A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology.
J Biomol Screen
; 8(5): 522-32, 2003 Oct.
Article
in En
| MEDLINE
| ID: mdl-14567779
ABSTRACT
To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen trade mark technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biochemistry
/
Receptors, Tumor Necrosis Factor
/
Drug Evaluation, Preclinical
Type of study:
Diagnostic_studies
/
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
J Biomol Screen
Journal subject:
BIOLOGIA MOLECULAR
Year:
2003
Document type:
Article
Affiliation country:
Switzerland