Homogeneous High-Throughput Screening (HTS) Assays for Serine/Threonine Kinases Using beta-Galactosidase Enzyme Fragment Complementation

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. ( Journal of Biomolecular Screening 2008:737-747)

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Faculty Opinions recommendation of High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029996.351440 ◽

2005 ◽

Author(s):

Burckhard Seelig

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

In Vitro Evolution ◽

Double Emulsions ◽

Beta Galactosidase

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Enzyme Fragment Complementation: A Flexible High Throughput Screening Assay Technology

Assay and Drug Development Technologies ◽

10.1089/154065802761001356 ◽

2002 ◽

Vol 1 (1) ◽

pp. 97-104 ◽

Cited By ~ 69

Author(s):

Richard M. Eglen

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Fragment Complementation

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Evaluation of the InteraX™ System Technology in a High-Throughput Screening Environment

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104272568 ◽

2005 ◽

Vol 10 (5) ◽

pp. 485-494 ◽

Cited By ~ 6

Author(s):

Frank H. Büttner ◽

Renate Kumpf ◽

Susanne Menzel ◽

Dominique Reulle ◽

Martin J. Valler

Keyword(s):

High Throughput ◽

Protein Interactions ◽

High Throughput Screening ◽

Egf Receptor ◽

Chimeric Proteins ◽

System Technology ◽

Protein Protein Interactions ◽

A Cell ◽

Well Assay ◽

Beta Galactosidase

The authors have developed a cell-based high-throughput screening (HTS)-compatible assay tomeasure EGFRdimerization using the InteraX TMenzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins with complementing deletionmutants of the beta galactosidase enzyme, each fused to the extracellular and transmembrane part of EGFR. On binding of EGF, EGF receptor dimerizes and an active beta galactosidase is built. The authors used this homogeneous 384-well assay to screen about 20,000 diverse compounds. From 2 independent primary screen runs 239 hits were identified. For run 1, amean S/Bratio of 4.26 and ameanZβ factor of 0.74were obtained, for run 2 amean S/Bratio of 3.88 and amean Zβ factor of 0.71 were obtained. After hit confirmation, repeated 4 times, 112 hits remainedwith a confirmation rate of 48.9%. Thirty of the 112 could be identified as cytotoxic. Fifty-one of the remaining 82 compounds could be shown to be inhibitors of the beta galactosidase enzymeitself. In summary, 31 compounds remained as potential EGFRdimerization or EGF stimulation inhibitors. The authors conclude that the InteraX TMsystemtechnology is HTS capable and can detect smallmolecule inhibitors capable of inhibiting protein-protein interactions.

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