Discovery of an Enzyme-Activated Fluorogenic Probe for In Vivo Profiling of Acylaminoacyl-Peptide Hydrolase

Acylaminoacyl-peptide hydrolase (APEH), a serine peptidase that belongs to the prolyl oligopeptidase (POP) family, catalyzes removal of N-terminal acetylated amino acid residues from peptides. As a key regulator of protein N-terminal acetylation, APEH was involved in many important physiological processes while its aberrant expression was correlated with progression of various diseases such as inflammation, diabetics, Alzheimer’s disease (AD), and cancers. However, while emerging attention has been attracted in APEH-related disease diagnosis and drug discovery, the mechanisms behind APEH and related disease progression are still unclear; thus, further investigating the physiological role and function of APEH is of great importance. To date, enzyme-activated fluorescent probes targeting POPs have been extensively reported and adopted in relevant medical research and applications. Nevertheless, as an important member of the POP family, APEH was rarely referred in the field of bioimaging while the fluorescent probe for in vivo sensing of APEH activity has not been reported yet. Thus, acquiring an efficient APEH-targeted probe is in urgent need. Herein, an enzyme-activated fluorogenic probe for in vivo profiling of APEH was first discovered via a substrate mimic-based strategy. By combination of stimulated molecular docking-based preliminary screening and experiment-based secondary screening, the optimal probe (named as TMN-AcA), which displayed high binding affinity, sensitivity, and specificity toward APEH, was screened out. Owing to the superior properties of TMN-AcA, endogenous APEH activity in various cell lines and transplanted tumor could be visualized while tissue distribution of APEH was revealed. Most importantly, APEH was first demonstrated to be a potential biomarker of multiple-organ injury via TMN-AcA-based bioimaging and immunohistochemistry (IHC) analysis while the newly developed probe could serve as a vital tool for APEH-related disease diagnosis and biological function study.