Development of time-resolved fluorescent based [EU]-GTP binding assay for selection of human Histamine 3 receptor antagonists/inverse agonist: a potential target for Alzheimer's treatment.

Background: The histamine H3 receptor is an attractive G protein-coupled receptor drug target that regulates neurotransmission in the central nervous system and plays a crucial role in cognitive and homeostatic functions. This receptor exhibits molecular, pharmacological, and functional heterogeneity that affects the preclinical development of effective antagonists. The range of assay technologies like radio isotope based [35S] GTPγS binding assay, luminescent based reporter gene assay (In-direct cAMP measurement) for binding and signaling have been developed in High Throughput Screening (HTS) laboratories for the identification of hit or lead compounds acting on H3 receptor.

Purpose: The [35S] GTPγS binding assay still remains a useful and a simple technique to demonstrate receptor activation and is one of the few functional, cell-free assays that has set the standards in the field of research. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentation.

Methods: Herein, we have developed and optimized a membrane based non-radioactive assay using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence technology.

Results: The characterization of H3 agonist or antagonist with HTRF platform has revealed a rank order potency (pEC50 & P K B) comparable to that from isotopic functional studies measured by liquid scintillation counter (LSC). Lastly, the Eu-GTP binding assay has been found to be highly robust (Z' factor 0.84) with high percentage over basal counts.

Conclusion: This assay can be utilized as a component of cascade for the screening of H3 receptor ligands.