An offline-addition format for identifying GPCR modulators by screening 384-well mixed compounds in the FLIPR.
Although fluorescence imaging plate reader (FLIPR)-based assays have been widely used in high-throughput screening, improved efficiencies in throughput and fidelity continue to be investigated. This study presents an offline compound addition protocol coupled with a testing strategy using mixtures of compounds in a 384-well format to identify antagonists of the neurokinin-1 receptor expressed in the human astrocytoma cell line (U373 MG). Substance P evoked a concentration-dependent increase in intracellular cellular Ca(2+) with an EC(50) value of 0.30 +/- 0.17 nM, which was inhibited by neurokinin-1 (NK1) antagonists L-733,060 and L-703,606. Test compounds, as mixtures of 10 compounds/well, were added to the cells offline using an automated dispensing unit and incubated prior to performing the assay in the FLIPR. Using the offline protocol, a higher through put of ~200,000 compounds was achieved in an 8-h working day, and several novel structural classes of compounds were identified as antagonists for the NK1 receptor. These studies demonstrate that the offline compound addition format using a mixture of compounds in a 384-well FLIPR assay provides an efficient platform for screening and identifying modulators for G-protein-coupled receptors.
Gopalakrishnan
Sujatha M
SM
Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. sujatha.m.gopalakrishnan@abbott.com
Mammen
Betsy
B
Schmidt
Martin
M
Otterstaetter
Bernd
B
Amberg
Willi
W
Wernet
Wolfgang
W
Kofron
James L
JL
Burns
David J
DJ
Warrior
Usha
U
D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018040:Receptors, Neurokinin-1 / Q000378:metabolism
D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1