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Rationally Engineered hCES2A Near-Infrared Fluorogenic Substrate for Functional Imaging and High-Throughput Inhibitor
Yufan Fan1, Tiantian Zhang1, Yunqing Song1
1Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Analytical Chemistry
|October 2, 2023
Summary
Researchers developed a new near-infrared probe, TP-HTCF, for highly sensitive and specific detection of human carboxylesterase 2A (hCES2A) in living systems. This probe enables advanced imaging and drug discovery for this important endoplasmic reticulum enzyme.
Area of Science:
- Biochemistry
- Chemical Biology
- Molecular Imaging
Background:
- Human carboxylesterase 2A (hCES2A) is an endoplasmic reticulum enzyme crucial for drug metabolism and toxin activation.
- Existing hCES2A substrates lack specificity and accurate localization, hindering in situ imaging and drug discovery.
Purpose of the Study:
- To design and synthesize a highly specific near-infrared (NIR) fluorogenic substrate for hCES2A.
- To develop a practical tool for sensing hCES2A activity in living cells and organisms.
Main Methods:
- Rational ligand design and scaffold screening to identify the HTCF fluorophore.
- Synthesis and evaluation of HTCF esters, with TP-HTCF identified as the optimal substrate.
- Development of a TP-HTCF-based biochemical assay for high-throughput screening of inhibitors.
- In situ imaging of hCES2A in living cells, liver slices, and tumor-xenograft mice.
Main Results:
- TP-HTCF demonstrated ultrahigh sensitivity, excellent specificity, and a high signal-to-noise ratio for hCES2A.
- Hydrolysis of TP-HTCF by hCES2A released HTCF, emitting bright fluorescence around 670 nm.
- The TP-HTCF assay enabled high-throughput screening of hCES2A inhibitors.
- High-resolution imaging of hCES2A was achieved in various biological systems.
Conclusions:
- A rational strategy for developing specific fluorogenic substrates for ER-resident enzymes was established.
- TP-HTCF serves as a valuable tool for sensing hCES2A activity in living systems.
- This work advances in situ functional imaging and drug discovery related to hCES2A.
