Pang, Z.; Leung, V. H.; Wang, C. C.; Attarpour, A.; Rinaldi, A.; Shen, H.; Glynn, C.; Sigua, L. H.; Yender, M.; Selke, A.; Xu, S.; Wu, P.; Goubran, M.; Cravatt, B. F.; Ye, L.

As our understanding of biological systems reaches single-cell and high spatial resolutions, it becomes imperative that pharmacological approaches match this precision to understand drug actions. This need is particularly urgent for the targeted covalent inhibitors that are currently re-entering the stage for cancer treatments. Therefore, we developed 3D volumetric clearing-assisted tissue click chemistry (vCATCH) to image in vivo target-bound drugs by leveraging the unique kinetics of click reactions. In mice, vCATCH overcomes limitations of existing methods to achieve high-throughput volumetric chemical labeling through simple and passive incubation steps, thus enabling unbiased cellular resolution drug imaging in entire animals. We combined vCATCH with HYBRiD imaging and virtual reality to visualize and quantify in vivo targets of two clinical cancer drugs, afatinib and ibrutinib, which recapitulated their known pharmacological distribution and revealed previously unreported tissue and cell type engagement potentially linked to off-target effects. vCATCH provides a body-wide, unbiased platform to map covalent drug engagements at unprecedented scale and precision.