2D Strategy for the Construction of an Enzyme-Activated NIR Fluorophore Suitable for the Visual Sensing and Profiling of Homologous Nitroreductases from Various Bacterial Species

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–NO 2 ) 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–NO 2 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–NO 2 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.

Exploring Multifaceted Roles of Nitroreductases in Bacterial, Animal and Plant Biosystems and their Detection using Fluorescent Probes

“…[29] These NTRs are considered vital for the reduction of nitro groups present in 5-nitroimidazole drugs (metronidazole, tinidazole, and ornidazole) intracellularly and results in the production of active radical intermediates, which finally obstruct colonization of bacteria by hindering DNA synthesis (Figure 1). [30] For that reason, they are essential in the antibacterial activity of nitroimidazole antibiotics, such as chloramphenicol. [31] Some of the bacteria are now found to develop resistance against 5-nitroimidazole drugs, which makes the treatment of bacterial infections a challenging task.…”

Hypoxia-Directed and Self-Immolative Theranostic Agent: Imaging and Treatment of Cancer and Bacterial Infections

“…A hypoxia-responsive luminescent and CEST-MRI agent was also developed for in vitro and in vivo tumor detection and imaging . Nitroreductase from various bacterial species has recently been isolated using an in-gel assay and used to construct an enzyme-activated NIR fluorophore . A lanthanide-based luminescent probe has also been synthesized that permits the detection of nitroreductase in live bacteria .…”

“… 26 Nitroreductase from various bacterial species has recently been isolated using an in-gel assay and used to construct an enzyme-activated NIR fluorophore. 27 A lanthanide-based luminescent probe has also been synthesized that permits the detection of nitroreductase in live bacteria. 28 A multifunctional luminogen probe with aggregation-induced emission characteristics was reported recently for selective imaging and photodynamic killing of both cancer cells and Gram-positive bacteria.…”

Visual Sensing of β-Glucosidase From Intestinal Fungus in the Generation of Cytotoxic Icarisid II

“…On the basis of the special catalytic characteristics of β-Glc, several fluorescent carbon dots have been developed as fluorescent biosensors for the sensitive determination of Glucosidase, including α-Glc and β-Glc (Kalaiyarasan, et al, 2019;Kong, et al, 2020;Liu, et al, 2020;Wang, et al, 2020). While, enzymatic fluorescent probes have been widely used to sense biological enzymes, exhibiting the advantages of high sensitivity, high selectivity and facilitate in vivo/in vitro imaging (Feng, et al, 2021;Li, et al, 2020;Liu, et al, 2021;Ning, et al, 2019;Tian, et al, 2021;Yan, et al, 2022;Zhang, et al, 2021). Using the excited-state intramolecular proton transfer (ESIPT) phenomenon, flavonol derivatives have been used as fluorescent indicators for β-Glc activity (Reszka, et al, 2020).…”