Development Of Inhibitors Against Firefly Bioluminescence

Bioluminescence is the production and emission of visible light by a chemical reaction within a living organism. It occurs widely in marine vertebrates and invertebrates, as well as in some insects, fungi, microorganisms and terrestrial invertebrates. Bioluminescence is a special type of chemiluminescence, which converts biochemical energy to light, without dependence on the absorption of light. In such a case, the conversion efficiency from chemical energy to luminous energy is close to 100%.Bioluminescence technology is widely used in fundamental research and life science because of its characteristics of high efficiency, high sensitivity, visualibilty and non-invasive property. For example, bioluminescence imaging (BLI) has extensively been applicable in monitoring cells and biomolecular processes in living subjects, including pathogen detection, tumor growth and responses to therapy patterns of gene regulation, measurements of protein-protein interactions and ADMET (absorption, distribution, metabolism, excretion and toxicity) process in living subjects. As the most thoroughly studied bioluminescence systems, firefly luciferase/luciferin system has the advantage of steady and strong luminous, long wavelength and high permeability in tissues. However, the firefly luciferase/luciferin system can lead to "false positives" in luciferase/luciferin-based high throughput screening, which really bothers medicinal chemists.In this thesis, based on the recent research progress on firefly luciferase inhibitors, we design and synthesised a series of carboxyl chalcones as firefly luciferase inhibitors. Also, the carboxvl chalcone inhibitors were evaluated for their inhibition potency in vitro and in vivo. The inhibition kinetics and mode of inhibition were also explored for better understanding the inhibition mechanisms. Besides, we also report here 2-phenylnaphthalenes and triazoles as potent firefly luciferase inhibitors.This thesis consists of three parts:1. A series of carboxyl chalcones were designed, synthesised and evaluated in enzyme level, in cellulo and in vivo. To figure out its inhibition mode and mechanism of inhibition, we further explored its influence on substrates kinetics behaviour of aminoluciferin and ATP. As a "privileged" scaffold in drug development, the chalcones frequently popped out in bioassays directed at various targets. However, most of the popped out compounds turned out to be "false positives". Here in this paper, the most potent carboxyl chalcones 3i inhibited firefly luciferase with an IC50 value of 0.20μM. It also showed good potential as quenching agent in dual luciferase reporter gene assay. We anticipate that these compounds will contribute to the broader utilization of bioluminescence in life science research while circumventing or at least reducing the futile efforts on "false positives". Moreover, the brand new inhibition mode of these chalcones can be helpful for further investigation into the amazing luciferin-luciferase system and might help to expand its application in various areas since our compounds exhibited good potency both in cellulo and in vivo.2. A series 2-phenylnaphthalenes were evaluated for their inhibition potency in vitro and in vivo. The most potency compound 2 showed inhibition potency with 0.13 μM in enzyme level, and exhibited systemic inhibition effects in transgenic mice. Enzymatic kinetics study was conducted for better understanding its inhibition mode and mechanism.3. A series of triazoles were evaluated for their inhibition potency in vitro and in vivo. The best compound 1a showed potency of 3.28μM in enzyme level. Also, enzymatic kinetics study was conducted for better understanding its inhibition mode and mechanism. As the triazole core is a "privileged" scaffold for various targets, this finding might alert researchers for "false positives" that might emerge in high throughput screenings.