title: Reassessment of the pharmacology of Sphingosine-1-phosphate S1P3 receptor ligands using the DiscoveRx PathHunter™ and Ca2+ release functional assays.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 Ca(2+) release and PathHunter™ assays.
  
  
  RESULTS
  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 Ca(2+) 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 Ca(2+) release assay.
  
  
  CONCLUSIONS
  Comparison of data from the PathHunter™ assay with binding and functional Ca(2+) 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.
authors.affiliation: Novartis Institutes for Biomedical Research, Wimblehurst Road, Horsham, West Sussex, UK.
authors.forename: D M
authors.initials: DM
authors.lastname: Riddy
authors.forename: C
authors.initials: C
authors.lastname: Stamp
authors.forename: D A
authors.initials: DA
authors.lastname: Sykes
authors.forename: S J
authors.initials: SJ
authors.lastname: Charlton
authors.forename: M R
authors.initials: MR
authors.lastname: Dowling
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D001667:Binding, Competitive; D001681:Biological Assay; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D000068876:Fingolimod Hydrochloride; D006801:Humans; D007189:Indans / Q000378:metabolism; D008024:Ligands; D010755:Organophosphates / Q000378:metabolism; D010069:Oxadiazoles / Q000378:metabolism; D011409:Propylene Glycols / Q000378:metabolism; D011869:Radioligand Assay; D049349:Receptors, Lysosphingolipid / Q000819:agonists / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D000071557:beta-Arrestins
chemical_list: C532995:2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino)ethanol; D019390:Arrestins; D007189:Indans; D008024:Ligands; D010755:Organophosphates; D010069:Oxadiazoles; D011409:Propylene Glycols; D049349:Receptors, Lysosphingolipid; C574440:SIP3 protein, human; D000071557:beta-Arrestins; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine; D002118:Calcium