guanosine-diphosphate and plerixafor

Chemokine receptors have been implicated in several disease processes such as acute and chronic inflammation, cancer, and allograft rejection and are therefore targets for drug development. The chemokine receptors CCR5 and CXCR4 are of particular interest as they serve as entry cofactors for human immunodeficiency virus. These receptors are members of the G protein-coupled receptor (GPCR) family. In this respect, assessing GPCR activation by GTP binding is an important tool to study the early stage of signal transduction. The assay normally utilizes the non-hydrolysable GTP analogue guanosine 5'-gamma-[35S]thiotriphosphate. In order to avoid the problems involved in working with radioactivity, a new non-radioactive version of the assay was developed using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence. The assay was optimized for CXCR4 and CCR5 and validated for screening of chemokine antagonists using the small molecule CXCR4 antagonist AMD3100 and CCR5 antagonists.

Topics: Anti-HIV Agents; Benzylamines; Calcium; CCR5 Receptor Antagonists; Cell Line; Cell Membrane; Chemokines; Cyclams; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Europium; Fluorescence; Guanosine Diphosphate; Guanosine Triphosphate; Heterocyclic Compounds; Humans; Receptors, CCR5; Receptors, CXCR4; Reproducibility of Results; Saponins; Signal Transduction; Sodium Chloride; Temperature; Time Factors