A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology.
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.
Wilson
Janet
J
Serono Pharmaceutical Research Institute, 14 Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland.
Rossi
Claudia Pena
CP
Carboni
Susanna
S
Fremaux
Christèle
C
Perrin
Dominique
D
Soto
Claudio
C
Kosco-Vilbois
Marie
M
Scheer
Alexander
A
D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D016827:CD8 Antigens / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D053278:OX40 Ligand; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011933:Reagent Kits, Diagnostic; D053262:Receptors, OX40; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity
D016827:CD8 Antigens; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D053278:OX40 Ligand; D010455:Peptides; D011933:Reagent Kits, Diagnostic; D053262:Receptors, OX40; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C505716:TNFRSF4 protein, human; C089251:TNFSF4 protein, human; D004121:Dimethyl Sulfoxide