ABSTRACT
Chalcone refers to an aromatic ketone and an enone that constitutes the central core for various important biological compounds in drug discovery. Moreover, the firefly luciferase (Fluc) as the bioluminescent reporter has been widely used in life science research and high-throughput screening (HTS). However, Fluc might suffer from direct inhibition by HTS compounds resulting in the occurrence of "false positives." In the current research, we discovered a series of chalcone compounds as Fluc inhibitors with favorable potency both in vitro and in vivo. Moreover, our compound 3i showed remarkable systemic inhibition in transgenic mice. Both enzymatic kinetics study and cocrystal structure demonstrated that compound 3i is competitive for substrate aminoluciferin, while noncompetitive for ATP. Besides, compound 3i exhibited excellent selectivity as a promising quenching agent in a simulated dual-luciferase reporter assay. We believed that our research would contribute to improving scientists' awareness of the Fluc inhibitors, pay attention to the bias results, and even expand the utilization of bioluminescence in life science research.
Subject(s)
Chalcones/pharmacology , Enzyme Inhibitors/pharmacology , Luciferases, Firefly/antagonists & inhibitors , Luminescence , Animals , Cell Line, Tumor , Chalcones/chemistry , Enzyme Inhibitors/chemistry , Female , Fireflies , Luciferases, Firefly/isolation & purification , Luciferases, Firefly/metabolism , Luminescent Measurements , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, Transgenic , Molecular StructureABSTRACT
To enhance the efficiency of firefly luciferase/luciferin bioluminescence imaging, a series of N-cycloalkylaminoluciferins (cyaLucs) were developed by introducing lipophilic N-cycloalkylated substitutions. The experimental results demonstrate that these cyaLucs are effective substrates for native firefly luciferase (Fluc) and can produce elevated bioluminescent signals in vitro, in cellulo, and in vivo. It should be noted that, in animal studies, N-cyclobutylaminoluciferin (cybLuc) at 10 µM (0.1 mL), which is 0.01% of the standard dose of d-luciferin (dLuc) used in mouse imaging, can radiate 20-fold more bioluminescent light than d-luciferin (dLuc) or aminoluciferin (aLuc) at the same concentration. Longer in vivo emission imaging using cybLuc suggests that it can be used for long-time observation. Regarding the mechanism of cybLuc, our cocrystal structure data from firefly luciferase with oxidized cybLuc suggested that oxidized cybLuc fits into the same pocket as oxyluciferin. Most interestingly, our results demonstrate that the sensitivity of cybLuc in brain tumor imaging contributes to its extended application in deep tissues.
Subject(s)
Brain/metabolism , Firefly Luciferin/analogs & derivatives , Firefly Luciferin/chemistry , Luminescent Agents/chemistry , Animals , Cell Line, Tumor , Firefly Luciferin/metabolism , Humans , Luciferases/chemistry , Luminescent Agents/chemical synthesis , Luminescent Agents/metabolism , Luminescent Measurements/methods , Male , Mice, Inbred BALB CABSTRACT
In this article, four novel substrates with long halftime have been designed and synthesized successfully for luxAB bacterial bioluminescence. After in vitro and in vivo biological evaluation, these molecules can emit obvious bioluminescence emission with known bacterial luciferase, thus indicating a new promising approach to developing the bacterial bioluminescent system.
Subject(s)
Bacterial Proteins/metabolism , Animals , In Vitro Techniques , Kinetics , Luminescence , Mice , Mice, Nude , Proton Magnetic Resonance Spectroscopy , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Substrate SpecificityABSTRACT
The luciferase reporter gene assay system is broadly applied in various biomedical aspects, including signaling pathway dissection, transcriptional activity analysis, and genetic toxicity testing. It significantly improves the experimental accuracy and reduces the experimental error by the addition of an internal control. In the current research, we discovered some specific ions that could selectively inhibit firefly luciferase while having a negligible effect on renilla luciferase in vitro in the dual-reporter gene assay. We showed that these ionic compounds had a high potential of being utilized as quench-and-activate reagents in the dual-reporter assay. Furthermore, results from kinetic studies on ion-mediated quenching effects indicated that different ions have distinct inhibition modes. Our study is anticipated to guide a more affordable design of quench-and-activate reagents in biomedicine and pharmaceutical analysis.
Subject(s)
Fireflies/enzymology , Ions/metabolism , Luciferases, Firefly/metabolism , Luciferases, Renilla/metabolism , Luminescent Agents/metabolism , Renilla/enzymology , Animals , Enzyme Assays , Fireflies/genetics , Genes, Reporter , Luciferases, Firefly/antagonists & inhibitors , Luciferases, Firefly/genetics , Luciferases, Renilla/antagonists & inhibitors , Luciferases, Renilla/genetics , Luminescence , Renilla/geneticsABSTRACT
A novel fluorogenic probe for tert-butoxy radicals based on the hydrazino-naphthalimide system is reported. Interestingly, different regioisomers exhibited significantly different optical properties toward ROS, which suggested 4-hydrazinyl naphthalimide as a potential new platform for the in vitro and in cellulo detection of alkoxyl radicals.