Development of an Improved IP₁ Assay for the Characterization of 5-HT_2C Receptor Ligands

Abstract

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor is a member of the serotonin 5-HT(2) subfamily of G-protein-coupled receptors signaling predominantly via the phospholipase C (PLC) pathway. Stimulation of phosphoinositide (PI) hydrolysis upon 5-HT(2C) receptor activation is traditionally assessed by measuring inositol monophosphate (IP(1)) using time-consuming and labor-intensive anion exchange radioactive assays. In this study, we have developed and optimized a cellular IP(1) assay using homogeneous time-resolved fluorescence (HTRF), a fluorescence resonance energy transfer (FRET)-based technology (Cisbio; Gif sur Yvette, France). The measurement is simple to carry out without the cumbersome steps associated with radioactive assays and may therefore be used as an alternative tool to evaluate PI hydrolysis activated by 5-HT(2C) agonists. In Chinese hamster ovary (CHO) cells stably expressing 5-HT(2C) receptors, characterization of 5-HT(2C) agonists with the HTRF platform revealed a rank order of potency (EC(50), nM) comparable to that from intracellular calcium mobilization studies measured by the fluorometric imaging plate reader (FLIPR). A similar rank order of potency was seen with conventional radioactive PI assay with the exception of 5-HT. Lastly, the new assay data correlated better with agonist-induced calcium responses in FLIPR (R(2) = 0.78) than with values determined by radioactive IP(1) method (R(2) = 0.64). Our study shows that the HTRF FRET-based assay detects IP(1) with good sensitivity and may be streamlined for high-throughput (HTS) applications.

Monitoring Gq-coupled receptor response through inositol phosphate quantification with the IP-One assay

“…The data obtained indicated a strong correlation between the two techniques, whatever the cell type used. This was confirmed by a recent study performed on the 5-HT 2C receptor [68]. EC 50 values obtained with a series of reference agonists confirmed the good correlation between the two techniques.…”

“…All antagonists were identified by both IP-One and calcium assays with a good correlation between the IC 50 values obtained with the two techniques. This high degree of correlation was confirmed on the 5-HT 2C receptor using SB-206553 as an antagonist [68]. Positive allosteric modulators (PAM) have been also investigated using the IP-One assay.…”

“…One showed a strong correlation in agonist potencies between the two assays on the 5-HT 2C receptor [68], while another study showed a significant decrease in agonist potencies for some GPCRs when using the IP-One assay [63]. On the four GPCR models tested, agonist potencies were highly correlated between FFAR1 and NPSR receptors, and EC 50 values of agonists for the muscarinic M 1 receptor and the vasopressin V1 b receptor were significantly higher in the IP-One assay than in the calcium assay.…”

“…Two studies compared how agonists behaved in IP-One and calcium assays ( Table 2) [63,68]. One showed a strong correlation in agonist potencies between the two assays on the 5-HT 2C receptor [68], while another study showed a significant decrease in agonist potencies for some GPCRs when using the IP-One assay [63].…”

“…Like the IPOne assay, CDS requires long incubation times to allow cytoskeleton changes to occur. Overall, these data indicate [55] EtB Ala-Endothelin 70 nM 93 nM n.d. [55] CCK1 CCK8 sulfated 2 nM 43 nM n.d. [55] P2Y1 ATP 1.6 µM 1 1 µM n.d. [55] V1a Vasopressin 1 nM 0.4 nM 0.6 nM [55] OT Ocytocin 13 nM 7 nM 3 nM [55] mGlu1 Quisqualate 113 nM 75 nM 21 nM [55] mGlu5 Quisqualate 13 nM 9 nM 1 nM [55] M1 Acetylcholine 56 nM 42 nM n.d. [55] M1 Carbachol 28 µM 4 0 µM n.d. [ [68] 5HT2c RO 60-0175 7 nM 9 nM 10 nM [68] 5HT2c 5-CT 89 nM n.d. 17 nM [68] 5HT2c WAY-161503 0.4 nM 8 nM 3 nM [68] 5HT2c WAY-161504 62 nM 1.3 µM n.d. [68] 5HT2c WAY-163907 6.5 µM 1.2 µM n.d. [68] n.d.: Not determined.…”