Profluorogenic reductase substrate for rapid, selective, and sensitive visualization and detection of human cancer
William C Silvers1, Bijeta Prasai, David H Burk
1Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, United States.
Abstract:
Achieving the vision of identifying and quantifying cancer-related events and targets for future personalized oncology is predicated on the existence of synthetically accessible and economically viable probe molecules fully able to report the presence of these events and targets in a rapid and highly selective and sensitive fashion. Delineated here are the design and evaluation of a newly synthesized turn-on probe whose intense fluorescent reporter signature is revealed only through probe activation by a specific intracellular enzyme present in tumor cells of multiple origins. Quenching of molecular probe fluorescence is achieved through unique photoinduced electron transfer between the naphthalimide dye reporter and a covalently attached, quinone-based enzyme substrate. Fluorescence of the reporter dye is turned on by rapid removal of the quinone quencher, an event that immediately occurs only after highly selective, two-electron reduction of the sterically and conformationally restricted quinone substrate by the cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1). Successes of the approach include rapid differentiation of NQO1-expressing and -nonexpressing cancer cell lines via the unaided eye, flow cytometry, fluorescence imaging, and two-photon microscopy. The potential for use of the turn-on probe in longer-term cellular studies is indicated by its lack of influence on cell viability and its in vitro stability.
Insights
A new fluorescent probe detects cancer by activating in response to a specific enzyme (hNQO1) found in tumor cells. This rapid, selective probe offers potential for personalized oncology diagnostics and imaging.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Personalized oncology requires sensitive and selective molecular probes.
- Current probes often lack the necessary speed and specificity for real-time cancer event detection.
Purpose of the Study:
- To design and evaluate a novel "turn-on" fluorescent probe for cancer detection.
- To develop a probe activated by a specific enzyme overexpressed in tumor cells.
Main Methods:
- Synthesis of a novel naphthalimide-based fluorescent probe.
- Incorporation of a quinone-based substrate quenched by photoinduced electron transfer.
- Activation mechanism involving selective reduction by human NAD(P)H:quinone oxidoreductase 1 (hNQO1).
Main Results:
- The probe exhibits quenched fluorescence that is restored upon hNQO1-mediated reduction.
- Rapid and selective differentiation of hNQO1-expressing cancer cells demonstrated.
- Successful application in unaided eye observation, flow cytometry, fluorescence imaging, and two-photon microscopy.
Conclusions:
- The developed turn-on probe is a sensitive and selective tool for detecting cancer-associated hNQO1 activity.
- The probe's stability and lack of cytotoxicity support its use in cellular studies.
- This approach holds promise for advancing personalized oncology diagnostics and imaging.
