The use of dynamic mass redistribution (dmr) in the european lead factory (elf): a case study

G. L. Baillie, O. L. Watt, S. McElroy. University of Dundee, Newhouse, United Kingdom.

Introduction: The ELF is a public-private partnership aimed at identifying hit compounds against molecular targets submitted by European academics and SMEs. These are screened against the Joint European Compound Library (JECL), comprising ∼300,000 compounds from seven pharma companies and ∼150,000 compounds synthesised for the project. A key deliverable is a list of up to 50 hit compounds, over which the programme owner gains the rights to exploit for drug development or as new chemical probes. Many target classes are accepted by the ELF with this case study focussing on a Gi/Gq linked GPCR which is indicated in neuropathic pain. To provide orthogonal corroborative evidence of target engagement for hits from a high throughput screen (HTS), a DMR assay was developed and validated using a series of pharmacological agents.

Methods: A PerkinElmer EnSpire plate reader with a Corning EPIC® module was used with DMR-specific 384 well microplates. This provides a real time, label free measurement of the movement of cellular components in response to activation/inhibition of specific pathways such as those mediated by GPCRs. CHO cells stably expressing the target GPCR were tested in the DMR assay and an intracellular Ca²⁺-release assay, as used for the HTS, as a comparable control.

Results: Three reference agonists produced a rapid, dose-dependent, transient increase in DMR signal, reaching a maximum at ∼4 minutes. The %effect relative to unstimulated control was calculated for the 4-minute response and the agonist potencies determined, which were comparable with those obtained in the Ca²⁺-release assay. The agonist’s signalling pathways were investigated in both assays using PTX to inhibit coupling through Gαi and/or YM-254890 to inhibit Gαq (table 1). Responses in DMR appeared to be mainly modulated via Gi-proteins, while responses in the Ca²⁺-release assay were mainly via Gq proteins. There was also a marked reduction in potency of the ligands in DMR vs. Ca²⁺-release assays.

Table 1: pEC50 and max effect values ± SD (n=5) from DMR assays

*p<0.05 compared to vehicle

Conclusion: A DMR assay was successfully developed which interrogated a different coupling pathway for the same GPCR target. This is the first time within the ELF that DMR has been used to provide a robust orthogonal assay for validating hit compounds.