Development of a bioluminescent nitroreductase probe for preclinical imaging

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.publishedVersio

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging.

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important

Imaging of NTR activity in cells and in <i>in vivo</i> cancer model with NCL.

<p>(A) Concentration-dependent uncaging of NCL in MDA-MB-231 NTR+luc+ cancer cells in comparison with luciferin. (B) Selectivity of NTR imaging by NCL in the same cells in comparison with NTR-luc+ cells. The dashed line indicates background (cells only), *P = 0.0001. (C) <i>In vivo</i> imaging of NTR activity in subcutaneous NTR+ and NTR- xenografts (n = 5). Total luminescence over 1 h from IP injection of luciferin (1.5 mg) and NCL (1.9 mg). d) Representative images of mice 15 min post injection of luciferin or NCL.</p

<i>In vivo</i> activation of NCL probe by luciferase and nitroredictase expressing <i>E</i>.<i>coli</i> in a mouse model of thigh infection.

<p>(A). Luminescence over 4 h from <i>E</i>. <i>coli luc+</i> infected quadriceps (5 × 10⁴–5 × 10⁷ bacteria) after IP injection of 0.8 mg NCL probe (200 μL of 10 mM solution in PBS). (B). Luminescence over 4 h from <i>E</i>. <i>coli luc+</i> infected quadriceps (5 × 10⁷ bacteria) following IP injection of 0.8 mg of probe or 0.63 mg of luciferin (200 μL of 10 mM solution in PBS). (C). Luminescence imaging of mice over 24 h bearing 5 × 10⁷ bacteria, treated with various (0.08, 0.8 and 1.6 mg) NCL probe concentrations (200 μL of 1, 10 and 20 mM solutions of NCL in PBS). As a positive control, mice were injected with equal amounts of <i>E</i>. <i>coli</i> MG1655 expressing lux luciferase that doesn't require exogenous substrate for light production [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131037#pone.0131037.ref044" target="_blank">44</a>]. The signal was collected over 24 h, n = 3 per group.</p