Bioluminescent Assays for High-Throughput Screening

Abstract

In the development of high throughput screening (HTS) as a central paradigm of drug discovery, fluorescence has generally been adopted as the favored methodology. Nevertheless, luminescence has maintained a prominent position among certain assay formats, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a wider range of applications due to their sensitivity, broad linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by the development of several new assay designs for diverse targets such as kinases, cytochrome p450s, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for HTS, highlighting new detection capabilities brought about by engineering luciferase genes, enzymes, and substrates. In genetic reporter applications, modifications to the luciferase genes have improved assay sensitivity by substantially increasing expression efficiency and enhanced response dynamics by reducing expression lifetime. The performance of assays based on detection of ATP and luciferin has been enhanced by modifications to the luciferase enzyme that increase its chemical and physical stability. Detection of ATP allows rapid analysis of cell metabolism and enzymatic processes coupled to ATP metabolism. Because luciferins are not naturally associated with mammalian physiology, assays for luciferin detection utilize synthetic derivatives designed to yield luminescence only when coupled with specific target enzymes. Finally, new methods for modulating the specific activity of luciferases are leading to the development of intracellular biosensors for dynamic detection of physiological processes.

Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

“…Ataluren | multisubstrate adduct inhibitor | protein stability | reporter gene assay | X-ray crystallography F irefly luciferase from Photinus pyralis (FLuc) is an ATP-dependent luciferase widely used as a reporter enzyme for cell-based gene expression assays, principally due to the high sensitivity and large dynamic range bioluminescence affords (1,2). However, as an enzyme that catalyzes the bimolecular reaction between two small molecule substrates, D-luciferin and ATP, it is prone to inhibition by a variety of low-molecular-weight heterocyclic compounds typically found in screening collections (3,4).…”

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

“…The use of luciferase as a reporter gene allows for a high dynamic range of detection of signaling activity 23 . Indeed, the sensitivity of the assay makes it possible to detect osmotic stress-induced GC production from single larvae 18 .…”

“…However, such assays might suffer from lower sensitivities due to background effects caused by the excitation light, which also poses limits on the detection of fluorescent compounds. Additionally, they might provide less kinetic resolution because of the generally higher stability of fluorescent proteins 23 . Furthermore, they present challenges in terms of imaging equipment, data analysis and data storage that might limit their use for smaller research labs.…”

The Potential of Non-Ionic Surfactant Against P-Glycoprotein Efflux Transporters for Drug Development System

“…Besides solubility and gastric stability, metabolic processes as well as transporter-mediated efflux play an essential role in influencing oral bioavailability of drugs [2]. P-glycoprotein (P-gp), also known as MDR1 and ABCB1, is a 170 kDa integral plasma membrane protein that functions as an ATP-dependent drug efflux pump and plays an essential role in multi-drug resistance and certain adverse drug-drug interactions [3]. Its efflux mechanism involves the protein binding to the ATP and requires energy derived by the hydrolysis of ATP to ADP in the presence of adenosine-triphosphatase enzyme (ATPase) [4].…”