Gene-directed enzyme prodrug therapy (GDEPT) is a promising candidate as an alternative anticancer strategy to selectively treating malignant tumor cells. GDEPT is a suicide gene therapy that targets a vector encoding a foreign prodrug-activating enzyme in tumor cells via capitalizing on a bacterial- or viral-based delivery system to achieve such site-specific sensitization. Expression of the exogenous enzyme leads to local conversion of a subsequently delivered nontoxic prodrug substrate into an active therapeutic metabolite, which is followed by tumor cell cytotoxicity. As malignant tumor cell-specific promoters control gene expression, the enzyme can be preferentially predisposed to such. One GDEPT system that is an attractive approach is the nitroreductase (NTR)-CB1954 combination. The Escherichia coli NTR gene, nfsB, encodes an oxygen-insensitive FMN-dependent enzyme that when in the presence of a reducing cofactor, such as NAD(P)H, activates CB1954 to generate potent interstrand DNA cross-links that induce apoptosis in proliferating and quiescent cells. The NTR-CB1954 enzyme-prodrug pair entails the bioreduction of the aromatic nitro group(s) of CB1954 to reactive intermediates via a ping-pong bi-bi redox reaction mechanism. The 4-hydroxylamine intermediate undergoes further non-enzymatic reduction with cellular thioesters, and thus becomes more cytotoxic upon conversion into a bifunctional alkylating agent. Tumor cells after transduction with nsfB have been found to be 100-10,000-fold more sensitive to nitroaromatic substrates due to high NTR expression. Unfortunately, in practice, there are no means for visually identifying malignant tumor cells in real time as well as for determining whether they have been transduced. To circumvent these problems, we have developed a caged optical reporter that affords the visualization of NTR suicide gene therapy delivery. The OFF-ON near-infrared (NIR) fluorescent construct is a d-PeT-regulated nitroaromatic-appended smart probe that provided 8-fold fluorescence enhancement at 720 nm in solutio/in vitro under normoxic conditions upon application to glioblastoma U87 cell line, of which upregulated NTR expression was established ex vitro via labeling surrogate biomarker carbonic anhydrase IX (CAIX). The ability to potentially afford the visualization of nfsB-transduced malignant tumor cells in vivo (here via mimicking upregulated NTR expression) paves the way for their fluorescence-guided resection in the clinic.

Caged optical reporter for visualizing nitroreductase suicide gene therapy delivery

ACS Spring 2022

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