Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging

Abstract

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.

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection

“…Work from our laboratory and others has demonstrated the utility of caged luciferins in vivo (28)(29)(30) for measuring transient small molecules (31)(32)(33)(34), enzyme and transporter activities (34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46), protein-protein and cell-cell interactions (42,47,48), and copper (49). Indeed, previous work from our laboratory utilized a Cu-dependent oxidation reaction to uncage luciferin for in vivo copper imaging (50), a first demonstration of a general activitybased sensing (ABS) strategy which we envisioned expanding to other essential metals in biology by changing the reaction trigger.…”

Artificial Fluorogenic Substrates in Microfluidic Devices for Bacterial Diagnostics in Biotechnology

“…The conversion of fluorogenic compounds during cultivation answers if bacteria are viable under the condition of interest [3,4,10]. The role of enzymes in virulence and bacterial survival is of high interest for basic research as well as future oriented therapeutic strategies [7,9,11].…”

“…Novel applications with those chemical compounds for bacterial detection [4][5][6], metabolic characterization [2,7,8], drug development [9], preclinical studies [10], and screening for antibiotic resistance [11] are reported.…”

“…Important scaffolds are from the fluorochrome family of xanthene dyes as fluorescein and rhodamine derivatives or the family of coumarins as 4-methylumbelliferone, resorufin, and 7-hydroxycoumarin as reviewed elsewhere [12] and also other fluorescent scaffolds as, e.g., oxy-luciferin [10] or artificial fluorochromes as Cy3 or Cy5.5 [11].…”

“…A recent application of previously synthesized artificial β-lactamase substrates containing green fluorescent Tokyo Green has Artificial Fluorogenic Substrates been presented for Mycobacterium tuberculosis detection in microfluidic droplets [6]. Smart substrate design enables diagnostic concepts with coupled enzymatic reactions as shown for a caged luciferin that requires conversion by nitroreductases prior reaction with luciferase for metabolic characterization of living bacteria [10]. Furthermore, enzymatic cleavage of quencher moieties can be used for analytical FRET biosensor construction [11].…”