Development Of Imaging Probes And Inhibitors For Firefly Bioluminescence

Bioluminescence is a particular type of luminescence in a living organism, which refers to a process that converts biochemical energy to light energy under the catalysis of enzymes without dependence on theabsorption of light. Bioluminescence imaging (BLI) has been widely-used in biomedical research and other fields because of the advantages of noninvasive property, quantitative, high sensitivity, simple operation, and real-time detection.Bioluminescence is widespread invarious living organisms, such as bacteria, insects, and marine organisms. The firefly luciferin-luciferase reaction is considered as the most well-studied and widely-used bioluminescent system. Firefly luciferase can catalyze theoxidation of D-Luciferin (LH2) into oxyluciferin in the presence of ATP, Mg2+ and oxygen, and then emit yellow to green light with an emission wavelength varying from 550 to 620 run. Renilla luciferase/coelenterazine system, another important bioluminescent system, was widely-used as a reporter gene. Renilla luciferase catalyzes coelenterazine oxidation to release the blue light in the presence of oxygen with an emission wavelength around 480nm. The dual luciferase reporter gene system combiningfirefly and renilla luciferases can reduce error and improve experimental accuracy.In this thesis, we designed and evaluated three bioluminescent probes for nitroreductase imaging in vitro and in vivo. Moreover, a series of carboxyl chalcone inhibitors were designed and evaluated for their inhibition activity against firefly luciferase. Besides, we evaluated 22 ionic compounds as inhibitors of firefly luciferase, which may be used in dual luciferase reporter gene system.This thesis consists of three parts:1. Three bioluminescent probes for nitroreductase (NTR) imaging were designed and evaluated in vitro, in cellulo and in vivo. Tumor hypoxia is a prevalent feature in the development and progression of solid tumors. The NTR level is directly related to the degree of hypoxia. In the presence of electron donor NADH or NADPH, the nitro compound will be reduced to the corresponding amino compound by nitroreductase. In this study, a probe showed good selectivity and sensitivity to nitroreductase with the detection limit of as low as 1 ng/mL. Animal experiments also displayed that the probe was able to determine different levels of the tumor. As a result, this probe which can visualize nitroreductase is a promising bioluminescent probe.2. A series of carboxyl chalcones were designed, synthesized and evaluated against recombinant firefly(Photinus pyralis) luciferase in vitro and in vivo. Because the structure of chalcones has abundant flexibility, they are capable of binding to a variety of targets and exhibit a broad range of biological activity. Previous studies exhibited that carboxyl chalcones had significant inhibition of firefly luciferase activity, possibly resulting in "false positive" results in luciferase/luciferin-based high throughput screening. In this study, the most potent carboxyl chalcones 3i revealed significant inhibition on recombinant firefly luciferase activity with an IC50 value of 0.69 μM. Not only these carboxyl chalcone can remind researchers the possible "false positive" results, but also help us get insights into the firefly luciferase/luciferin system and expand its scope of application.3. We simulated the standard protocol of the dual luciferase reporter gene assay to evaluate 22 ionic compounds’ inhibitory activity on purified recombinant firefly (Photinus pyralis) luciferase and recombinant renillareniformis luciferase. One of the essential applications of firefly luciferase inhibitors was considered as a quenching agent. In the dual reporter assay system, a selective quenching agent could selectively inhibit firefly luciferase while have a negligible effect on renilla luciferase. We revealed that some ionic compounds, such as NaSCN, KI, Na2S2O3, NaN3, could selectively inhibit Fluc rapidly while having little effect on Rluc. These ionic compounds are expected as the novel quenching agents, which will be utilized to explore a simple and practical noninvasive detection method in the dual luciferase assay.