Hydrolysis-Resistant Ester-Based Linkers for Development of Activity-Based NIR Bioluminescence Probes

Area of Science:

• Biomedical Engineering
• Chemical Biology
• Molecular Imaging

Background:

• Activity-based sensing (ABS) probes with near-infrared (NIR) bioluminescence are valuable for cancer biomarker research due to sensitivity and deep tissue penetration.
• Existing NIR substrates lack suitable attachment sites for ABS triggers, hindering probe development.
• Standard linkers often suffer from hydrolysis, leading to high background signals.

Purpose of the Study:

• To design and synthesize a novel, hydrolysis-resistant linker for NIR bioluminescence probes.
• To overcome limitations of existing linkers in activity-based sensing applications.
• To develop the first NIR bioluminescent probe for nitroreductase (NTR) activity.

Main Methods:

• Rational design of an ester-based linker incorporating an isopropyl shielding arm.
• In vitro assessment of linker hydrolysis resistance against spontaneous and enzyme-mediated cleavage.
• In cellulo validation and application in developing a novel NIR probe for NTR activity.

Main Results:

• The new linker demonstrated significantly enhanced resistance to hydrolysis (140.5-fold spontaneous, 67.8-fold esterase-mediated).
• Minimal ester cleavage was observed with various hydrolyzing enzymes.
• The developed NIR probe successfully visualized elevated NTR expression in hypoxic lung cancer cells and a mouse model.

Conclusions:

• The novel hydrolysis-resistant linker is a significant advancement for developing robust NIR bioluminescence probes.
• This technology enables sensitive detection of biomarkers like NTR in deep tissues, relevant to tumor hypoxia and cancer progression.
• The developed NTR probe holds promise for improved diagnostics and understanding of aggressive cancer phenotypes.