Reassessment of the pharmacology of sphingosine-1-phosphate S1P(3) receptor ligands using the DiscoveRx PathHunter and Ca(2+) release functional assays.

Authors: Riddy D, Stamp C, Sykes D, Charlton S and Dowling M.
Publisher/Year: Br J Pharmacol 167(4):868-80.
Pub Med ID/Journal ID: PMID:22577868



DiscoverRx's PathHunter assay measures GPCR agonist potency, via the recruitment of β-arrestin, independent of the subtype of G(α) protein activated. This assay is frequently used in drug discovery although little is known about the agonist pharmacology generated. Here we have compared agonist potency, efficacy and affinity values obtained in PathHunter assays with those from more established radioligand binding and functional techniques.


Using cells expressing the human sphingosine-1-phosphate S1P(3) receptor at four different densities, we compared pharmacological affinity and efficacy values of four structurally distinct ligands - FTY720-P, VPC24191, CYM5442 and the endogenous agonist S1P - obtained from competition binding, functional Ca2+ release and PathHunter assays.


The pK(i) values for S1P were significantly different (9.34 ± 0.10 and 8.92 ± 0.15) in clones expressing different receptor levels using the binding assay. In the PathHunter and Ca2+ assays, S1P and CYM5442 were full agonists, FTY720-P was a partial agonist, while the efficacy of VPC24191 could not be detected in PathHunter assays. VPC23019, previously described as a S1P(1/3) receptor antagonist, behaved as an S1P(3) receptor partial agonist in the Ca2+ release assay.


Comparison of data from the PathHunter assay with binding and functional Ca2+ assays suggest that PathHunter assays measured a different agonist-bound receptor conformation. While this assay has great utility in drug discovery, care must be taken as high-efficacy, low-affinity agonist compounds would not be detected. Therefore highly amplified, more traditional assays are necessary to identify agonists with low efficacy.