GPCR Mediator Characterization on a Label-Free Platform
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GPCR Mediator Characterization on a Label-Free Platform Edmond Massuda, James Keefer, Hao Chen, Scott Perschke - Caliper Discovery Alliances & Services, Hanover Maryland USA. Results (cont) Conclusions • • Label-free technology such as SRU BIND provides a comprehensive platform for screening and characterization of GPCR ligands (agonists, antagonists etc). • • Many GPCR expressing cell lines have been utilized for screening assays at Caliper Life Sciences using BIND technology. Outsourcing is available. • • Label free assays can help define compound potency, efficacy, and allosteric effects. • • Label-free assays can identify functionally selective agonists and cellular signaling pathways. • • Kinetic responses vary based on proteins coupled to the targeted receptor. Abstract Materials and Methods G-protein coupled receptors (GPCRs) are among the most therapeutically-productive drug target families for CNS diseases. GPCRs respond to extracellular ligands and activate intracellular signal transduction pathways which typically lead to changes in cellular morphology. Using a label-free platform, SRU BIND ® , we have characterized a panel of 15 clonal cell lines, each expressing a unique GPCR. We have analyzed the morphological response to agonist stimulation in these cell lines, specifically with respect to the magnitude, rate and duration, which are influenced by the G-protein coupling. Agonist and antagonist assays have been developed in 384-well format for each target. Compared to traditional assays which measure the GPCR functional response through only a single metric such as changes in cAMP or Ca 2+ , the ability of BIND ® to measure the holistic cellular response allows for the detection and measurement of multiple downstream pathways. This is evident, for example, by the display of biphasic responses in some cell lines. The technical platform and methodologies enable screening for novel receptor mediators and can potentially identify ligands acting through non-classical signaling pathways. By using pathway blockers in combination with agonist stimulation of the cloned GPCR, we have begun to characterize these pathways. Introduction BIND is a label-free assay technology that facilitates detection of biomolecular interactions including biochemical and cellular. BIND enables highly sensitive measurements of changes in mass in proximity to the biosensor surface, which allows for precise monitoring of cellular adherence. Biosensors (in 96, 384, or 1536 format) with a nanostructured optical grating reflect light at a wavelength that is shifted in proportion to the mass in proximity to the surface. The optical grating reflects a narrow range of the wavelengths of light upon illumination, which is utilized to determine the “Peak Wavelength Value” or “PWV”. As the average cellular mass near the biosensor surface increases, the PWV increases. Cellular responses to GPCR agonists often result in a change of cellular interaction with, or attachment to, the biosensor. Increases (+PWV) and decreases (–PWV) in cellular mass in proximity to surface can be detected using kinetic and endpoint read modes. Kinetic measurements allow for real time comprehensive data collection on each well of the plate, several times per minute. Using BIND technology, we have characterized a panel of cell lines expressing unique cloned GCPR receptors. Agonist and antagonist assays, in 384-well format, have been developed for each cell line. Cells expressing cloned GPCRs were purchased from PerkinElmer or ChanTest. All data was collected using a SRU BIND Reader. Compound addition and mixing was done with a Caliper Sciclone ALH 3000. Assays were run in either kinetic or endpoint mode. Typical assays are performed in a 80 μL volume in a BIND Sensor CA2-384M plate. Cells are plated on the CA2 mixed extracellular matrix overnight in complete media to reach 80%-90% confluence. Media is replaced with HBSS for two hours prior to running the assay. At time zero, a baseline measurement is taken, then samples or controls are added to wells. For antagonist assays, the inhibition is determined with a reference agonist at the EC80. The holistic cellular morphological response endpoint is determined at the response peak time-point and compared to control wells to assess the compound effect. For pathway blocking experiments, most blockers were pre-incubated with cells for two hours. Results Figure 2. GPCR responses in CHO cells expressing a unique cloned receptor, as measured by the change in PWV versus time. The left figure shows typical GPCR responses based on the native G-protein mechanism of action. Note that magnitude of the response depends on the expressed receptor and its G-protein coupling. On the right, the biphasic responses of the 5-HT1A and CB2 cell lines are atypical, suggesting a blend of the GPCR mechanisms shown on the left graph. Figure 5. The sensitivity of Alpha2A compound pharmacology on 3 platforms was compared in agonist and antagonist assays. EC 50 and IC 50 values were similar with the 3 methods, although there were some exceptions. Emax is defined as the maximum effect. In the plot, a panel of compounds of interest were screened at 10 μM on calcium flux and BIND Alpha 2A assays. Several partial agonists were detected with BIND (x-axis) but not with calcium flux (y-axis). Figure 6. Label-free pathway blocker analysis on Alpha2A in Aequorin/CHO cells. Blockers were used to determine how the various cellular signaling pathways contribute to the change in PWV and kinetic response profile detected by BIND after agonist stimulation of Alpha2A. The table shows the function, concentration, and treatment time of the blockers. The blocker concentrations selected were based on literature. Kinetic traces represent each blocker or control after agonist addition (100 nM UK 14,304). GW5074, which inhibits MEK/ERK, and Pertussis toxin (PTX), which inhibits Gi signaling, blocked the response detected on the SRU by UK 14,304. Wortmannin prevented the desensitization of the response. Figure 7. Alpha1A agonist assay: quality control and validation. Z’ experiments are run with every assay. The results for the this assay are typical of label-free GPCR assay quality Figure 4. GPCR coupling and pharmacology with BIND. Figure 1. SRU BIND Technology. Cellular morphology shift reveals GPCR activation. The measured signal or BIND Response is quantifiable and allows for pharmacology studies. In SEM image on right, HEK cells respond to ATP. Diagram courtesy of SRU. J. Brown GSK Neuroscience Alpha1A Agonist Signal Transduction Pathway Blocker Effect on Alpha2A Alpha1A Antagonist 5-HT1A Agonist 5-HT1A Antagonist References: 1. Lomasney et al. (1990) Proc. Natl. Acad. Sci. 87, 5094-5098 2. Katsushi Shibata et al. (1996) Br. J. Pharmacol., 118, 1403-1408 3. Tate K. M. et al. (1991) Eur. J. Biochem., 196, 357-361 4. Xie D.-W et al. (1995) Neuropsychopharmacology 12, 263-268 Acknowledgements: Michael Getman - SRU Biosystems Mark Welborn, John Gravel, Audrey Kues and Candice Lewis - Caliper Discovery Alliances & Services Receptor Coupling Ref Agonist EC 50 (nM) Ref Antagonist IC 50 (nM) Alpha1a Gq/11 A61603 0.65 WB 4101 4.2 Alpha2a Gi UK 14,304 19 Rauwolscine 7.4 Alpha2b Gi UK 14,304 59 Rauwolscine 16 AT-1 Gq/11 + Gi Angiotensin II 2.1 Saralasin 320 Beta1 Gs Isoproterenol 2.0 Atenolol 300 C5A Gi C5A 1.1 N/A N/A CXCR2 Gi IL-8 3.9 SB 225002 3800 CB1 Gi Win 55,212-2 940 O-2050 34 CB2 Gi Win 55,212-2 9.2 - - DA2 Gi/o Gi/q Dopamine 2.6 (±)-Sulpiride 23 H1 Gq/11 2-pyridylethylamine 1900 Pyrilamine 10 5-HT1A Gi/o 5-Carboxamidotryptamine 1.0 WAY-100,635 10 NK2 Gq/11 (Gs?) [bAla8]-NKA (4-10) 2.3 GR 159897 9.1 ORL-1 Gi, Gi/o, Go Nocicepn 0.84 Trap 101 300 Vaso2 Gs [Arg8]-vasopressin 3.9 Vasopressin analog 1400 Alpha2A Agonists (EC 50 nM, Emax %) Alpha2A Antagonists (IC 50 nM) NE: No Effect *Computer Estimate Figure 3. Adrenergic Alpha1A and serotonin 5-HT1A pharmacology. Oxymetazoline is a known partial agonist. For the antagonist assay, A61603 or 5-CT was used at the EC80. Results are representative of at least 2 experiments performed with duplicate data points. Additional pharmacology results for each assay can be found at: www.CaliperLS.com/products/contract-research/in-vitro/gpcrs/gpcr-functional-pair.htm UK 14,304 addition No ATP ( + ) ATP Agonist addition Ca-flux % Max Response BIND % Maximum Response
Transcript of GPCR Mediator Characterization on a Label-Free Platform
GPCR Mediator Characterization on a Label-Free PlatformEdmond Massuda, James Keefer, Hao Chen, Scott Perschke - Caliper Discovery Alliances & Services, Hanover Maryland USA.
Results (cont)
Conclusions • • Label-free technology such as SRU BIND provides a comprehensive platform for screening and characterization of GPCR ligands (agonists, antagonists etc). • • Many GPCR expressing cell lines have been utilized for screening assays at Caliper Life Sciences using BIND technology. Outsourcing is available. • • Label free assays can help define compound potency, efficacy, and allosteric effects. • • Label-free assays can identify functionally selective agonists and cellular signaling pathways. • • Kinetic responses vary based on proteins coupled to the targeted receptor.
Abstract
Materials and Methods
G-protein coupled receptors (GPCRs) are among the most therapeutically-productive drug target families for CNS diseases. GPCRs respond to extracellular ligands and activate intracellular signal transduction pathways which typically lead to changes in cellular morphology. Using a label-free platform, SRU BIND®, we have characterized a panel of 15 clonal cell lines, each expressing a unique GPCR. We have analyzed the morphological response to agonist stimulation in these cell lines, specifically with respect to the magnitude, rate and duration, which are influenced by the G-protein coupling. Agonist and antagonist assays have been developed in 384-well format for each target. Compared to traditional assays which measure the GPCR functional response through only a single metric such as changes in cAMP or Ca2+, the ability of BIND® to measure the holistic cellular response allows for the detection and measurement of multiple downstream pathways. This is evident, for example, by the display of biphasic responses in some cell lines. The technical platform and methodologies enable screening for novel receptor mediators and can potentially identify ligands acting through non-classical signaling pathways. By using pathway blockers in combination with agonist stimulation of the cloned GPCR, we have begun to characterize these pathways.
IntroductionBIND is a label-free assay technology that facilitates detection of biomolecular interactions including biochemical and cellular. BIND enables highly sensitive measurements of changes in mass in proximity to the biosensor surface, which allows for precise monitoring of cellular adherence. Biosensors (in 96, 384, or 1536 format) with a nanostructured optical grating reflect light at a wavelength that is shifted in proportion to the mass in proximity to the surface. The optical grating reflects a narrow range of the wavelengths of light upon illumination, which is utilized to determine the “Peak Wavelength Value” or “PWV”. As the average cellular mass near the biosensor surface increases, the PWV increases. Cellular responses to GPCR agonists often result in a change of cellular interaction with, or attachment to, the biosensor. Increases (+PWV) and decreases (–PWV) in cellular mass in proximity to surface can be detected using kinetic and endpoint read modes. Kinetic measurements allow for real time comprehensive data collection on each well of the plate, several times per minute. Using BIND technology, we have characterized a panel of cell lines expressing unique cloned GCPR receptors. Agonist and antagonist assays, in 384-well format, have been developed for each cell line.
Cells expressing cloned GPCRs were purchased from PerkinElmer or ChanTest. All data was collected using a SRU BIND Reader. Compound addition and mixing was done with a Caliper Sciclone ALH 3000. Assays were run in either kinetic or endpoint mode. Typical assays are performed in a 80 μL volume in a BIND Sensor CA2-384M plate. Cells are plated on the CA2 mixed extracellular matrix overnight in complete media to reach 80%-90% confluence. Media is replaced with HBSS for two hours prior to running the assay. At time zero, a baseline measurement is taken, then samples or controls are added to wells. For antagonist assays, the inhibition is determined with a reference agonist at the EC80. The holistic cellular morphological response endpoint is determined at the response peak time-point and compared to control wells to assess the compound effect. For pathway blocking experiments, most blockers were pre-incubated with cells for two hours.
Results
Figure 2. GPCR responses in CHO cells expressing a unique cloned receptor, as measured by the change in PWV versus time. The left figure shows typical GPCR responses based on the native G-protein mechanism of action. Note that magnitude of the response depends on the expressed receptor and its G-protein coupling. On the right, the biphasic responses of the 5-HT1A and CB2 cell lines are atypical, suggesting a blend of the GPCR mechanisms shown on the left graph. Figure 5. The sensitivity of Alpha2A compound pharmacology on 3 platforms was compared in agonist and antagonist assays. EC50 and IC50
values were similar with the 3 methods, although there were some exceptions. Emax is defined as the maximum effect. In the plot, a panel of compounds of interest were screened at 10 μM on calcium flux and BIND Alpha 2A assays. Several partial agonists were detected with BIND (x-axis) but not with calcium flux (y-axis).
Figure 6. Label-free pathway blocker analysis on Alpha2A in Aequorin/CHO cells. Blockers were used to determine how the various cellular signaling pathways contribute to the change in PWV and kinetic response profile detected by BIND after agonist stimulation of Alpha2A. The table shows the function, concentration, and treatment time of the blockers. The blocker concentrations selected were based on literature. Kinetic traces represent each blocker or control after agonist addition (100 nM UK 14,304). GW5074, which inhibits MEK/ERK, and Pertussis toxin (PTX), which inhibits Gi signaling, blocked the response detected on the SRU by UK 14,304. Wortmannin prevented the desensitization of the response.
Figure 7. Alpha1A agonist assay: quality control and validation. Z’ experiments are run with every assay. The results for the this assay are typical of label-free GPCR assay quality
Figure 4. GPCR coupling and pharmacology with BIND.
Figure 1. SRU BIND Technology. Cellular morphology shift reveals GPCR activation. The measured signal or BIND Response is quantifiable and allows for pharmacology studies. In SEM image on right, HEK cells respond to ATP. Diagram courtesy of SRU.
J. Brown GSK Neuroscience
Alpha1A Agonist Signal Transduction Pathway Blocker Effect on Alpha2A Alpha1A Antagonist
5-HT1A Agonist 5-HT1A Antagonist
References: 1. Lomasney et al. (1990) Proc. Natl. Acad. Sci. 87, 5094-5098 2. Katsushi Shibata et al. (1996) Br. J. Pharmacol., 118, 1403-1408 3. Tate K. M. et al. (1991) Eur. J. Biochem., 196, 357-361 4. Xie D.-W et al. (1995) Neuropsychopharmacology 12, 263-268
Acknowledgements:Michael Getman - SRU Biosystems Mark Welborn, John Gravel, Audrey Kues and Candice Lewis - Caliper Discovery Alliances & Services
Receptor Coupling Ref Agonist EC50 (nM) Ref Antagonist IC50 (nM)Alpha1a Gq/11 A61603 0.65 WB 4101 4.2Alpha2a Gi UK 14,304 19 Rauwolscine 7.4Alpha2b Gi UK 14,304 59 Rauwolscine 16
AT-1 Gq/11 + Gi Angiotensin II 2.1 Saralasin 320Beta1 Gs Isoproterenol 2.0 Atenolol 300C5A Gi C5A 1.1 N/A N/A
CXCR2 Gi IL-8 3.9 SB 225002 3800CB1 Gi Win 55,212-2 940 O-2050 34CB2 Gi Win 55,212-2 9.2 - -DA2 Gi/o Gi/q Dopamine 2.6 (±)-Sulpiride 23H1 Gq/11 2-pyridylethylamine 1900 Pyrilamine 10
5-HT1A Gi/o 5-Carboxamidotryptamine 1.0 WAY-100,635 10NK2 Gq/11 (Gs?) [bAla8]-NKA (4-10) 2.3 GR 159897 9.1
ORL-1 Gi, Gi/o, Go Nociceptin 0.84 Trap 101 300Vaso2 Gs [Arg8]-vasopressin 3.9 Vasopressin analog 1400
Alpha2A Agonists (EC50 nM, Emax %)
Alpha2A Antagonists (IC50 nM)NE: No Effect*Computer Estimate
Figure 3. Adrenergic Alpha1A and serotonin 5-HT1A pharmacology. Oxymetazoline is a known partial agonist. For the antagonist assay, A61603 or 5-CT was used at the EC80. Results are representative of at least 2 experiments performed with duplicate data points.
Additional pharmacology results for each assay can be found at:www.CaliperLS.com/products/contract-research/in-vitro/gpcrs/gpcr-functional-pair.htm
UK 14,304 addition
No ATP (+) ATP
Agonistaddition
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BIND % Maximum Response
