Development of scintillation-proximity assays for alpha adrenoceptors

Gobel, Jeff; Saussy, David L.; Goetz, Aaron S.

doi:10.1016/s1056-8719(00)00072-1

Cited by 27 publications

(15 citation statements)

References 3 publications

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“…It is well recognized from other membrane receptor screening settings that assay sensitivity can be improved by choosing a radiolabeled ligand concentration at or below its K d to permit effective competition by an unlabeled ligand [32] . In the present study, therefore, we selected 0.04 nmol/L as the concentration for [ 125 I] hNMU-25, which is well below the K d value (0.3 nmol/L) of unlabeled ligands [5] .…”

Section: Discussionmentioning

confidence: 99%

Identification of non-peptidic neuromedin U receptor modulators by a robust homogeneous screening assay

Meng

Binkert

et al. 2008

Acta Pharmacologica Sinica

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Aim: To develop a homogeneous binding assay for high-throughput screening (HTS) of hit compounds at human neuromedin U receptor (hNMU-R) 1 and to identify non-peptidic small molecule hNMU-R modulators through functional assessments and structure-activity relationship (SAR) analyses. Methods: Membrane preparations of Chinese hamster ovary cells (CHO-K1) stably expressing hNMU-R1, [ 125 I]hNMU-25, and wheat germ agglutinin-coupled microbeads were used to develop an HTS assay based on scintillation proximity assay (SPA) technology. This method was applied to a large-scale screening campaign against a diverse library of 36 000 synthetic compounds or natural products and subsequent confirmation studies. CHO-K1 cells stably expressing full-length hNMU-R1 or hNMU-R2 and a calcium-sensitive dye were employed to functionally measure intracellular calcium mobilization upon ligand stimulation. Preliminary SAR was determined based on limited structural modifications. Results: The K i value (0.7 nmol/L) of hNMU-25 (the natural ligand) at hNMU-R1 measured by the SPA method was consistent with that reported in the literature, and the Z' factor for this HTS assay was 0.81. A total of 100 hits, showing more than 30% competitive inhibition on [ 125 I]hNMU-25 binding to hNMU-R1, were identified initially, 3 of which were confirmed thereafter to have reasonable hNMU-R1-binding affinities and similar chemical structures. Based on their common molecular skeleton, 203 analogs were synthesized and tested. Among the 16 analogs that retained variable hNMU-R1-binding abilities, 2 elicited calcium influx in both hNMU-R1 and hNMU-R2-expressing cells, but none displayed antagonist activity. Conclusion: The homogeneous hNMU-R1 binding assay is an efficient and robust tool for screening potential hNMU-R modulators. Two non-selective hNMU-R agonists discovered are of low molecular weight nature with novel chemical structures. The preliminary SAR investigation suggests that both the triphenyl and guanidinol groups are crucial to the bioactivities observed.

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Section: Discussionmentioning

confidence: 99%

Identification of non-peptidic neuromedin U receptor modulators by a robust homogeneous screening assay

Meng

Binkert

et al. 2008

Acta Pharmacologica Sinica

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“…Although similar SPA approaches have been employed to study ligand-binding characteristics of a variety of G protein-coupled receptors [6,[13][14][15] , its application in α4β2 nAChR is novel and may be expandable to other ligand-gated ion channels.…”

Section: Discussionmentioning

confidence: 99%

“…In the assay system reported here, no signal shift was observed for more than 48 h, indicating a very stable interaction among various reagents. It was learned from other membrane receptor screening settings that assay sensitivity can be improved by choosing a radiolabeled ligand concentration at or below its K d to permit effective competition by an unlabeled ligand [14] . In the present study, we selected a relatively higher concentration of [ ]cytisine bound to the receptor to a minimum (<10%).…”

Section: Discussionmentioning

confidence: 99%

A robust homogeneous binding assay for alpha4beta2 nicotinic acetylcholine receptor1

Hui

Gao

Xie

et al. 2005

Acta Pharmacologica Sinica

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Aim: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel α4β2 nicotinic acetylcholine receptor (nAChR) modulators. Methods: Membrane preparation of HEK293 cells expressing α4β2 nAChR, [ 3 H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. Results: IC 50 values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [ 3 H]cytisine binding to α4β2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to α4β2 nAChR (K i <2 µmol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. Conclusions: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential α4β2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.

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“…DMSO, MeOH up to 20% (v/v)) [29]. Immobilization of the receptor is based on either the interaction of glycoproteins and glycolipids with wheat germ agglutinin (WGA) [30][31][32][33], by capturing anti-receptor protein antibodies on an anti-IgG coated surface [34], via the streptavidin-biotin interaction [34] or via antibodies directed against the receptor [34,35].…”

Section: Scintillation Proximity Assay (Spa)mentioning

confidence: 99%