a clear, complete picture,” said Hanfeng Lin, first author of the study and graduate student in Wang’s 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 validate COOKIE-Pro’s effectiveness, researchers tested two known drugs: spebrutinib and ibrutinib. They found that spebrutinib was over ten times more potent against an off-target protein than its intended target. For ibrutinib, COOKIE-Pro identified known off-targets accurately.
“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.”
The research team also demonstrated that COOKIE-Pro could be used for large-scale screening by applying it to 16 different inhibitor fragments at once.
Other contributors include Bin Yang, Lang Ding, Matthew V. Holt, Sung Yun Jung and Bing Zhang from Baylor College of Medicine; Meng C. Wang from Howard Hughes Medical Institute; and Yen-Yu Yang from Thermo Fisher Scientific.
Funding came from several sources including grants from the National Institutes of Health (R01-CA250503 and R01-CA268518), Cancer Prevention and Research Institute of Texas (CPRIT grant RP220480), as well as support through a Michael E. DeBakey Professorship in Pharmacology.
