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gpcrBIAS℠

DiscoverX provides the largest portfolio of GPCR assays and now for the first time, a unique service that utilizes state of the art tools developed by Professor Terry Kenakin* for characterization of ligand bias.  With the appropriate β-Arrestin, Internalization, and 2nd messenger assays to quantify selective response and statistical tools to scale these effects, harnessing bias to produce selective ligands is now made simple.

Service Details

Service: Compound profiling for second messenger, β-arrestin, or internalization assays from a choice of >150 GPCRs.  Application of ligand bias analysis to identify statistically significant biased interactions.

Deliverables: Study report containing detailed profile of compounds in two or more signaling pathways. Analysis of activity relative to natural ligands to derive a biased index value.

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*Professor Terry Kenakin is in the Department of Pharmacology, UNC School of Medicine, Chapel Hill, NC. Prior to joining UNC, he spent 32 years in drug discovery where his work involved the use of quantitative receptor pharmacology and theory to advance new programs. Terry has written numerous books on pharmacology and is Editor-in-Chief of the Journal of Receptors and Signal Transduction and Co-Editor-in-Chief of Current Opinion in Pharmacology.

 


gpcrBIASAssay Panel

Listed below are the assays currently available for screening and profiling.  Choose a single GPCR from the list below and two assays to study your ligand bias.


 
Target Gene ▲Common NameArrestincAMPCalciumInternalization
ADCYAP1R1Adenylate cyclase activating polypeptide receptor 1 (PAC1)XXXX
ADORA1Adenosine receptor A1 (A1)XX  
ADORA3Adenosine receptor A3 (A3)XX  
ADRA1BAdrenergic receptor alpha 1B (α1B-adrenoceptor)X X 
ADRA2AAdrenergic receptor alpha 2A (α2A-adrenoceptor)XX  
ADRA2CAdrenergic receptor alpha 2C (α2C-adrenoceptor)XX X
ADRB1Adrenergic receptor beta 1 (β1-adrenoreceptor)XX  
ADRB2Adrenergic receptor beta 2 (β2-adrenoreceptor)XX X
AGTR1Angiotensin II receptor, type 1 (AT1)X X 
AGTRL1Apelin receptor (APJ)XX X
AVPR1AVasopressin receptor 1A (V1A)X XX
AVPR1BVasopressin receptor 1B (V1B)X XX
AVPR2Vasopressin receptor 2 (V2)XX X
BDKRB1Bradykinin receptor B1 (B1)X X 
BDKRB2Bradykinin receptor B2 (B2)X XX
BRS3Bombesin receptor, subtype 3 (BB3)X X 
C3AR1Complement component 3a receptor 1 (C3A)XX X
C5AR1Complement component 5a receptor 1 (C5A)XX X
C5L2G-Protein Coupled Receptor 77 (C5L2)X  X
CALCRCalcitonin receptor (CT)XX  
CALCRL-RAMP1Calcitonin receptor-like (Calcitonin receptor-like receptor)XX  
CALCRL-RAMP3Calcitonin receptor-like (Calcitonin receptor-like receptor)XX  
CALCR-RAMP3Calcitonin receptor (CT)XX  
CCKARCholecystokinin A receptor (CCK1)X XX
CCKBRCholecystokinin B receptor (CCK2)X XX
CCR1Chemokine (C-C motif) receptor 1 (CCR1)XX X
CCR10Chemokine (C-C motif) receptor 10 (CCR10)XX X
CCR2Chemokine (C-C motif) receptor 2 (CCR2)X  X
CCR4Chemokine (C-C motif) receptor 4 (CCR4)X  X
CCR5Chemokine (C-C motif) receptor 5 (CCR5)XX X
CCR6Chemokine (C-C motif) receptor 6 (CCR6)XX X
CCR7Chemokine (C-C motif) receptor 7 (CCR7)XX X
CHRM1Cholinergic receptor, muscarinic 1 (M1)X XX
CHRM2Cholinergic receptor, muscarinic 2 (M2)XX X
CHRM3Cholinergic receptor, muscarinic 3 (M3)X XX
CHRM4Cholinergic receptor, muscarinic 4 (M4)XX X
CHRM5Cholinergic receptor, muscarinic 5 (M5)X XX
CMKLR1Chemokine-like receptor 1 (CMKLR1)XX X
CNR1Cannabinoid receptor 1 (CB1)XX  
CNR2Cannabinoid receptor 2 (CB2)XX  
CRHR1Corticotropin releasing hormone receptor 1 (CRF1)XX X
CRHR2Corticotropin releasing hormone receptor 2 (CRF2)XX X
CRTH2prostaglandin D2 receptor 2 (PTGDR2)XX  
CXCR1Chemokine (C-X-C motif) receptor 1 (CXCR1)XX X
CXCR2Chemokine (C-X-C motif) receptor 2 (CXCR2)XX X
CXCR3Chemokine (C-X-C motif) receptor 3 (CXCR3)XX X
CXCR4Chemokine (C-X-C motif) receptor 4 (CXCR4)XX X
CXCR5Burkitt lymphoma receptor (CXCR5)XX  
CXCR6Chemokine (C-X-C motif) receptor 6 (CXCR6)XX X
CXCR7Chemokine (C-X-C motif) receptor 7 (CXCR7)X  X
DRD1Dopamine receptor D1 (D1)XX X
DRD2LDopamine Receptor D2 (long isoform) (D2L)XX  
DRD2SDopamine Receptor D2 (short isoform) (D2S)XX  
DRD4Dopamine Receptor D4 (D4)XX  
DRD5Dopamine receptor D5 (D5)XX  
EBI2G protein-coupled receptor 183 (GPR183)XX X
EDG1Sphingosine 1-phosphate receptor 1 (S1P1)XX X
EDG2Lysophosphatidic acid receptor 1 (LPA1)  XX
EDG3Sphingosine 1-phosphate receptor 3 (S1P3)X XX
EDG4Lysophosphatidic acid receptor 2 (LPA2)X X 
EDG5Sphingosine 1-phosphate receptor 2 (S1P2)X  X
EDG7Lysophosphatidic acid receptor 3 (LPA3)X XX
EDNRAEndothelin receptor type A (ETA)X X 
EDNRBEndothelin receptor type B (ETB)X  X
F2RL1Coagulation factor II (thrombin) receptor-like 1 (PAR2) (PAR2)X XX
F2RL3Coagulation factor II (thrombin) receptor-like 3 (PAR4) (PAR4)X XX
FFAR1Free fatty acid receptor 1 (FFA1)X X 
FPR1Formylpeptide receptor 1 (FPR1)XXXX
FPRL1Formylpeptide receptor-like 1 (FPR2/ALX)XX X
FSHRFollicle stimulating hormone receptor (FSHR)XX  
GALR1Galanin receptor 1 (GALR1)XX X
GALR2Galanin receptor 2 (GALR2)X X 
GCGRGlucagon receptor (glucagon)XX X
GHSRGhrelin receptor (ghrelin)X X 
GIPRGastric inhibitory polypeptide receptor (GIP)XX  
GLP1RGlucagon-like peptide receptor 1 (GLP-1)XX X
GLP2RGlucagon-like peptide receptor 2 (GLP-2)XX X
GPBAR1G protein-coupled bile acid receptor 1 (GPBA)XX  
GPR1G-protein coupled receptor 1 (GPR1)X  X
GPR103pyroglutamylated RFamide peptide receptor (QRFPR)X X 
GPR109AHydroxycarboxylic acid receptor 2 (HCA2)XX  
GPR109BHydroxycarboxylic acid receptor 3 (HCA3)XX  
GPR120G-protein coupled receptor 120, long isoform (FFA4)X  X
GPR35G-protein coupled receptor 35 (GPR35)XX X
GPR92Lysophosphatidic acid receptor 5 (GPR92)X X 
GRPRGastrin releasing peptide receptor (BB2)X XX
HCRTR1Orexin receptor 1 (OX1)X XX
HCRTR2Orexin receptor 2 (OX2)X XX
HRH1Histamine receptor H1 (H1)X X 
HRH2Histamine receptor H2 (H2)XXX 
HRH3Histamine receptor H3 (H3)XX  
HTR1A5-hydroxytryptamine receptor 1A (5HT1A)XX  
HTR1B5-hydroxytryptamine receptor 1B (5HT1B)XX  
HTR1F5-hydroxytryptamine receptor 1F (5HT1F)XX  
HTR2A5-hydroxytryptamine receptor 2A (5HT2A)X XX
HTR2C5-hydroxytryptamine receptor 2C (5HT2C)X XX
HTR5A5-hydroxytryptamine receptor 5A (5HT5A)XX  
LHCGRChoriogonadotropin receptor (LH)XX  
LTB4RLeukotriene B4 receptor (BLT1)XXXX
MC1RMelanocortin receptor 1 (MC1)XX  
MC3RMelanocortin receptor 3 (MC3)XX X
MC4RMelanocortin receptor 4 (MC4)XX  
MC5RMelanocortin receptor 5 (MC5)XX  
MCHR1Melanin concentrating hormone receptor 1 (MCH1)XX X
MCHR2Melanin-concentrating hormone receptor 2 (MCH2)X X 
MLNRMotilin receptor (motilin)X XX
MRGPRX1MAS-related GPR, member X1 (MRGPRX1)X X 
MRGPRX2MAS-related GPR, member X2 (MRGX2)X X 
MTNR1AMelatonin receptor 1A (MT1)XX X
MTNR1BMelatonin receptor 1B (MT2)XX X
NMBRNeuromedin B Receptor (BB1)X XX
NMU1RNeuromedin U receptor 1 (NMU1)X XX
NPBWR1Neuropeptides B/W receptor 1 (NPBW1)XX  
NPBWR2Neuropeptides B/W receptor 2 (NPBW2)XX  
NPFFR1Neuropeptide FF receptor 1 (NPFF1)XX  
NPY2RNeuropeptide Y receptor Y2 (Y2)XXXX
NTSR1Neurotensin receptor 1 (NTS1)X XX
OPRD1Opioid receptor delta (δ)XX X
OPRK1Opioid receptor kappa (κ)XX X
OPRL1Opioid receptor-like receptor (NOP)XX X
OPRM1Opioid receptor mu (μ)XX X
OXER1Oxoeicosanoid (OXE) receptor 1 (OXE)XX X
OXTROxytocin Receptor (OT)X XX
P2RY1Purinergic receptor P2Y, G-protein coupled, 1 (P2RY1)X XX
P2RY11Purinergic receptor P2Y, G-protein coupled, 11 (P2Y11)X   
P2RY2Purinergic receptor P2Y, G-protein coupled, 2 (P2Y2)X XX
P2RY4Pyrimidinergic receptor P2Y, G-protein coupled, 4 (P2Y4)X  X
PPYR1Pancreatic polypeptide receptor 1 (Y4)XX  
PRLHRProlactin releasing hormone receptor (PRRP)X XX
PROKR1Prokineticin receptor 1 (PKR1)XXXX
PROKR2Prokineticin receptor 2 (PKR2)X  X
PTAFRPlatelet activating receptor (PAF)X XX
PTGER3Prostaglandin E receptor 3 (subtype EP3) (EP3)XXX 
PTGER4Prostaglandin E receptor 4 (subtype EP4) (EP4)X  X
PTGFRProstaglandin F receptor (FP) (FP)X XX
PTGIRProstaglandin I2 (prostacyclin) receptor (IP) (IP1)XX X
PTHR1Parathyroid hormone receptor 1 (PTH1)XXXX
PTHR2Parathyroid hormone receptor 2 (PTH2)XXXX
RXFP3Relaxin family peptide receptor 3 (RXFP3)XX X
RXFP4Relaxin family peptide receptor 4 (RXFP4)XX  
SCTRSecretin receptor (secretin)XX X
SSTR2Somatostatin receptor 2 (SST2)XX X
SSTR3Somatostatin receptor 3 (SST3)XX  
SSTR4Somatostatin receptor 4 (SST4)XX  
SSTR5Somatostatin receptor 5 (SST5)XX  
TACR1Tachykinin receptor 1 (NK1)X XX
TACR2Tachykinin receptor 2 (NK2)X XX
TACR3Tachykinin receptor 3 (NK3)XXXX
TBXA2RThromboxane A2 receptor (TP)X XX
TRHRThyrotropin-releasing hormone receptor (TRH1)X XX
TSHR(L)Thyroid Stimulating Hormone Receptor (TSH)XX  
UTR2Urotensin II receptor (UT)X XX
VIPR1Vasoactive intestinal peptide receptor 1 (VPAC1)XXXX
VIPR2Vasoactive intestinal peptide receptor 2 (VPAC2)XXXX
XCR1Chemokine (C motif) XC receptor 1 (XCR1)XX  

Assay Concept

GPCR Signaling Bias
 


It has been found that receptors are not simply switches that uniformly activate cell signaling pathways but rather are microprocessors able to bias activation to some signaling pathways at the expense of others (Kenakin 1995). This phenomena changes the overall quality of efficacy possessed by an agonist. Ligand bias can lead to therapeutic opportunities or potential liabilities and is illustrated with analogs of angiotensin above.


References

Kenakin, T.P, Morgan, P.H. (1989) The theoretical effects of single and multiple transducer receptor coupling proteins on estimates of the relative potency of agionist. Mol. Pharmacol. 35:214-222

Kenakin T (1995) Agonist-receptor efficacy. II. Agonist-trafficking of receptor signals.  Trends Pharmacol Sci 16:232–238.

Tschammer N, Bollinger S, Kenakin T, Gmeiner P. (2011) Histidine 6.55 is a major determinant of ligand-biased signaling in dopamine D2L receptor. Mol Pharmacol. 79(3):575-85.

White, K.L., Scopton, A.P., Rives, M.-L., Bikbulatov, R.V., Polepally, P.R., Brown, P.J., Kenakin, T., Javich, J.A.,  Zjawiony, J.K., Roth, B.L. (2013) Identification of novel functionally selective kappa opioid receptor scaffolds Mol. Pharm. 85: 83-90.

Data Analysis & Interpretation 

Experiment

Dose response curves are performed in quadruplicate to measure GPCR responses for 2nd messenger signaling, ß-Arrestin recruitment, and receptor internalization. Experiments with the natural ligand and test substance are performed in parallel.

 

Model

Based on work published in ACS Chemical Neuroscience, (Kenakin et al. 2012) dose response curves are fitted with the Black and Leff Operational model to account for inherent effects within cell type, system, or readout.
 

Deliverable

Data is analyzed to deliver mean bias with a 95% confidence limit. A bias plot is used to visualize the results of the study.
 
 
Formula Definition
Where Α is the sample concentration, τ defines the operational efficacy for an agonist, ΚΑ is the equilibrium dissociation constant of the agonist‐receptor complex, Em is the maximal response capability of the assay, n is the “transducer slope” for the function linking agonist concentration to measured response.  Note that Em and n are global variables common for all agonists in the assay.

References

Kenakin T, Watson C, Muniz-Medina V, Christopoulos A, Novick S (2012) A Simple Method for Quantifying Functional Selectivity and Agonist Bias ACS Chem. Neurosci. 3:193–203

Black JW, Leff P, Shankley NP, and Wood J (1985) An operational model of pharmacological agonism: the effect of E/[A] curve shape on agonist dissociation constant estimation. Br J Pharmacol 84:561–571.

Panel Features & Benefits

  • Better prediction of therapeutic efficacy
  • Obtain the next level of agonist characterization
  • Easy to interpret statistically relevant quantitative scale for biased signaling
  • Determine molecular mode of action for agonists at the level of ligand-receptor interaction