Profiling Ligands Of GPCR Targets Using An Optical Biosensor With Dynamic Mass Redistribution Technology Paul Lee, Ph.D. Lead Discovery Amgen Inc. Thousand Oaks, CA 2008 Label-Free Summit, Corning NY October 5-7, 2008
Outline of Presentation GPCR signaling pathways and assays commonly used Overview of Dynamic Mass Redistribution (DMR) Technology Evaluation of 12 compounds on two GPCRs in a double-blind fashion Summary Acknowledgements
GPCR Ligands and Biological Responses Marinissen et al 2001 TIPS 22:368-376
Common Issues with GPCR Cellular Assays High receptor density is often required to achieve sufficient signal response Multiple assay formats are required to evaluate the different GPCR classes (eg camp and Ca 2+ flux assays) Potential interference issues from fluorescent labels or dyes G αq Ca 2+ Release Ca 2+ flux assay promiscuous coupling G α16 Cell Overexpress Target Receptors G αi camp Inhibition camp assay G αs camp Accumulation
DMR Assay Addresses Some of the Issues with Conventional GPCR Cellular Assays Screen with endogenous receptors Does not require high receptor level Does not require engineered cells No promiscuous coupling Single assay format evaluates all three major GPCR signaling pathways Direct measurement of a cellular response, no interference issues from fluorescent labels or dyes G αq Ca 2+ Release Endogenous receptors G αi camp Inhibition Dynamic Mass Redistribution DMR Assay G αs camp Accumulation
Dynamic Mass Redistribution (DMR) (A) (B) Cell-Based Assays Cell Cell Mass Redistribution Surface Neg DMR Waveguide 150 nm Pos DMR Detection Zone Substrate Epic Biosensor Broadband Source Reflected wavelength DMR refers to the orderly relocation of intracellular proteins and molecules upon receptor stimulation or signaling pathway activation. DMR within a cell (A) results in changes in the local index of refraction (wavelength shift) (B). Courtesy of Corning Inc.
GPCR Signaling Pathway Analysis Using DMR Technology 500 nm Serotonin Gi 50 nm PGE 2 Forskolin Buffer Gs 500 nm Oxotremorine Gq Preincubation with 1 μm forskolin (solid lines) potentiates Gi-coupled GPCR responses, attenuates Gs-coupled GPCR responses, and has no effect on Gq-coupled GPCR responses in most cases.
Evaluation of 12 Compounds on Two GPCRs Using DMR Technology in a Double-blind blind Study GPCRa Agonist GPCRb Agonist Compound 1 2 3 4 5 6 7 8 9 10 11 12 CHO-GPCRa CHO-GPCRb Compound Conc. (nm) 10,000 10,000 3333.33 1111.11 370.37 123.46 41.15 13.72 4.57 1.52 0.51 0.17 0.06 0.02 0.00 0.00 The twelve compounds were tested in an Epic Optical Biosensor in a 384 well format to determine their pharmacological profile
Agonist Induced DMR in CHO-GPCRa and CHO-GPCRb Cells 250 200 150 100 50 0-50 Cpd 1-induced kinetic response on CHO-GPCRa [Cpd1] 600 nm 200 nm 67 nm 23 nm 7.5 nm 0 nm 750 650 550 450 350 250 150 50-50 Cpd 2-induced kinetic response on CHO-GPCRb [Cpd2] 5 um 625 nm 40 nm 10 nm 0 nm The plates seeded with 12k cells per well a day earlier were incubated in the optical biosensor for 2 hr before taking a 5 min baseline measurement. Then 10 μl of compound was added to the cells and DMR responses monitored continuously for 45 min.
Determination of GPCRa and GPCRb Coupled Signaling Pathways Cpd 1 to CHO-GPCRa Cpd 2 to CHO-GPCRb 50 nm Cpd1 Forskolin Buffer 500 nm Cpd2 Buffer Cpd 1 induced DMR response was enhanced in the presence of forskolin, suggesting GPCRa is a Gi-coupled GPCR. For CHO-GPCRb cells, forskolin preincubation had no effect on Cpd 2 induced DMR response, suggesting GPCRb is a Gq-coupled GPCR.
DMR Profiles of the 12 Compounds on CHO-GPCRa Cells in Agonist Mode Cpd1 Cpd2 Cpd3 Cpd4 Cpd5 Cpd6 Cpd7 Cpd8 Cpd9 Cpd10 Cpd11 Cpd12 Cpd 1, 3 and 6 induced dose-dependent positive DMR responses on CHO-GPCRa cells, whereas Cpd 4, 8 and 9 induced negative DMR responses.
DMR Profiles of the 12 Compounds on CHO-GPCRa Cells in Antagonist Mode Cpd1 Cpd2 Cpd3 Cpd4 Cpd5 Cpd6 Cpd7 Cpd8 Cpd9 Cpd10 Cpd11 Cpd12 Cpd 1, 3, 4, 6, 8 and 9 inhibited Cpd 1 (50 nm) induced DMR responses in a dose-dependent manner.
DMR Profiles of the 12 Compounds on CHO-GPCRb Cells in Agonist Mode Cpd1 Cpd2 Cpd3 Cpd4 Cpd5 Cpd6 Cpd7 Cpd8 Cpd9 Cpd10 Cpd11 Cpd12 Cpd 2, 5 and 7 induced dose-dependent positive DMR responses on CHO-GPCRb cells, while cpd 3, 4 and 9 also induced positive DMR responses at a lower intensity.
DMR Profiles of the 12 Compounds on CHO-GPCRb Cells in Antagonist Mode Cpd1 Cpd2 Cpd3 Cpd4 Cpd5 Cpd6 Cpd7 Cpd8 Cpd9 Cpd10 Cpd11 Cpd12 Cpd 2, 3, 4, 5, 6, 7, 9 10, 11 and 12 inhibited Cpd 2 (500 nm) induced DMR responses in a dose-dependent manner.
Highlight of Compound 8 Induced DMR Responses in the Two Cell Lines Agonist Mode Antagonist Mode CHO-GPCRa CHO-GPCRb Cpd 8 is Amisulpride which is a known selective dopamine D2/D3 receptor antagonist.
Highlight of Compound 9 Induced DMR Responses in the Two Cell Lines Agonist Mode Antagonist Mode CHO-GPCRa CHO-GPCRb Cpd 9 is Haloperidol which is a known nonselective dopamine receptor antagonist.
Effects of Compound 3, 4 and 9 on Parental CHO-K1 Cells Cpd3 Cpd4 Cpd9 Cpd 3, 4 and 9 appear active in both CHO-GPCRa and CHO-GPCRb cell lines. Testing these compounds in the parental CHO-K1 cell line showed Cpd 3 and 4 induced a DMR positive response, whereas Cpd 9 produced a weak DMR negative response at 10 μm. These 3 compounds may contribute to DMR via other receptors in addition to GPCRa and GPCRb.
Comparison of DMR Responses to camp and Ca 2+ Flux Results in CHO-GPCRa and CHO-GPCRb Cells CHO-GPCRa (camp assay) CHO-GPCRa (DMR) CHO-GPCRb (Aqn assay) CHO-GPCRb (DMR) IC50 nm (vs IC50 nm (vs IC50 nm (vs IC50 nm (vs ompound Name EC50 nm 15 nm DA) EC50 nm 50 nm DA) EC50 nm 67 nm Carb) EC50 nm 500 nm Carb) 1 Dopamine 2.2 ± 0.7 20 ± 7 2 Carbachol 32 ± 2 66 ± 32 3 PD 128907 0.7 ± 0.2 3500 ± 1867 2098 ± 141 4 Spiperone 3 ± 2 N-DMR 5 ± 4 10520 ± 8641 4782 ± 1978 5 Oxotremorine 9 ± 1 2 ± 2 6 R(+)-7-OH-DPAT 0.3 ± 0.1 1 ± 3 534 ± 126 6280 ± 2917 7 Pilocarpine 391 ± 245 344 ±122 8 Amisulpride 122 ± 56 N-DMR 86 ± 25 9 Haloperidol 9 ± 5 N-DMR 24 ± 8 2290 ± 1250 3916 ± 1470 10 Telenzepine 10 ± 3 4 ± 2 11 Scopolamine 0.6 ± 0.1 2 ± 2 12 Atropine 0.7 ± 0.3 5 ± 2 13 ATP For GPCRa, DMR results agree with camp data in general. Cpd 3 appears much less potent in the DMR assay and it is also active on the parental cells. On GPCRb, DMR results are in accord to the Ca 2+ flux data for agonists and antagonists tested. Cpd 4, 6 and 9 are active in both assays at high concentrations.
ATP Induced DMR Responses in CHO-GPCRa and CHO-GPCRb Cell Lines Cpd 13 on CHO-GPCRa Cpd 13 on CHO-GPCRb EC 50 = 127 ± 18 nm EC 50 = 232 ± 52 nm
Conclusions The universal nature of the DMR technology and live cell real-time measurement make assay development for different classes of GPCRs simple and uniform. This technology enables rapid and simultaneous analysis of multiple GPCRs that couple to Gs, Gi and Gq signaling pathways. In a single assay platform, DMR response profiles provide information on the compound selectivity and G protein signaling mechanism. We demonstrated that the DMR assay, a label-free, non invasive technology, successfully identified agonists and antagonists for two GPCRs in a double-blind study. The potency ranking of known agonists and antagonists tested in the two GPCRs is similar between the DMR response and camp or Ca 2+ flux assay except for one compound.
Conclusions The ability to detect endogenous GPCR activation allows for analysis of cellular responses under physiologically relevant settings. 1. Potentially allows analysis in primary cells, and studies in bio- and disease-relevant samples without manipulation 2. IP issues can be avoided along with cost saving associated with time and effort in developing bioengineered cell line. It enables orphan GPCR screen. It is in 384-well high throughput screening format. In summary, DMR technology represents a generic platform amenable to pharmacological evaluation of cellular responses to GPCR activation in a label-free live cell assay environment.
Acknowledgements Amgen Inc. Carlo van Staden Jenny Ly John Salon Corning Life Science Alice Gao Arron Xu Ye Fang Ron Verkleeren