HTS Instrument Discovers Low Affinity Inhibitors of the Inositol Phosphate IP Signaling Pathway

EJ Dell, JL Tardieu, F Degorce



G-protein coupled receptors (GPCRs) are transmembrane proteins which play a key role in the signal transduction of extracellular stimuli. GPCRs are associated with a complex assembly of intracellular proteins regulating a large variety of downstream effectors. The production of inositol 1,4,5-triphosphate (IP3) is one such second messenger, which is produced in response to the activation of Gq-coupled receptors. However, IP3’s very short half-life make its assessment too challenging for drug screening assays and the monitoring of calcium release, triggered by IP3, has been extensively used as a downstream readout of this signaling pathway. An alternate way to monitor IP3 is to measure the accumulation of inositol monophosphate (IP1), which is a downstream metabolite of IP3. Monoclonal antibodies raised against IP1 lead to the optimization of a homogeneous time-resolved fl uorescence assay (HTRF®), taking advantage on the fact that IP1 is stable and accumulates in cells. The HTRF® IP-One assay has been compared to existing methods and has been shown to lead to similar compound potency data. Moreover, the end point accumulation of IP1 allows for the discrimination of slow acting compounds that remain unseen by calcium sensing

Epigenetic screening, HTRF® Technology, HTRF microplate readers