Oxidoreductase-Facilitated Visualization and Detection of Human Cancer Cells

Abstract

quinone oxido-reductase isozyme 1 (hNQO1) results in fast trigger group removal to yield a highly fluorescent green-energy-range reporter that possesses a high molar absorptivity; there is a 136-fold increase in brightness for the enzymatically produced reporter versus probe precursor, a value 4 times greater than previously reported for the hNQO1 analyte. The novel probe is taken up and activated rapidly within only hNQO1-positive human cancer cells; addition of an hNQO1 inhibitor prevents the selective activation of the probe. Comparison of cytosolic fluorescence intensity in positive cells versus background in negative cells yields a quantitative metric (positive-to-negative ratio, PNR) for judging hNQO1 activity. We show it is possible to determine hNQO1 presence in previously studied colorectal cancer cells and the unexplored ovarian cancer cell line NIH:OVCAR-3, with respective PNR values of 926 and 34 being obtained. Even with 10 min probe incubation, ready discrimination of positive cells from negative cells is achieved. Cell viability is unaffected by probe presence, thereby highlighting the practicality of probe use in live-cell imaging applications.

An Activatable Photosensitizer Targeting Human NAD(P)H: Quinone Oxidoreductase 1

“…We measured a K M and V max of 0.61(±0.08) μ m and 0.42(±0.06) μmol min −1 mg hNQO1 −1 , respectively, and a k cat value of 13(±9) s −1 (Figure S9, Supporting Information). The catalytic efficiency ( k cat / K M ) of the enzyme‐mediated activation of 1 was calculated to be 21×10 3 m −1 s −1 , which is comparable to previously developed hNQO1‐targeted fluorescent chemosensors that employ the same quinone substrate as 1 …”

“…PN exhibits a high singlet oxygen quantum yield (≈1) and has good photostability in aqueous solution . Moreover, its electronic framework is similar to previously used fluorophores, and so we hypothesized its singlet oxygen production would be quenched by the nearby electron‐deficient quinone via photon‐induced electron‐transfer (PeT) mechanism . Upon hNQO1‐mediated reduction, the quinol product is expected to initiate intramolecular cyclization at the central amide carbonyl carbon, producing dihydrocoumarin and releasing native PN (Figure , and Figure S1, Supporting Information).…”

“…Rather, its high level has been mainly exploited for diagnostics and alternative therapeutic applications. For example, an array of fluorescent chemosensors have been developed to differentiate cancer cells from healthy cells, and in some instances identify metastases in animal models . Several groups have also developed hNQO1‐bioactivated anticancer agents (e.g., prodrugs) to enhance the cancer selectivity of DNA alkylators or other chemotherapeutic agents .…”

Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging

“…30 Probes 3 , 6 , and 7 target hNQO1 via a quinone propionic acid motif, whose redox potential was tuned to quench the naphthalimide fluorophore by PET. 31–33 Upon two-electron reduction by hNQO1, the resultant hydroquinone undergoes lactonization spurred by the gem -dimethyl substituents, in a manner akin to the trimethyl lock moiety ( cf : EC 3.1.1, probe 32 ). In probes 3 and 6 , the lactonization directly restores fluorescence, whereas in probe 7 , an additional auto-immolative rearrangement step is necessary.…”

Trimethyl Lock: A Multifunctional Molecular Tool for Drug Delivery, Cellular Imaging, and Stimuli‐Responsive Materials

“…In the presence of NQO1, the quinone TML moiety is reduced, and subsequentl actonization releases the fluorescent dye. [147][148][149] For compound 107,e nhanced releaseo ft he fluorophore is achieved by incorporation of as elf-immolative p-aminobenzyl alcohol unit linked through ac arbamate;t his makes the release more entropically favorable by generating CO 2 during the process. [143] More recent contributions to the field of rhodamine-based NQO1-sensitive latent fluorophores based on quinone TML moieties have been reported by the McCarley group, who utilize hydroxymethyl rhodamine green (HMRG, 98,S cheme 23) and morpholinourea rhodamine (MURh, 100)f or the design of fluorogenic probes 97 and 99, respectively.…”