LinkLight™ Assay Technology: A Versatile Cell‐based ‐protein Interaction Assay Platform

50 Williams Parkway, Unit A‐2 East Hanover, NJ 07936 USA 973‐585‐6777 (O) 973‐585‐6776 (Fax) [email protected] http://bioinvenu.com/ LinkLight™ Assay Technology Utilizing protein-protein interactions as functional signal readout

 Developed in 2006 in Sanofi‐Aventis  Two provisional patent applications were filed in 2007 & 2008)  Patent issued in 2014

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.

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. Examples of GPCR LinkLight™ Assays

TM CMKLR1 LinkLightTM Stable cells GPR1 LinkLight Assay CCKAR LinkLightTM Assay DRD2 LinkLight Assay Monoclonal Stable cells (Transient Expression) 500000 125000 20000 1500000 400000 100000 15000 1000000 75000 300000

RLU 10000 RLU RLU 50000 RLU 200000 500000 25000 5000 100000

0 0 0 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -12 -10 -8 -6 -4 -12 -10 -8 -6 -4 log[ peptide] (M) log [Chemerin peptide] (M) log[Bradykinin] (M) log[Dopamine] (M)

ADORA2B LinkLight Assay CXCR7 LinkLight Assay DRD1 LinkLight Assay ENDRB LinkLight Assay (Transient Expression) (Transient Expression) (Transient Expression) (Transient Expression)

100000 500000 400000 300000

80000 400000 300000 200000 60000 300000 200000 RLU RLU RLU RLU 200000 40000 100000 100000 20000 100000 0 0 0 0 -12 -10 -8 -6 -4 -12 -10 -8 -6 -4 -12 -10 -8 -6 -4 -12 -10 -8 -6 -4 log[Dopamine HCl] (M) log[Endothelin 2] (M) log[NECA] (M) log[SDF-1] (g/ml) GPR84 LinkLight Assay AVPR2 ß-arrestin LinkLight™ Assay GALR1 LinkLight Assay GLP1R LinkLight Assay (Transient Expression) (Transient Expression) (Transient Expression) 60000 400000 100000 50000

80000 40000 300000 40000 60000 30000 200000 RLU RLU RLU 40000 RLU 20000 20000 100000 20000 10000

0 0 0 0 -12 -10 -8 -6 -4 -12 -10 -8 -6 -4 -8 -6 -4 -2 0 -11 -10 -9 -8 -7 -6 -5 -4 log[Galanin] (M) log[GLP1] (M) log[Capric acid] (M) log[8-AVP], M TEV linked GPCR ADRB2 does not alter G‐protein signaling pathway

cAMP Assay LinkLight™ Assay 14000 20000 Isoproterenol Metaproterenol Isoproterenol Salmeterol Betaxolol 12000 Procaterol Formoterol Metaproterenol 15000 Procaterol 10000 BRL 37344 Fenoterol 8000 Propranolol BRL 37344 10000 Betaxolol CGP 12177A RLU 6000 RLU Albuterol 4000 5000 2000

0 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4 log[Compound] (M) log[Compound] (M)

CRE-Luc Assay FLIPR Assay 25000 15000

20000 10000 15000 RLU 10000 RFU 5000 5000

0 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -12 -11 -10 -9 -8 -7 -6 -5 -4 log[Isoproterenol] (M) log[isoproterenol] (M)

Assay LinkLight™ cAMP FLIPR Cre‐Luc

Isoproterenol EC50 3.5 nM 9.1 nM 7.2 nM 2.0 nM

Other TEV linked GPCRs such as GPR43, EDG1, GPR1 have also been demonstrated no interference to G‐protein signaling

CMKLR1 LinkLight™ Assay Parameters

Chemerin vs. Peptide 1 Serum effect Cell Number Per well 4 400000 4.0×10 10% 4 3.0×10 Peptide 1 15K 2.5% Chemerin 4 300000 1.0% 3.0×10 10K 0.25% 5K 4 2.0×10 0.1% 4 2.5K 200000 0% 2.0×10 RLU RLU RLU

1.0×10 4 100000 1.0×10 4

0 0 0 -12 -11 -10 -9 -8 -7 -6 -5 -4 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -12 -11 -10 -9 -8 -7 -6 -5 -4 log [M], Compound log[M], Chemerin peptide 1 log [M], Chemerin Peptide 1

DMSO Concentration Incubation Time

4 5% 4 3.010 4.0×10 1.0 hr 1% 1.5 hr 0.5% 3.0×10 4 4 2.0 hr 2.010 Assay buffer 3.0 hr

RLU 4 RLU 2.0×10 1.010 4 1.0×10 4

0 0 -12 -11 -10 -9 -8 -7 -6 -5 -4 -12 -11 -10 -9 -8 -7 -6 -5 -4 log [M], Chemerin peptide 1 log [M], Chemerin Peptide 1 GPCR LinkLight™ Assay Identify biased ligands

ADRB2 LinkLight ADRB2 LinkLight antagonist assay agonist assay 30000 Isoproterenol (100nM)+ICI11855 40000 Isoproterenol (100nM)+Propranolol ICI118551(antagonist) Isoproterenol (100nM)+SR59230A propranolol (antagonist) 30000 SR59230A (antagonist) 20000 Betaxolol (antagonist) 20000 RLU RLU 10000 10000

0 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -14 -12 -10 -8 -6 -4 log[M], compound log[cpd], M

Some of well‐known ADRB2 antagonists showed partial agonist activity in GPCR LinkLight™ assays

GPCR LinkLight™ Assay Identify biased ligands

G protein Signaling b‐arrestin Signaling cAMP assay LinkLight™ assay Pharmacological Research 51:275-218 (2005) 23000 SSR148708A (Conivaptan) 22000 SSR137367 (Tolvaptan) 21000 20000

(10 nM AVP) 19000

RLU 18000 17000 16000 15000 14000 -12 -11 -10 -9 -8 -7 -6 -5 log[compound], M

Some of vasopressin 2 antagonists showed weak agonist activity in LinkLight™ assays

Comparison of Cell Based Protein‐Protein Interaction Assay Technologies Technology Advantage Disadvantage

Instantaneous, reversible, Sensitivity limited by donor‐ BRET acceptor distance, orientation, temporal & specific signal readout spectrum overlap, FRET donor/acceptor ratio, difficult to operate, unable to generate stable cells

Quick signal readout, specific, easy Fragment spontaneous self‐ EFC & PFC to operate. complementation can result in (Enzyme or protein fragment high background, false complementation) interactions. PathHunter™ sensitive, easy to operate. Off‐target hits due to long Tango™ transcription/translation process, irreversible (transcriptional based) Sensitive, specific, & quick signal Irreversible LinkLight™ readout, Multiplex ability, easy to operate & low cost of detection reagents.

Current GPCR β-arrestin LinkLight Cell Lines

GPCR β-arrestin-2 LinkLight™ cells Cat. # GPCR β-arrestin-2 LinkLight™ cells Cat. #

GPR1 1001 CXCR7 (CMKOR1) 1027

ADRB2 (b2AR, b2) 1002 HTR2C (5HT2C) 1028

CMKLR1 (ChemR23) 1003 LPAR3 (EDG7) 1029

FFA2 (GPR43) 1004 EDNRB (ETb) 1030

AVPR2 (V2R, V2) 1005 ADORA2B (A2B) 1031

HTR2A (5HT2a) 1006 GPBAR1 (TGR5) 1032

GPR109A (HM74a) 1007 NTSR1 (NT1) 1033

GPR119 (GPCR2) 1008 GPR84 (EX33) 1034

NMUR1 (GPR66) 1009 P2YR4 (P2Y4) 1035

S1P1 (EDG1) 1010 BDKRB1 (B1) 1036

BDKRB2 (B2) 1012 S1PR2 (EDG5) 1037

CCKAR (CCK1) 1013 P2YR12 () 1041

CCKBR (CCK2) 1014 MLNR (GPR38) 1042

GALR1 (GALNR1) 1018 LPAR4 (P2Y9, GPR23) 1043

GLP1R 1019 O3FAR1 (GPR120) 1044

DRD2 (D2) 1020 GPR183 (EBI2) 1045

S1PR3 (EDG3) 1021 FFAR3 (GPR41) 1046

S1PR4 (EDG6) 1022 FFAR1 (GPR40) 1047

S1PR5 (EDG8) 1023 KISS1R (GPR54) 1048

LPAR2 (EDG4) 1024 ADORA1 (A1AR, RDC7) 1049

DRD1 (D1) 1025 GPR55 1050

DRD3 (D3) 1026 CCR6 1051 NHR LinkLight™ Assay Examples of NHR LinkLight™ Assay

ER LinkLight Assay (Transient Expression) 40000

30000

20000 RLU

10000

0 -10 -8 -6 -4 -2 log[-estradiol] (M)

RXR LinkLight Assay (Transient Expression) 5000

4000

3000

RLU 2000

1000

0 -10 -8 -6 -4 -2 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 INSR TransLightTM Assay Transient Expression 25000

20000

15000

RLU 10000

5000

0 -14 -12 -10 -8 -6 -4 log[Insulin] (mg/ml) ERBB4 TransLightTM Assay Transient Expression 150000

100000 RLU 50000

0 -14 -12 -10 -8 -6 -4 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.

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!

Thank You!

50 Williams Parkway, Unit A‐2 East Hanover, NJ 07936 USA 973‐585‐6777 (O) 973‐585‐6776 (Fax) [email protected] http://bioinvenu.com/