LinkLight Assay Technology: A Versatile Cell based Protein protein Interaction Assay Platform
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1 LinkLight Assay Technology: A Versatile Cell based Protein protein Interaction Assay Platform 5 Williams Parkway, Unit A 2 East Hanover, NJ 7936 USA (O) (Fax) Haifeng.eishingdrelo@bioinvenu.com
2 LinkLight Assay Technology Utilizing protein-protein interactions as functional signal readout Developed in 26 in Sanofi Aventis Two provisional patent applications were filed in 27 & 28) Patent issued in 214 LinkLight technology consists of two components: a protein A is fused to a Tobacco Etch Virus (TEV) protease and a protein B is fused to an inactive permuted protein containing a TEV protease cleavage site. Upon interaction between protein A and B, inactive permuted protein is cleaved, the cleaved fragments spontaneously refold, and active reporter protein is reconstituted.
3 GPCR LinkLight Assay Utilizing GPCR & β arrestin interactions as functional signal readout Simple Assay Procedure 1. Seed cells in 384 wel plate, culture for 24 hours. 2. Change to serum free medium if necessary, add ligands, incubate for one hour. 3. Add luciferase detection reagent, count signals on a luminescent reader. A GPCR is linked with a TEV protease. β-arrestin 2 is linked with a permuted luciferase (pluc) containing a TEV cleavage site. Upon GPCR recruits β-arrestin 2, the permuted luciferase is cleaved. The cleaved fragment spontaneously refolds to form active luciferase. Highly sensitive luminescent signals produced with the active luciferase are easily detected.
4 Examples of GPCR LinkLight Assays 125 CMKLR1 LinkLight TM Stable cells 2 GPR1 LinkLight TM Assay Monoclonal Stable cells 15 CCKAR LinkLight TM Assay 5 DRD2 LinkLight Assay log[chemerin peptide] (M) log [Chemerin peptide] (M) log[bradykinin] (M) log[dopamine] (M) 1 ADORA2B LinkLight Assay 5 CXCR7 LinkLight Assay 4 DRD1 LinkLight Assay 3 ENDRB LinkLight Assay log[neca] (M) 4 GALR1 LinkLight Assay log[sdf-1 ] (g/ml) GLP1R LinkLight Assay log[dopamine HCl] (M) 5 GPR84 LinkLight Assay log[endothelin 2] (M) 6 AVPR2 ß-arrestin LinkLight Assay log[galanin] (M) log[glp1] (M) log[capric acid] (M) log[8-avp], M
5 TEV linked GPCR ADRB2 does not alter G protein signaling pathway 2 Isoproterenol Salmeterol Formoterol Procaterol Fenoterol BRL CGP 12177A Albuterol LinkLight Assay Metaproterenol Betaxolol log[compound] (M) 14 Isoproterenol 12 Procaterol Metaproterenol 1 8 BRL Propranolol 6 Betaxolol 4 2 camp Assay log[compound] (M) 25 CRE-Luc Assay 15 FLIPR Assay RFU log[isoproterenol] (M) log[isoproterenol] (M) Assay LinkLight camp FLIPR Cre Luc Isoproterenol EC nm 9.1 nm 7.2 nm 2. nm Other TEV linked GPCRs such as GPR43, EDG1, GPR1 have also been demonstrated no interference to G protein signaling
6 CMKLR1 LinkLight Assay Parameters Peptide 1 Chemerin Chemerin vs. Peptide % 2.5% 1.%.25%.1% % Serum effect Cell Number Per well 15K 1K 5K 2.5K log [M], Compound log[m], Chemerin peptide log [M], Chemerin Peptide % DMSO Concentration 5% hr Incubation Time % Assay buffer hr 2. hr 3. hr log [M], Chemerin peptide log [M], Chemerin Peptide 1
7 GPCR LinkLight Assay Identify biased ligands ADRB2 LinkLight antagonist assay ADRB2 LinkLight agonist assay 3 2 Isoproterenol (1nM)+ICI11855 Isoproterenol (1nM)+Propranolol Isoproterenol (1nM)+SR5923A 4 3 ICI118551(antagonist) propranolol (antagonist) SR5923A (antagonist) Betaxolol (antagonist) log[m], compound log[cpd], M Some of well known ADRB2 antagonists showed partial agonist activity in GPCR LinkLight assays
8 GPCR LinkLight Assay Identify biased ligands G protein Signaling camp assay Pharmacological Research 51: (25) (1 nm AVP) 23 SSR14878A (Conivaptan) b arrestin Signaling LinkLight assay SSR (Tolvaptan) log[compound], M Some of vasopressin 2 antagonists showed weak agonist activity in LinkLight assays
9 Comparison of Cell Based Protein Protein Interaction Assay Technologies Technology Advantage Disadvantage BRET FRET EFC & PFC (Enzyme or protein fragment complementation) PathHunter Tango (transcriptional based) LinkLight Instantaneous, reversible, temporal & specific signal readout Quick signal readout, specific, easy to operate. sensitive, easy to operate. Sensitive, specific, & quick signal readout, Multiplex ability, easy to operate & low cost of detection reagents. Sensitivity limited by donoracceptor distance, orientation, spectrum overlap, donor/acceptor ratio, difficult to operate, unable to generate stable cells Fragment spontaneous selfcomplementation can result in high background, false interactions. Off target hits due to long transcription/translation process, irreversible Irreversible
10 Current GPCR β-arrestin LinkLight Cell Lines GPCR β-arrestin-2 LinkLight cells Cat. # GPCR β-arrestin-2 LinkLight cells Cat. # GPR1 11 CXCR7 (CMKOR1) 127 ADRB2 (b2ar, b2) 12 HTR2C (5HT2C) 128 CMKLR1 (ChemR23) 13 LPAR3 (EDG7) 129 FFA2 (GPR43) 14 EDNRB (ETb) 13 AVPR2 (V2R, V2) 15 ADORA2B (A2B) 131 HTR2A (5HT2a) 16 GPBAR1 (TGR5) 132 GPR19A (HM74a) 17 NTSR1 (NT1) 133 GPR119 (GPCR2) 18 GPR84 (EX33) 134 NMUR1 (GPR66) 19 P2YR4 (P2Y4) 135 S1P1 (EDG1) 11 BDKRB1 (B1) 136 BDKRB2 (B2) 112 S1PR2 (EDG5) 137 CCKAR (CCK1) 113 P2YR12 (P2Y12) 141 CCKBR (CCK2) 114 MLNR (GPR38) 142 GALR1 (GALNR1) 118 LPAR4 (P2Y9, GPR23) 143 GLP1R 119 O3FAR1 (GPR12) 144 DRD2 (D2) 12 GPR183 (EBI2) 145 S1PR3 (EDG3) 121 FFAR3 (GPR41) 146 S1PR4 (EDG6) 122 FFAR1 (GPR4) 147 S1PR5 (EDG8) 123 KISS1R (GPR54) 148 LPAR2 (EDG4) 124 ADORA1 (A1AR, RDC7) 149 DRD1 (D1) 125 GPR55 15 DRD3 (D3) 126 CCR6 151
![NHR LinkLight Assay Examples of NHR LinkLight Assay 4 ER LinkLight Assay 3 2 1-1 -8-6 -4-2 log[ -estradiol] (M) 5 RXR LinkLight Assay 4 3 2 1-1 -8-6 -4-2 log[rentinoic acid] (M) A nuclear hormone](/docs-images/77/76659943/images/11-3.jpg "receptor (NHR) is linked with the TEV protease. A nuclear receptor co activator 1 interaction domain (NCOA 1) is linked with a permuted luciferase containing a TEV cleavage sequence.")
11 NHR LinkLight Assay Examples of NHR LinkLight Assay 4 ER LinkLight Assay log[ -estradiol] (M) 5 RXR LinkLight Assay log[rentinoic acid] (M) A nuclear hormone receptor (NHR) is linked with the TEV protease. A nuclear receptor co activator 1 interaction domain (NCOA 1) is linked with a permuted luciferase containing a TEV cleavage sequence. Upon NHR migrating into the nucleus and forming a complex with NCOA 1, the permuted luciferase is cleaved. The cleaved fragment spontaneously refolds to form active luciferase.
![RTK LinkLight Assay Examples of RTK LinkLight Assay 25 INSR TransLight TM Assay Transient Expression 2 15 1 5-14 -12-1 -8-6 -4 log[insulin] (mg/ml) 15 ERBB4 TransLight TM Assay Transient Expression 1](/docs-images/77/76659943/images/12-3.jpg "5-14 -12-1 -8-6 -4 log[nrg] (mg/ml) Upon ligand binding, Receptor Tyrosine Kinases (RTK) interact with a phosphotyrosine binding (PTB) domain protein.")
12 RTK LinkLight Assay Examples of RTK LinkLight Assay 25 INSR TransLight TM Assay Transient Expression log[insulin] (mg/ml) 15 ERBB4 TransLight TM Assay Transient Expression log[nrg] (mg/ml) Upon ligand binding, Receptor Tyrosine Kinases (RTK) interact with a phosphotyrosine binding (PTB) domain protein. The interactions between RTK TEV and PTB pluc result in cleavage of permuted luciferase. The cleaved fragment spontaneously refolds to form active luciferase.
13 BioInvenu Offers GPCR LinkLight assay cell lines, transient or division arrested cells GPCR LinkLight assay open system including reporter host cell lines and cloning vectors for customers to establish their own target assays NHR LinkLight assay cell lines, transient or division arrested cells RTK LinkLight assay cell lines, transient or division arrested cells Customer assay developments, compound profiling, & screening services. LinkLight assay detection reagent is a fraction of other assay s cost!
14 5 Williams Parkway, Unit A 2 East Hanover, NJ 7936 USA (O) (Fax) info@bioinvenu.com Thank You!
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