Directionally Modified Fluorophores for Super-Resolution Imaging of Target Enzymes: A Case Study with Carboxylesterases

Abstract

In the need for improving the labeling quality of super-resolution imaging, multifarious fluorescent labeling strategies have sprang up. Among them, a small molecule inhibitor-probe (SMI-probe) shows its advancement in fine mapping due to its smaller size and its specific binding to a specific site. Herein, we report a novel protocol of mechanism-guided directional modification of fluorophores into fluorescent inhibitors for enzyme targeting, which could half the size of the SMI-probe. To confirm the feasibility of the strategy, carboxylesterase (hCE) inhibitors are designed and developed. Among the constructed molecule candidates, NIC-4 inhibited both isoforms of hCE1 and hCE2, with IC50 values of 4.56 and 4.11 μM. The CE-targeting specificity of NIC-4 was confirmed by colocalizing with an immunofluorescent probe in fixed-cell confocal imaging. Moreover, NIC-4 was used in live-cell super-resolution microscopy, which indicates dotlike structures instead of the larger staining with the immunofluorescent probe. Moreover, it enables the real-time tracking of dynamic flow of carboxylesterases in live cells.

Computer-aided molecular design and optimization of potent inhibitors disrupting APC‒Asef interaction

“…The carbamate in the N-terminus is highly specific for carboxylesterase hydrolysis, and elastase easily recognizes and hydrolyzes residues A and G; thus, we decided to remove these segments ( Fig. 1 B) 32 , 33 , 34 . Considering potential drug-like properties, other residues (Y and E) adjacent to the C-terminus containing some unfavorable polar groups in side chains are eliminated for further molecular modifications.…”

Shining a light on liver health: advancements in fluorescence-enhanced enzyme biosensors for early disease detection

“…(D) Structurally diversified carbamates designed and developed serve as inhibitors or substrates of hCEs in this study. Adapted with permission from (Jia et al, 2021). (E) Inhibition properties of NIC series.…”

“…Inhibition types for NIC-3/4 were defined by the Lineweaver−Burk analysis. Adapted with permission from (Jia et al, 2021). (F) Time-lapse SIM images of different areas of hCE1 movement, representatively.…”

“…Successful application of the probe was demonstrated in the detection and imaging of CE activity in zebrafish (Figure 4C). In a separate study in 2021, the Han research group introduced a series of CE fluorescent probes utilizing carbamate as a foundational moiety named NIC−1, NIC-2, NIC-3, and NIC-4 (Figure 4D), subsequently employing them for superresolution microscopy imaging of CE activity in live cells (Jia et al, 2021). Results underscored the efficacy of the NIC-4 as a CE inhibitor, whereby methyl substitution of nitrogen in the carbamate moiety effectively impeded the proton transfer process, thereby hindering nucleophilic attacks necessary for CE hydrolysis (Figure 4E).…”

A Space‐Dependent ‘Enzyme‐Substrate’ Type Probe based on ‘Carboxylesterase‐Amide Group’ for Ultrafast Fluorescent Imaging Orthotopic Hepatocellular Carcinoma

“…Since then, a series of probes that employed the carboxylic ester bonds as responsive sites have been reported and was used to investigate CE activity at physiological levels in ex vivo and in vivo (Table S1, Supporting Information). [26][27][28][29][30][31][32][33] Despite their wide use, the carboxylic ester recognition moieties easily suffer from the potential mutual interference of other esterases, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), [34][35][36] which may seriously compromise the accurate measurement of CE activity in vivo. Recently, a probe with improved selectivity was designed and developed by Yoon's group based on carbamate unit as response group (Figure 1A), because author noticed irinotecan (CPT-11) as a precursor of an anticancer drug containing a carbamate unit could be a substrate catalyzed by CE.…”