Scientists at Scripps Research have developed a technique to image, across different tissues, and with higher precision than ever before, where in the body drugs bind to their targets. The new method, called CATCH (clearing-assisted tissue click chemistry), attaches fluorescent tags to drug molecules and uses chemical techniques to improve the fluorescent signal. In their published paper in Cell (“In situ identification of cellular drug targets in mammalian tissue”), the researchers reported on their use of the CATCH method with several different experimental drugs, to reveal where—even within individual cells—the drug molecules hit their targets. They hope that the new technique could become a routine tool in drug development.
“This method ultimately should allow us, for the first time, to see relatively easily why one drug is more potent than another, or why one has a particular side effect while another one doesn’t,” said study senior author Li Ye, PhD, assistant professor of neuroscience at Scripps Research and the Abide-Vividion chair in chemistry and chemical biology. The study’s first author, Zhengyuan Pang, is a graduate student in the Ye lab. The study also was a close collaboration with the laboratory of Ben Cravatt, PhD, the Gilula chair of chemical biology at Scripps Research.
Understanding where drug molecules bind their targets to exert their therapeutic effects—and side effects—is a basic part of drug development. However, drug-target interaction studies traditionally have involved relatively imprecise methods, such as bulk analyses of drug-molecule concentration in entire organs, the authors pointed out. And as they further stated, “The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions.” While imaging-based methods, such as positron emission tomography (PET), are now used widely to profile small-molecule distribution in vivo, “ … they also lack sufficient resolution to differentiate drug-binding states at the cellular level to precisely identify drug-target interactions,” the team continued. “An ideal method should allow in situ visualization of target-bound drugs at single-cell resolution, while at the same time, being compatible with multiplexed molecular characterization of their drug-target interactions.”
The new CATCH method developed by Ye and colleagues involves the insertion of tiny chemical handles into drug molecules. These distinct chemical handles don’t react with anything else in the body, but do allow the addition of fluorescent tags after the drug molecules have bound to their targets. In part, because human or animal tissue tends to diffuse and block the light from these fluorescent tags, the scientists combined the tagging process with a technique that makes tissue relatively transparent. “CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types,” the researchers noted.
For their reported initial study, the team optimized the CATCH method to evaluate covalent drugs that bind irreversibly to their targets with stable, covalent bonds. This irreversibility of binding makes it particularly important to verify that such drugs are hitting their intended targets. The scientists first evaluated several covalent inhibitors of an enzyme in the brain called fatty acid amide hydrolase (FAAH).
FAAH inhibitors have the effect of boosting levels of cannabinoid molecules, including anandamide—also known as the “bliss molecule”—and are being investigated as treatments for pain and mood disorders. The scientists were able to image, at the single-cell level, where these inhibitors hit their targets within large volumes of mouse brain tissue, and could easily distinguish their different patterns of target engagement.
In one experiment, they showed that an experimental FAAH inhibitor called BIA-10-2474, which caused one death and several injuries in a clinical trial in France in 2016, engages unknown targets in the midbrain of mice even when the mice lack the FAAH enzyme—offering a clue to the source of the inhibitor’s toxicity.
In other experiments demonstrating the unprecedented precision and versatility of the new CATCH method, the scientists showed that they could combine drug-target imaging with separate fluorescent-tagging methods to reveal the cell types to which a drug binds. They also could distinguish drug-target engagement sites in different parts of neurons. Finally, they could see how modestly different doses of a drug often strikingly affect the degree of target engagement in different brain areas. “Collectively, by complementing competitive and direct labeling strategies, CATCH can reveal dose-dependent, quantitative target engagement across heterogeneous brain regions that are not easily accessible by traditional lysate-based methods,” the team stated. “Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods.”
The proof-of-principle study is just the beginning, Ye emphasized. He and his team plan to develop CATCH further for use on thicker tissue samples, ultimately perhaps whole mice. “…as the first step toward volumetric in situ drug imaging, we demonstrated that CATCH could be applied in thick brain sections (~500 mm),” they wrote. “Expanding this capacity to whole brain, or even whole animal, would represent another technical milestone to allow unbiased screen drug targets across different tissue and cell types.”
Additionally, they plan to extend the basic approach to more common, noncovalently-binding drugs and chemical probes. On the whole, Ye said, he envisions the new method as a basic tool not only for drug discovery but even for basic biology. “In summary, by bringing unprecedented resolving power to small-molecule drug-target imaging in situ, we believe that CATCH offers a valuable tool for both drug discovery and basic chemical biology research,” the authors concluded.