Biology Department of WuXi AppTec (Shanghai) Co., Ltd. ADORA2A (Adenosine A2A Receptor) Cell Line Name: HEK293/A2A/Gα15 Species/Source: Human/Recombinant Gene Synonyms: ADORA2A ; A2aR; ADORA2; RDC8 Accession Number: NM_000675 G coupling Gs Functional Assay-Calcium Flux Assay ADORA2A NECA 120 Agonist NECA CPA 2+ 100 Measurement: Intracellular Ca 80 Detection method: FLIPR Fluorimetry 60 Incubation 25℃ 40 Activity (%) Activity 20 Agonist effect EC50: 5.4 nM 0 -20 -2 0 2 4 6 Log [Compound] nM ADORA2A 120 Antagonist CGS-15943 CGS-15943 100 2+ ZM241385 Measurement: Intracellular Ca 80 Detection method: FLIPR Fluorimetry 60 40 Incubation 25℃ 20 Inhibition (%) Inhibition Stimulant NECA: 40 nM 0 Antagonist effect IC50: 0.22 nM -20 -4 -2 0 2 4 Log [Compound] nM ADORA2A (Adenosine A2A Receptor) Cellular Background: HEK293 Species/Source: Human/Recombinant Gene Synonyms: ADORA2A ; A2aR; ADORA2; RDC8 Accession Number: NM_000675 Radio ligand binding Assay ADORA2A Ligand [3H] CGS 21680 125 NECA Ligand concentration 6 nM 100 CPA ZM241385 Kd (nM) 47.5 nM 75 CGS15943 Non specific NECA (10 μM) 50 Incubation 120 min/RT Inhibition % 25 Detection method Scintillation counting 0 Reference: NECA (IC50:82.0 nM) -4 -3 -2 -1 0 1 2 3 4 5 Log [Compound] nM 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, China Email: [email protected]; www.wuxiapptec.com Page 1 of 44 Biology Department of WuXi AppTec (Shanghai) Co., Ltd. Alpha1A (Alpha-1-adrenergic receptor) Cell Line Name: HEK293/Alpha1A Species/Source: Human/Recombinant Gene Synonyms: ADRA1A; ADRA1C; ADRA1L1; ALPHA1AAR Accession Number: NM_000680 (isoform 1) G coupling Gq Functional Assay-Calcium Flux Assay Alpha1A 120 Agonist (-)Epinephrine (-)-Epinephrine (E4642) 100 A-61603 2+ Measurement: Intracellular Ca 80 60 Detection method: FLIPR Fluorimetry 40 (%) Activity Incubation 25℃ 20 0 Agonist effect EC50: 4.0 nM -2 -1 0 1 2 3 4 Log [Compound] nM Alpha1A 5-Methylurapidil Antagonist 5-Methylurapidil 120 WB-4104 Measurement: Intracellular Ca2+ 100 Prazosin 80 BMY7378 Detection method: FLIPR Fluorimetry 60 Incubation 25℃ 40 Inhibition (%) Inhibition 20 Stimulant (-)Epinephrine : 6.3 nM 0 -2 0 2 4 6 Antagonist effect IC50: 3.6 nM Log [Compound] nM Radio ligand binding Assay Ligand [3H]prazosin Alpha1A 125 WB4101 Ligand concentration 0.2 nM 100 Epinephrine Prazosin Kd (nM) 0.09 nM 75 Non specific epinephrine (0.1 mM) 50 % Inhibition % 25 Incubation 60 min/RT 0 Detection method Scintillation counting -3 -2 -1 0 1 2 3 4 5 Reference: WB 4101 (IC50:0.28 Log [Compound] nM nM) 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, China Email: [email protected]; www.wuxiapptec.com Page 2 of 44 Biology Department of WuXi AppTec (Shanghai) Co., Ltd. Alpha2A (Alpha-2-adrenergic receptor) Cell Line Name: HEK293/Alpha2A/Gα15 Species/Source: Human/Recombinant Gene Synonyms: ADRA2A; ADRA2; ADRAR; ZNF32; ADRA2R; ALPHA2AAR Accession Number: NM_000681 (isoform 1) G coupling Gi Functional Assay-Calcium Flux Assay Alpha2A UK 14304 Agonist UK14304 120 Epinephrine (E4250) 100 Measurement: Intracellular Ca2+ 80 Detection method: FLIPR Fluorimetry 60 ℃ 40 Incubation 25 (%) Activity 20 Agonist effect EC50: 0.14 nM 0 -20 -2 0 2 Log [Compound] nM Alpha2A 125 Antagonist Yohimbine hydrochloride Yohimbin 100 RX 821002 2+ Measurement: Intracellular Ca 75 Prazosin Detection method: FLIPR Fluorimetry 50 25 Incubation 25℃ (%) Inhibition 0 Stimulant UK14304 : 4.3 nM -25 -2 -1 0 1 2 3 4 Antagonist effect IC50: 32.6 nM Log [Compound] nM Radio ligand binding Assay Alpha2A Ligand [3H]RX 821002 125 Epinephrine Ligand concentration 0.5 nM 100 Prazosin Yohimbin 75 Kd (nM) 0.46 nM RX821002 Non specific epinephrine (0.1 mM) 50 UK14304 Incubation 60 min/RT Inhibition % 25 0 Detection method Scintillation counting -2 -1 0 1 2 3 4 5 Reference: Rx81002(IC50:2.3 nM) Log [Compound] nM 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, China Email: [email protected]; www.wuxiapptec.com Page 3 of 44 Biology Department of WuXi AppTec (Shanghai) Co., Ltd. Beta1 (Beta-1 Adrenergic Receptor) Cell Line Name: HEK293/Beta1/Gα15 Species/Source: Human/Recombinant Gene Synonyms: ADRB1; RHR; B1AR; ADRB1R; BETA1AR Accession Number: NM_000684 G coupling Gs Functional Assay-Calcium Flux Assay Beta1 (-)Epinephrine 120 Isoproterenol Agonist (-)Epinephrine 100 Salbutamol hemisulfate Measurement: Intracellular Ca2+ 80 60 Detection method: FLIPR Fluorimetry 40 Activity (%) Activity Incubation 25℃ 20 0 Agonist effect EC50: 2.72 nM -20 -2 -1 0 1 2 3 4 5 Log [Compound] nM Beta1 120 Antagonist Carvedilol Carvedilol 100 (± )-Propranolol hydrochloride Measurement: Intracellular Ca2+ 80 Atenolol 60 ICI 118 551 hydrochloride Detection method: FLIPR Fluorimetry 40 20 Inhibition (%) Inhibition Incubation 25℃ 0 -20 Stimulant (-)-Epinephrine : 20 nM -2 -1 0 1 2 3 4 Log [Compound] nM Antagonist effect IC50: 4.75 nM Radio ligand binding Assay Beta1 125 Ligand [3H](-)CGP 12177 Atenolol Ligand concentration 0.3 nM 100 Salbutamol Propranolol 75 Kd (nM) 0.04 nM ICI118551 50 Non specific propranolol (2 µM) Isoproternol % Inhibition % 25 Incubation 120 min/RT 0 Detection method Scintillation counting -4 -3 -2 -1 0 1 2 3 4 5 Reference: Atenolol (IC50:857 nM) Log [Compound] nM 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, China Email: [email protected]; www.wuxiapptec.com Page 4 of 44 Biology Department of WuXi AppTec (Shanghai) Co., Ltd. Beta2 (Beta-2 Adrenergic Receptor) Cell Line Name: HEK293/Beta2/Gα15 Species/Source: Human/Recombinant Gene Synonyms: ADRB2; BAR; B2AR; ADRBR; ADRB2R; BETA2AR Accession Number: NM_000024 G coupling Gs/Gi Functional Assay-Calcium Flux Assay Beta2 Adrenaline Agonist Adrenaline 120 Isoproterenol 100 Salbutamol hemisulfate 2+ Measurement: Intracellular Ca 80 Detection method: FLIPR Fluorimetry 60 40 Incubation 25℃ (%) Activity 20 Agonist effect EC50: 0.11 nM 0 -20 -4 -3 -2 -1 0 1 2 3 4 Log [Compound] nM Beta2 Carvedilol Antagonist Carvedilol 125 (± )-Propranolol hydrochloride ICI 118551 Measurement: Intracellular Ca2+ 100 75 Detection method: FLIPR Fluorimetry 50 Incubation 25℃ 25 Inhibition (%) Inhibition Stimulant Adrenaline: 0.43 nM 0 -25 Antagonist effect IC50: 7.07 nM -2 -1 0 1 2 3 Log [Compound] nM Radio ligand binding Assay Beta2 Ligand [3H](-)CGP 12177 125 Atenolol Ligand concentration 0.3 nM 100 Salbutamol Propranolol Kd (nM) 0.1 nM 75 ICI118551 Non specific propranolol (2 µM) 50 Isoproternol Incubation 120 min/RT Inhibition % 25 Detection method Scintillation counting 0 Reference: ICI118551(IC50:3.3 nM) -4 -3 -2 -1 0 1 2 3 4 5 Log [Compound] nM 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, China Email: [email protected]; www.wuxiapptec.com Page 5 of 44 Biology Department of WuXi AppTec (Shanghai) Co., Ltd. CB1 (Cannabinoid Type 1 Receptor) Cell Line Name: CHO/CB1/Gα15 Species/Source: Human/Recombinant Gene Synonyms: CNR1; CB1; CNR; CB-R; CB1A; CB1R; CANN6; CB1K5 Accession Number: NM_016083 (isoform1) G coupling Gi Functional Assay-Calcium Flux Assay CB1 WIN55212-2 Agonist WIN55212-2 120 CP55940 2+ 100 Anandamide Measurement: Intracellular Ca 80 Detection method: FLIPR Fluorimetry 60 40 Incubation 25℃ (%) Activity 20 Agonist effect EC50:352 nM 0 -20 -1 0 1 2 3 4 5 6 Log [Compound] nM CB1 Antagonist Rimonabant hydrochloride Rimonabant 120 AM281 2+ Measurement: Intracellular Ca 100 AM630 Detection method: FLIPR Fluorimetry 80 60 Incubation 25℃ 40 20 Inhibition (%) Inhibition Stimulant WIN55212-2: 4000 nM 0 -20 Antagonist effect IC50: 12.5 nM -1 0 1 2 3 4 5 6 log [Con.] nM CB1 (Cannabinoid Type 1 Receptor) Cellular Background: CHO-K1 Species/Source: Human/Recombinant Gene Synonyms: CNR1; CB1; CNR; CB-R; CB1A; CB1R; CANN6; CB1K5 Accession Number: X54937 Radio ligand binding Assay Ligand [3H]CP 55940 CB1 125 Ligand concentration 0.4 nM CP55940 100 WIN55212-2 AM281 Kd (nM) 3.7 nM 75 Anandamide Non specific WIN 55212-2 (5 µM) 50 Incubation 120 min/37℃ Inhibition % 25 0 Detection method Scintillation counting -3 -2 -1 0 1 2 3 4 5 Reference: CP 55940 (0.81 nM) Log [Compound] nM 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, China Email: [email protected]; www.wuxiapptec.com Page 6 of 44 Biology Department of WuXi AppTec (Shanghai) Co., Ltd. CB2 (Cannabinoid Type 2 Receptor) Cell Line Name: CHO/CB2/Gα15 Species/Source: Human/Recombinant Gene Synonyms: CNR2; CB2; CX5; CB-2 Accession Number: NM_001841 G coupling Gi Functional Assay-Calcium Flux Assay CB2 120 Agonist CP55940 CP55940 2+ 100 WIN55215-2 Measurement: Intracellular Ca 80 Anandamide Detection method: FLIPR Fluorimetry 60 40 ℃ Activity (%) Incubation 25 20 Agonist effect EC50: 199 nM 0 -1 0 1 2 3 4 5 Log [Compound] nM Antagonist AM630 CB2 120 2+ AM630 Measurement: Intracellular Ca 100 AM281 Detection method: FLIPR Fluorimetry 80 Rimonbant 60 Incubation 25℃ 40 20 Stimulant CP55940: 300 nM (%) Inhibition 0 Antagonist effect IC50: 563 nM -20 0 1 2 3 4 5 Log [Compound] nM Radio ligand binding Assay Ligand [3H]CP 55940 CB2 125 Ligand concentration 0.5 nM CP55940 100 WIN55212-2 Anandomide Kd (nM) 5.1 nM 75 AM630 Non specific WIN 55212-2 (5 µM) 50 AM281 % Inhibition % 25 Incubation 120 min/37℃ 0 Detection method Scintillation counting -3 -2 -1 0 1 2 3 4 5 Reference: WIN55212-2(IC50: Log [Compound] nM 2.2nM) 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, China Email: [email protected]; www.wuxiapptec.com Page 7 of 44 Biology Department of WuXi AppTec (Shanghai) Co., Ltd. CCKA (Cholecystokinin A Receptor) Cell Line Name: HEK293/CCKA Species/Source: Human/Recombinant Gene