Nitroreductase (NTR) is an endogenous reductase overexpressed in hypoxic tumors; however, its precise detection in living cells and animals remains a considerable challenge. Herein, we developed three reaction-based probes and a related bioluminescence assay for the real-time NTR detection. The high sensitivity and selectivity of probe 3, combined with its remarkable potential of bioluminescence imaging, affords a valuable approach for in vivo imaging of NTR in a tumor model mouse.
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.
Two series of novel coelenterazine analogues (alkynes and triazoles) with imidazopyrazinone C-6 extended substitution have been designed and synthesized successfully for the extension of bioluminescent substrates. After extensive evaluation, some compounds display excellent bioluminescence properties compared with DeepBlueC in cellulo, thus becoming potential molecules for bioluminescence techniques.
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.
As the most convenient and efficient bioluminescence system, the firefly luciferase/luciferin complex has been widely used in life science research and high-throughput screening (HTS). Nonetheless, the interpretation of firefly luciferase-based assay data is often complicated by the occurrence of "false positives," in part because firefly luciferase (Fluc) is subject to direct inhibition by HTS compounds that might inadvertently act as inhibitors of its catalytic site. Here we report a series of 2-phenylnaphthalenes as Fluc inhibitors with suitable potency both in vitro and in vivo. Besides, our compound 5 showed significant systemic inhibition in transgenic mice. Enzymatic kinetics study reveals that compound 5 is competitive for substrate aminoluciferin and noncompetitive for the second substrate ATP. Furthermore, compound 5 exhibited good performance as a quenching agent in a dual-luciferase reporter assay. We anticipate that these Fluc inhibitors will contribute to the broader utilization of bioluminescence in life science research while circumventing or at least reducing the number of "false positives".Scheme 1 Mechanism of firefly bioluminescence and structure of D-luciferin, D-aminoluciferin, and resveratrol. This journal isScheme 2 Synthesis of 2-phenylnaphthalenes 4-14. Reagents and conditions: (a) bis(pinacolato)diboron, potassium acetate, PdCl 2 (dp.pf), dioxane, 60 C, 6 h. (b) Potassium fluoride PdCl 2 (dppf), dioxane, 90 C, 12h. (c) NaOH, EtOH, reflux then HCl. (d) Boron tribromide, DCM, À78 C, 12 h. 63452 | RSC Adv., 2015, 5, 63450-63457 This journal is
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