Imaging Heterogeneous Patterns of Aminopeptidase N Activity in Hierarchical Tissue Structures Through High-Resolution Whole-Organ 3D Mapping.

Enzymes play a crucial role in regulating physiological functions, and abnormal enzyme activity is associated with various pathological conditions. Precise imaging of enzyme activity in tissues, providing detailed spatial and quantitative information, advances our understanding of physiological and pathological processes. Despite their importance, there is still a lack of methods for high-resolution 3D imaging of enzyme activity across entire tissues. In this research, we report a methodology for high-resolution, whole-organ 3D mapping of enzyme activity, which combines tissue clearing with an activity-based covalent chemical probe. Focusing on aminopeptidase N (APN) as a representative target of peptidase, we developed ANA-o-BODIPY, an activity-based covalent fluorescent probe compatible with tissue clearing for imaging APN activity. Upon activation by APN, ANA-o-BODIPY produces a reactive intermediate, aza-quinone methide, which covalently binds to proximal proteins. This covalent probe is successfully utilized to record the location of APN activity during the tissue-clearing process. By combining the probe with tissue clearing, we have achieved high-resolution 3D mapping of APN activity across whole organs for the first time. Moreover, this advancement allowed us to visualize the heterogeneity of APN activity in individual tubular structures and to uncover the inhibitory effects of different APN inhibitors.