De Novo Design of a Highly Selective Nonpeptide Fluorogenic Probe for Chymotrypsin Activity Sensing in a Living System

Abstract

Chymotrypsin, an extensively known proteolytic enzyme, plays a substantial role in maintaining physiological functions, including protein digestion, immune response, and tissue repair. To date, intense attention has been focused on the invention of efficient and sensitive chemical tools for chymotrypsin activity measurement. Among them, the “nonpeptide”-based chymotrypsin probe design strategy utilizing the esterase activity of chymotrypsin has been well-developed due to its low cost and high atom-economy feature. However, the ester-bond-based nature of these probes make them possibly vulnerable to esterases and active chemicals. These defects strictly restricted the application of the previously reported probes, especially for imaging in living systems. Therefore, to acquire fluorogenic probes with sufficient stability and specificity for chymotrypsin sensing in a complicated biological environment, a more stable skeleton for nonpeptide-based chymotrypsin probe construction is urgently needed. Herein, a novel nonpeptide-based fluorogenic probe for specific chymotrypsin activity sensing was designed and synthesized by the substitution of an ester-based linker with a heptafluorobutylamide moiety. The acquired probe, named TMBIHF, showed high selectivity toward various enzymes and reactive chemicals, while it retained high sensitivity and catalytic efficiency toward chymotrypsin. Moreover, TMBIHF was successfully applied for monitoring chymotrypsin activity and pancreas development in live zebrafish, specific sensing of exogenous and endogenous chymotrypsin in nude mice, and visualizing chymotrypsin-like activity-dependent cellular apoptosis, thus providing an alternative and reliable way for chymotrypsin-targeted biosensor or prodrug construction.

Exploring the memory: existing activity-dependent tools to tag and manipulate engram cells

“…To examine the presence and study the physiological mechanisms underlying the functioning of engram cells, scientists created a class of tools known as activity-dependent tools. These are based on the definition of engram cells and the molecular mechanisms contributing to neural activity (Bai and Suzuki, 2020 ; Colecraft, 2020 ; Demchuk et al, 2020 ; Nectow and Nestler, 2020 ; Wei et al, 2021 ; Kasatkina and Verkhusha, 2022 ; Liu et al, 2022 ; Robison and Nestler, 2022 ; Soutschek and Schratt, 2023 ). A sensory experience triggers a sensory-evoked activity, which drives a synaptic input onto the neurons and initiates membrane depolarization and calcium influx into the cytoplasm (Cohen and Greenberg, 2008 ).…”