Esterase Specific Fluorescent Probe: Mechanistic Understanding Using QM/MM Calculation and Cell States Discrimination

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Esterases are crucial enzymes regulating homeostasis, protein metabolism, detoxification, and signal transmission.
• Esterase activity is vital for cell viability and cytotoxicity assays, necessitating efficient monitoring tools.
• Existing fluorescent probes for esterases often target specific cellular compartments and may have limitations in monitoring efficiency.

Purpose of the Study:

• To develop a novel fluorescent probe, PM-OAc, for ratiometric monitoring of mitochondrial esterase enzyme activity.
• To elucidate the mechanism of probe-esterase interaction and catalytic hydrolysis using computational methods.
• To demonstrate the probe's utility in distinguishing live from dead cells based on esterase activity.

Main Methods:

• Development of a unique fluorescent probe, PM-OAc.
• Utilizing time-dependent density functional theory (TD-DFT) for spectral analysis.
• Employing molecular dynamics (MD) simulations and QM/MM calculations to study substrate binding and catalytic mechanisms.
• Conducting fluorescent image-based analysis of cellular environments.

Main Results:

• The PM-OAc probe exhibits a bathochromic wavelength shift with esterase activity at alkaline pH (∼8.0) via an intramolecular charge transfer (ICT) process.
• Computational studies successfully elucidated the substrate binding and catalytic hydrolysis mechanism of the ester bond.
• The probe effectively differentiated between live and dead cells in fluorescence imaging, correlating with esterase activity.

Conclusions:

• The developed PM-OAc probe offers a unique and efficient ratiometric method for monitoring mitochondrial esterase activity.
• The probe's mechanism and substrate interaction are well-understood through advanced computational modeling.
• PM-OAc serves as a valuable tool for cell viability and cytotoxicity assessments, enabling clear distinction between live and dead cells.