Mark R Dowling

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 Ca(2+) 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 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. 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.

Slow receptor dissociation kinetics has been implicated in the long clinical duration of action of the muscarinic receptor antagonist tiotropium. However, despite the potential benefits of new drugs with slow dissociation kinetics, the rate parameters of new compounds are seldom measured due to technical difficulties and financial implications associated with radiolabeling multiple ligands. Here we describe the development and optimisation of a medium throughput assay which is capable of measuring the kinetic parameters of novel, unlabelled compounds. Radioligand binding studies were performed with membranes derived from CHO cells recombinantly expressing the human M ₃ muscarinic receptor. Initial characterisation of the radioligand [ ³ H]‐NMS yielded on and off rates of 4.1±0.2 × 10 ⁸ M ⁻¹ min ⁻¹ and 0.015±0.0005 min ⁻¹ , respectively. The specific binding of [ ³ H]‐NMS was measured over time in the presence and absence of several concentrations of unlabelled competitor compounds. These data were analysed using a competition kinetic model to provide on and off rates for the unlabelled competitor. Comparison of the kinetically derived Kd ( k _off / k _on ) with K _i values generated at equilibrium showed an excellent correlation ( r ² =0.99), providing good validation of the method. The on and off rates were also used in theoretical computer simulations to successfully predict the effect of incubation time on apparent IC ₅₀ values. This study demonstrates that a medium‐throughput competition kinetic binding assay can be used to determine accurate on and off rates of unlabelled compounds, providing the opportunity to optimise for kinetic parameters early in the drug discovery process. British Journal of Pharmacology (2006) 148 , 927–937. doi: 10.1038/sj.bjp.0706819