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A Homogenous 384-Well High Throughput Screen for Novel Tumor Necrosis Factor Receptor: Ligand Interactions Using Time Resolved Energy Transfer

Department of Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, Essex, United Kingdom, Lead Discovery Unit, Glaxo Wellcome Research & Development, Stevenage, Hertfordshire, United Kingdom

S. Turconi

Department of Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, Essex, United Kingdom

A. Miles-Williams

Department of Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, Essex, United Kingdom

H. Djaballah

Department of Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, Essex, United Kingdom

P. Hurskainen

Wallac Oy, Turku, Finland

J. Harrop

Department of Immunology, SmithKline Beecham Pharmaceuticals, Essex, United Kingdom

K. J. Murray

Department of Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, Essex, United Kingdom

A. J. Pope

Department of Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, Essex, United Kingdom

The herpes virus entry mediator (HVEM) receptor and its ligand, HVEM-L, are involved in both herpes simplex virus type-1 (HSV-1) herpes simplex virus type-2 (HSV-2) infection, and in T-cell activation such that antagonists of this interaction are expected to have utility in viral and inflammatory diseases. In this report we describe the configuration of a homogeneous 384-well assay based on time-resolved energy transfer from a europium chelate on the HVEM receptor to an allophycocyanin (APC) acceptor on the ligand. Specific time resolved emission from the acceptor is observed on receptor:ligand complex formation. The results of various direct and indirect labeling strategies are described. Several assay optimization experiments were necessary to obtain an assay that was robust to automation and file compound interference while sensitive to the effect of potential inhibitors. The signal was stable for more than 24 h at room temperature using the Eu³⁺ chelates, suggesting no dissociation of the lanthanide ion. The 384-well assay was readily automated and was able to identify more than 99.5% of known positive controls in the validation studies successfully. Screening identified both a series of known potent inhibitors and several structural classes of hits that readily deconvoluted to yield single compound inhibitors with the desired functional activity in secondary biological assays. The equivalence of the data in 384- and 1536-well formats indicates that routine implementation of 1536-well chelate-based energy transfer screening appears to be primarily limited by liquid handling rather than detection issues.

Journal of Biomolecular Screening, Vol. 4, No. 4, 205-214 (1999)
DOI: 10.1177/108705719900400408


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