Nitrooxidoreductase Rv2466c-Dependent Fluorescent Probe for Mycobacterium tuberculosis Diagnosis and Drug Susceptibility Testing

Abstract

Firstly, this study demonstrated that natural product-inspired coumarin-based nitrofuranyl calanolides (NFCs) can form the Rv2466c-mycothiol (MSH)-NFC (RvMN) ternary complex via NFC binding to W21, N51, and Y61 of Rv2466c and be specifically reduced by Rv2466c, which is accompanied by the generation of a high level of fluorescence. Additionally, the results unveiled that the acetylated cysteine−glucosamine (AcCys-GlcN) motif of MSH is sufficient to interact with Rv2466c and adopt the active conformation that is essential for fully reducing NFCs. Further clinical translational investigation in this Article indicated that the novel fluorescent NFC probe can serve as a much needed high-throughput and low-cost detection method for detection of living Mycobacterium tuberculosis (Mtb) and can precisely determine MIC values for a full range of available drugs. This method can greatly facilitate the development of phenotypic drug-susceptibility testing (pDST) that will allow the point-of-care treatment of tuberculosis (TB) within a week after diagnosis.

Derivatives of Natural Product Agrimophol as Disruptors of Intrabacterial pH Homeostasis in Mycobacterium tuberculosis

“…Since targeting pH IB represents a novel therapeutic strategy compared with existing antituberculosis drugs, we hypothesized that our bioactive candidates would also possess bactericidal activity against drug-resistant clinical isolates of Mtb . The antibacterial activities of 5a and 1 were then investigated against a multidrug-resistant (MDR- Mtb ) strain and an extensively drug-resistant (XDR- Mtb ) strain using 9,9-dimethyl-2-nitro-7-propyl-10,11-dihydrofuro­[2,3- f ]­pyrano­[2,3- h ]­chromen-5­(9 H )-one as a novel fluorescence indicator , (Figure B). Both 1 and 5a were active against MDR- and XDR- Mtb , with MIC 90 of 1.56 μM against MDR- Mtb and 6.25 μM against XDR- Mtb , respectively (Figures B,C).…”

“…9,9-Dimethyl-2-nitro-7-propyl-10,11-dihydrofuro-[2,3-f ]pyrano[2,3-h]chromen-5(9H)-one reagent was used as a fluorescence indicator to determine the bactericidal activity. 45 Briefly, 20 μL of indicator (1.0 μg/mL) was added to each well in 7H9 medium, and then the fluorescence was measured after an additional 24 h by a spectral scanning multimode reader (Infinite M200, Tecan) at an excitation wavelength of 390 nm and emission wavelength of 470 nm. The controls were the medium only (M) and the bacteria in medium without tested compound (B).…”

Improved Conventional and New Approaches in the Diagnosis of Tuberculosis

“…FLASH also distinguish live from dead cells, allowing the monitoring the drug susceptibility of clinical M. tuberculosis isolates. Mu et al developed a nitrooxidoreductase Rv2466c-dependent fluorescent probe ( Mu et al, 2019 ). The small molecule mycothiol (MSH) of M. tuberculosis binds to Rv2466c; its sulfhydryl group forms a disulfide bond with the Cys19 to activate Rv2466c, allowing the entrance of the coumarin-based nitrofuranyl calanolides (NFCs) into Rv2466c and interact with W21, N51, and Y61 of Rv2466c to form the Rv2466c-mycothiol-NFC ternary complex.…”

Traditional Medicinal Plants as a Source of Antituberculosis Drugs: A System Review

“…In particular, analogues bearing 2-nitrofurano group at the ring D position markedly improved the in vitro efficacy and reduced the mammalian cell toxicity, when compared with the parent compound (+)-calanolide A [55]. Recently, Mu et al demonstrated that the nitrofuranyl calanolides could be employed as novel fluorescent probes that can serve as a much needed high-throughput and low-cost detection method for detection of living M. tuberculosis and can precisely determine the MIC values for a full range of available drugs [178]. Thus, different modifications of the calanolide derivatives demonstrated three aspects (anti-HIV, anti-TB, and TB diagnosis) of potent usage in TB disease.…”