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Abstract
Nitroreductases (NTRs) mediate the reduction of nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives. The activity of NTRs in bacteria facilitates the metabolic activation and antibacterial activity of 5-nitroimidazoles. Therefore, NTR activity correlates with the drug susceptibility and resistance of pathogenic bacteria. As such, it is important to develop a rapid and visual assay for the real-time sensing of bacterial NTRs for the evaluation and development of antibiotics. Herein, an activatable near-infrared fluorescent probe (HC–NO2) derived from a hemicyanine fluorophore was designed and developed based on two evaluation factors, including the calculated partition coefficient (Clog P) and fluorescence wavelength. Using HC–NO2 as the special substrate of NTRs, NTR activity can be assayed efficiently, and then, bacteria can be imaged based on the detection of NTRs. More importantly, a sensitive in-gel assay using HC–NO2 has been developed to selectively identify NTRs and sensitively determine NTR activity. Using the in-gel assay, NTRs from various bacterial species have been profiled visually from the “fluorescence fingerprints”, which facilitates the rapid identification of NTRs from bacterial lysates. Thus, various homologous NTRs were identified from three metronidazole-susceptible bacterial species as well as seven unsusceptible species, which were confirmed by the whole-genome sequence. As such, the evaluation of NTRs from different bacterial species should help improve the rational usage of 5-nitroimidazole drugs as antibiotics.
Highlights
Nitroreductases (NTRs) mediate the reduction of nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives
Nitroreductases (NTRs) are biological enzymes of the flavin enzyme family that reduce nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives using nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor.[1−9] The hypoxic environment of the tumor tissue results in the overexpression of NTRs in tumor cells, highlighting the importance of NTR monitoring for clinical diagnosis and tumor therapy.[10,11]
The existence of NTRs in bacterial species has resulted in the development of novel antibacterial agents based on drug release activated by endogenous bacterial NTRs.[23−25] the expression and bioactivity of NTRs in various pathogenic bacterial species are essential for clinical infection therapy, for which an efficient analytic technique is required for endogenous bacterial NTR profiling and identification
Summary
Nitroreductases (NTRs) mediate the reduction of nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives. These observations indicate that HC−NO2 could serve as a potential off−on NIR fluorescent probe for NTRs. Based on the biological function of NTRs, an enzymatic reduction of HC−NO2 is expected (Figure 2a). (b) Fluorescence behavior of HC− NO2 toward various biological proteins in comparison with that toward NTRs. the coincubation of HC−NO2 and NTRs in the presence of NADH was analyzed using high-performance liquid chromatography (HPLC), where a peak corresponding to HC−NH2 was observed, indicating the enzymatic reduction and production of HC−NH2 (Figure S3).
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