Evaluation of the DiscoveRx PathHunter™ CHO Sphingosine-1-Phosphate 1 (S1P1) Lipid Receptor Beta-Arrestin Cell Line and its Suitability for High Throughput Screening

Jennifer Cryan, Sapna Desai, Sandra Arpino. GlaxoSmithKline, Stevenage, United Kingdom.

Rather than measuring classical functional responses of GPCRs, the Pathhunter™ β-arrestin assay works via Enzyme Fragment Complementation of two portions of the β-galactosidase enzyme, one of which is fused to β-arrestin and the other to the GPCR of interest. On receptor activation, functional enzyme is formed which can then generate a chemiluminescent signal. This allows the study of GPCR activation for any receptor that recruits β-arrestin, irrespective of whether the signalling cascade is known

The S1P1 assay has been configured so that it can run in both low volume (lv) 384 and 1536 well formats. Frozen cells are seeded in flasks overnight prior to use and are then prepared in a HBSS-based buffer to the required cell density. Cells are added to pre-stamped compound plates and are then incubated at 37°C/5% CO2 for 90 minutes followed by room temperature for 15 minutes. Detection mix is added and plates are again left at room temperature for 1 hour before the signal is detected using a Viewlux™.

During the evaluation, the technology was shown to be specific, with no response seen to compounds which would cause agonism of native receptors, or when S1P1 compounds were run against a specificity cell line (SST2). Positive assay results following overnight pre-incubation of pertussis toxin (PTX) indicates the technology is measuring β-arrestin recruitment and not Gαi-protein signalling. Compounds tested (26) showed a small range of potency differences with some showing no potency difference (4) and the others being up to half a log unit less potent in the presence of PTX. In antagonist mode, studies with three compounds have yielded pA2 values similar to fpKi values obtained.

The technology was also found to be suitable for HTS. Signal to noise is generally greater than 5 (384 format) and 4 (1536 format) with Z-primes >0.5 in both formats. Compound potencies remain stable over time and are comparable between 384 and 1536 formats.

In summary, this technology had a positive evaluation and is suitable for use in a HTS and compound profiling environment.