Aim: To develop a universal high-throughput screening assay based on Gα15/16-mediated calcium mobilization for the identification of novel modulators of Gprotein-coupled receptors (GPCR). Methods: In the present study, CHO-K1 or HEK293 cells were co-transfected with plasmids encoding promiscuous G-protein Gα15/16 and various receptors originally coupled to Gαs, Gαi, or Gαq pathways. Intracellular calcium change was monitored with fluorescent dye Fluo-4. Results:We found out for all the receptors tested, Gα15/16 could shift the receptors' coupling to the calcium mobilization pathway, and the EC 50 values of the ligands generated with this method were comparable with reported values that were obtained using traditional methods. This assay was validated and optimized with the δ-opioid receptor, which originally coupled to Gαi and was recently found to play important roles in neurodegenerative and autoimmune diseases. A largescale screening of 48 000 compounds was performed based on this system. Several new modulators were identified and confirmed with the traditional GTPγS binding assay. Conclusion: This cell-based calcium assay was proved to be robust and easy to automate, and could be used as a universal method in searching for GPCR modulators.
The chemokine receptor CXCR4 and its ligand, stromal cellderived factor-1 (SDF-1), play important roles in hematopoiesis regulation, lymphocyte activation, and trafficking, as well as in developmental processes, including organogenesis, vascularization, and embryogenesis. The receptor is also involved in HIV infection and tumor growth and metastasis. Antagonists of CXCR4 have been widely evaluated for drugs against HIV and tumors. In an effort to identify novel CXCR4 antagonists, we screened a small library of compounds derived from marine organisms and found bryostatin-5, which potently inhibits chemotaxis induced by SDF-1 in Jurkat cells. Bryostatin-5 is a member of the macrolactones, and its analogue bryostatin-1 is currently being evaluated in clinical trials for its chemotherapeutic potential. The involvement of bryostatins in the SDF-1/CXCR4 signaling process has never been reported. In this study, we found that bryostatin-5 potently inhibits SDF-1-induced chemotaxis but does not affect serum-induced chemotaxis. Further studies indicate that this inhibitory effect is not due to receptor antagonism but rather to bryostatin-5-induced receptor desensitization and down-regulation of cell surface CXCR4. We also show that these effects are mediated by the activation of conventional protein kinase C. [Cancer Res 2008;68(21):8678-86]
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