AB-NECA BDBM85525
B-NECA BDBM85777
BDBM85617 MPC-NECA
BDBM82537 NECA-N1-oxide
BDBM50003267 6-guanidino- NECA CHEMBL405144
2-substituted NECA derivatives, 8 BDBM85524
NECA BDBM86761 NSC_448222 CAS_35920-39-9
N-Ethylcarboxamidoadenosine [3H]NECA Adenosine, N6-Ethyl-carboxamido Adenosine-5 -(N-ethylcarboxamide) BDBM21241
N6-CyclohexylNECA 5-(6-Cyclohexylamino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide Cyclohexyl-NECA BDBM50021484
5-(6-Amino-2-cyclohex-1-enylethynyl-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide 2-substituted NECA derivatives, 12 BDBM50041894
5-[6-Amino-2-(3-hydroxy-but-1-ynyl)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide 2-substituted NECA derivatives, 5 BDBM50041906
5-[6-Amino-2-(4-hydroxy-pent-1-ynyl)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide 2-substituted NECA derivatives, 4 BDBM50041895
BDBM50034516 2-substituted NECA derivatives, 14 3-{4-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-ylethynyl]-phenyl}-propionic acid
BDBM50041901 2-substituted NECA derivatives, 2 5-[6-Amino-2-(5-cyano-pent-1-ynyl)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide
5-[6-Amino-2-(3-hydroxy-3-phenyl-but-1-ynyl)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide BDBM50041912 2-substituted NECA derivatives, 7
N-Ethylcarboxamidoadenosine NECA (2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-N-ethyl-3,4-dihydroxyoxolane-2-carboxamide CHEMBL464859 BDBM21220 Adenosine-5-(N-ethylcarboxamide) Adenosine, N6-Ethyl-carboxamido Adenosine analog, 3
5-(6-Amino-2-furan-2-ylethynyl-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide 2-substituted NECA derivatives, 10 BDBM50034540 5-{6-amino-2-[2-(furan-2-yl)ethynyl]-9H-purin-9-yl}-N-ethyl-3,4-dihydroxyoxolane-2-carboxamide
BDBM50034536 5-{6-amino-2-[2-(4-carbamoylphenyl)ethynyl]-9H-purin-9-yl}-N-ethyl-3,4-dihydroxyoxolane-2-carboxamide 2-substituted NECA derivatives, 13 5-[6-Amino-2-(4-carbamoyl-phenylethynyl)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide
2-substituted NECA derivatives, 11 BDBM50034533 5-{6-amino-2-[2-(1,3-thiazol-2-yl)ethynyl]-9H-purin-9-yl}-N-ethyl-3,4-dihydroxyoxolane-2-carboxamide 5-(6-Amino-2-thiazol-2-ylethynyl-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide
(2S,3R,5R)-5-[6-amino-2-(hex-1-yn-1-yl)-9H-purin-9-yl]-N-ethyl-3,4-dihydroxyoxolane-2-carboxamide 1, HENECA HE-NECA 5-(6-Amino-2-hex-1-ynyl-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide BDBM50004461 HENECA
5-[6-Amino-2-(5-phenyl-pent-1-ynyl)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide 2-substituted NECA derivatives, 3 BDBM50034513 5-[6-amino-2-(5-phenylpent-1-yn-1-yl)-9H-purin-9-yl]-N-ethyl-3,4-dihydroxyoxolane-2-carboxamide
- Bruns, RF; Lu, GH; Pugsley, TA Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol Pharmacol 29: 331-46 (1986)
- Macchia, M; Salvetti, F; Barontini, S; Calvani, F; Gesi, M; Hamdan, M; Lucacchini, A; Pellegrini, A; Soldani, P; Martini, C Fluorescent probes for adenosine receptors: synthesis and biology of N6-dansylaminoalkyl-substituted NECA derivatives. Bioorg Med Chem Lett 8: 3223-8 (1999)
- Macchia, M; Salvetti, F; Bertini, S; Di Bussolo, V; Gattuso, L; Gesi, M; Hamdan, M; Klotz, KN; Laragione, T; Lucacchini, A; Minutolo, F; Nencetti, S; Papi, C; Tuscano, D; Martini, C 7-Nitrobenzofurazan (NBD) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A(3) adenosine receptors. Bioorg Med Chem Lett 11: 3023-6 (2001)
- Camaioni, E; Di Francesco, E; Vittori, S; Volpini, R; Cristalli, G Adenosine receptor agonists: synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA. Bioorg Med Chem 5: 2267-75 (1997)
- ChEBML_30001 Inhibition of [3H]- NECA binding to adenosine receptor A2A
- ChEMBL_303281 (CHEMBL828274) Inhibition of [3H]NECA binding to adenosine A2a receptor
- ChEMBL_30713 (CHEMBL646480) Binding affinity for adenosine A2 receptor using [3H]- NECA
- ChEMBL_31030 (CHEMBL638319) Inhibition of [3H]- NECA binding to adenosine receptor A2
- ChEMBL_2223061 (CHEMBL5136395) Antagonist activity at human wild type A1R expressed in CHO-K1 cells assessed as inhibition of forskolin/ NECA-stimulated cAMP accumulation by measuring NECA-mediated cAMP stimulation at 1 uM incubated with NECA for 30 mins by LANCE cAMP detection assay
- ChEMBL_1991291 (CHEMBL4625026) Displacement of [3H]NECA from rat striatal A2A adenosine receptor
- ChEMBL_823419 (CHEMBL2046087) Displacement of [3H]NECA from human recombinant adenosine A3 receptor
- ChEMBL_940031 (CHEMBL2327714) Displacement of [3H]NECA from human recombinant adenosine A3 receptor
- ChEMBL_2223062 (CHEMBL5136396) Antagonist activity at human wild type A3R expressed in Flp-In-CHO cells assessed as inhibition of forskolin/ NECA-stimulated cAMP accumulation by measuring NECA-mediated cAMP stimulation at 1 uM incubated with NECA for 30 mins by LANCE cAMP detection assay
- ChEMBL_2223084 (CHEMBL5136418) Antagonist activity at human wild type A3R expressed in Flp-In-CHO cells assessed as inhibition of forskolin/ NECA-stimulated cAMP accumulation by measuring NECA-mediated cAMP stimulation at 10 uM incubated with NECA for 30 mins by LANCE cAMP detection assay
- ChEMBL_967979 (CHEMBL2401083) Antagonist activity at human adenosine A2A receptor expressed in HEK293 cells assessed as inhibition of NECA-induced cAMP accumulation incubated for 10 mins prior to NECA addition
- ChEBML_30176 [3H]NECA saturation binding in CHO cells expressing human Adenosine A3 receptor
- ChEMBL_27785 (CHEMBL645921) Displacement of [3H]NECA from A2-receptor of rat striatal membranes
- ChEMBL_687325 (CHEMBL1292001) Antagonist activity at human adenosine A2B receptor transfected in CHO cells assessed as inhibition of NECA-induced cAMP accumulation treated 15 mins before NECA challenge measured after 1 hr
- ChEBML_27478 Binding affinity against adenosine A2 receptor in rat striatal membranes using [3H]NECA
- ChEBML_30903 Inhibitory activity against Adenosine A2a receptor using [3H]NECA in rat striatal membranes
- ChEBML_31975 [3H]NECA saturation binding in CHO cells expressing human recombinant A2A adenosine receptor
- ChEMBL_1518654 (CHEMBL3619720) Displacement of [3H]-NECA from Adenosine A2A receptor in rat striatal membranes
- ChEMBL_1548619 (CHEMBL3757619) Displacement of [3H]NECA from adenosine A2A receptor in rat striatal membrane
- ChEMBL_1627002 (CHEMBL3869523) Displacement of [3H]NECA from adenosine receptor A2A in rat striatal membranes
- ChEMBL_1691295 (CHEMBL4041944) Displacement of [3H]NECA from adenosine receptor A2a in rat striatial membrane
- ChEMBL_1995264 (CHEMBL4629159) Displacement of [3H]-NECA from human A2A receptor expressed in CHO cells
- ChEMBL_27948 (CHEMBL649129) Displacement of [3H]-NECA from adenosine A2 receptor of rat striatal membrane
- ChEMBL_30005 (CHEMBL641296) Displacement of [3H]NECA from Adenosine A2A receptor of rat striatal membranes
- ChEMBL_30719 (CHEMBL646485) Displacement of [3H]-NECA from adenosine A2 receptor of rat striatal membrane
- ChEMBL_32144 (CHEMBL646354) Displacement of [3H]NECA from Adenosine A2A receptor in rat striatal membrane
- ChEMBL_436390 (CHEMBL904698) Displacement of [2H]NECA from human A3 receptor expressed in HeLa cells
- ChEMBL_90338 (CHEMBL696391) Inhibition of 1 uM NECA-stimulated cyclic AMP levels in human platelets
- ChEBML_27949 Inhibition of [3H]NECA specific binding against Adenosine A2 receptors of rat striatal membranes
- ChEBML_31377 Displacement of [3H]NECA from human adenosine A2A receptor in stably transfected CHO cells
- ChEBML_31864 Displacement of [3H]NECA from human adenosine A3 receptor in stably transfected HEK cells
- ChEMBL_1445860 (CHEMBL3376728) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_1452896 (CHEMBL3366212) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_1452897 (CHEMBL3366213) Displacement of [3H]NECA from human adenosine A3 receptor expressed in CHO cells
- ChEMBL_1465025 (CHEMBL3405041) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_1518656 (CHEMBL3619722) Displacement of [3H]-NECA from Adenosine A2A receptor in Wistar rat striatal membranes
- ChEMBL_1691303 (CHEMBL4041952) Displacement of [3H]NECA from adenosine receptor A2a in rat brain striatal membrane
- ChEMBL_27610 (CHEMBL875808) Maximal NECA stimulation of adenylate cyclase via A2 receptors in human platelet membranes
- ChEMBL_27611 (CHEMBL644330) Binding affinity against A2 adenosine receptors from human platelet membranes using [3H]NECA
- ChEMBL_27782 (CHEMBL645918) Binding affinity against Adenosine A2 receptor from human platelet membranes using [3H]NECA
- ChEMBL_27783 (CHEMBL645919) Binding affinity against adenosine A2 receptor from human platelet membranes using [3H]NECA
- ChEMBL_27929 (CHEMBL875263) Binding affinity for adenosine A2 receptor using [3H]NECA in rat striatal membrane
- ChEMBL_30174 (CHEMBL640451) Binding affinity against human adenosine A3 receptor using [3H]-NECA at 30 uM
- ChEMBL_30536 (CHEMBL643183) Inhibition of binding to Adenosine-2 receptor using [3H]NECA in rat striatum
- ChEMBL_30554 (CHEMBL649866) Inhibition of [3H]NECA binding to adenosine A2 receptor from rat striatal membranes
- ChEMBL_30568 (CHEMBL649880) Binding affinity against Adenosine-2 receptor with [3H]-NECA in rat striatal membranes.
- ChEMBL_30603 (CHEMBL642010) Inhibition of NECA-induced stimulation of adenylate cyclase at human adenosine A2B receptor
- ChEMBL_30709 (CHEMBL645452) Binding affinity for adenosine A2 receptor using [3H]NECA in rat striatal membrane
- ChEMBL_30725 (CHEMBL648804) Inhibition of [3H]NECA binding to Adenosine A2 receptor in rat brain striatum
- ChEMBL_325561 (CHEMBL860280) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_367222 (CHEMBL862969) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_376055 (CHEMBL864809) Displacement of [3H]NECA from human adenosine A3 receptor expressed in HeLa cells
- ChEMBL_445196 (CHEMBL894344) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_450736 (CHEMBL901019) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_471615 (CHEMBL941044) Displacement of [3]NECA from human adenosine A3 receptor expressed in HeLa cells
- ChEMBL_471790 (CHEMBL939998) Displacement of [3H]NECA from human adenosine A3 receptor expressed in CHO cells
- ChEMBL_566530 (CHEMBL955116) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_566532 (CHEMBL955118) Displacement of [3H]NECA from human adenosine A3 receptor expressed in CHO cells
- ChEMBL_587703 (CHEMBL1037717) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_587704 (CHEMBL1037718) Displacement of [3H]NECA from human adenosine A3 receptor expressed in CHO cells
- ChEMBL_629507 (CHEMBL1120796) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_964093 (CHEMBL2394446) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells
- ChEMBL_2164400 (CHEMBL5049261) Antagonist activity at human A2aR expressed in HEK293 cells assessed as reduction in NECA-induced cAMP production incubated for 20 mins in presence of 20 nM NECA by GloSensor cAMP detection assay
- ChEMBL_1646029 (CHEMBL3995085) Antagonist activity at human adenosine receptor A2b expressed in HEK293 cells assessed as inhibition of NECA-induced cAMP level preincubated for 15 mins followed by NECA stimulation for 15 mins by HTRF assay
- ChEMBL_1833780 (CHEMBL4333788) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced increase in cAMP accumulation preincubated for 10 mins followed by NECA addition by GloSensor cAMP assay
- ChEMBL_1833781 (CHEMBL4333789) Antagonist activity at human adenosine A2A receptor expressed in CHO cells assessed as inhibition of NECA-induced increase in cAMP accumulation preincubated for 10 mins followed by NECA addition by GloSensor cAMP assay
- ChEMBL_2164405 (CHEMBL5049266) Antagonist activity at human A2aR expressed in HEK293 cells assessed as reduction in NECA-induced cAMP production incubated for 20 mins in presence of 40 nM NECA by ultra LANCE cAMP detection assay
- ChEMBL_2164406 (CHEMBL5049267) Antagonist activity at human A2aR expressed in HEK293 cells assessed as reduction in NECA-induced cAMP production incubated for 20 mins in presence of 1 uM NECA by ultra LANCE cAMP detection assay
- ChEBML_1590507 Displacement of [3H]NECA from recombinant rat adenosine receptor A3 expressed in CHO cell membranes
- ChEBML_30704 Binding affinity against adenosine A2 receptor in rat striatal membranes using [3H]NECA as radioligand
- ChEMBL_1274846 (CHEMBL3090420) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells
- ChEMBL_1274849 (CHEMBL3090423) Displacement of [3H]NECA from human recombinant adenosine A2A receptor expressed in CHO cells
- ChEMBL_1471044 (CHEMBL3420489) Displacement of [3H]-NECA from human recombinant adenosine A2A receptor transfected in CHO cells
- ChEMBL_1518644 (CHEMBL3619587) Displacement of [3H]-NECA from Adenosine A2A receptor in Sprague-Dawley rat striatal membranes
- ChEMBL_1549259 (CHEMBL3757837) Displacement of [3H]NECA from human A2A adenosine receptor expressed in CHO cell membranes
- ChEMBL_27612 (CHEMBL644331) Maximal NECA stimulation of adenylate cyclase via Adenosine A2 receptor in human platelet membranes
- ChEMBL_27762 (CHEMBL643367) Maximal NECA stimulation of adenylate cyclase via adenosine A2 receptor in human platelet membranes
- ChEMBL_28671 (CHEMBL644726) Maximal NECA stimulation of adenylate cyclase via Adenosine A2 receptor in human platelet membranes
- ChEMBL_29333 (CHEMBL643147) Binding affinity for Adenosine A1 receptor in corpora striata of rats using [3H]NECA
- ChEMBL_29608 (CHEMBL640266) Binding affinity for Adenosine A1 receptor in corpora striata of rats using [3H]NECA
- ChEMBL_29997 (CHEMBL643008) Binding affinity for adenosine A2A receptor using [3H]-NECA in rat brain striatal membranes
- ChEMBL_303370 (CHEMBL839688) Inhibition of [3H]NECA binding to human adenosine A3 receptor expressed in HeLa cells
- ChEMBL_30560 (CHEMBL649872) Affinity for Adenosine A2 receptor determined by [3H]NECA binding to rat striatal membranes
- ChEMBL_30563 (CHEMBL649875) Binding affinity for Adenosine A2 receptor from rat striatum using [3H]NECA as radioligand
- ChEMBL_30569 (CHEMBL649881) Binding affinity at adenosine A2 receptor from rat striatal membranes by [3H]NECA displacement.
- ChEMBL_30703 (CHEMBL644778) Binding affinity against adenosine A2 receptor from rat striatum using [3H]NECA as radioligand.
- ChEMBL_30714 (CHEMBL646481) Binding affinity at adenosine A2 receptor from rat striatal membranes by [3H]NECA displacement.
- ChEMBL_30855 (CHEMBL882479) Binding affinity for Adenosine A2 receptor in corpora striata of rats using [3H]NECA
- ChEMBL_30856 (CHEMBL643728) Binding affinity for adenosine A2 receptor in corpora striata of rats using [3H]NECA
- ChEMBL_30867 (CHEMBL873059) Maximal NECA stimulation of adenylate cyclase via Adenosine A2 receptor in human platelet membranes.
- ChEMBL_31067 (CHEMBL640427) Radioligand binding assay for [3H]NECA affinity towards adenosine A2A receptor of rat striatum
- ChEMBL_31706 (CHEMBL644950) Binding affinity against human adenosine A3 receptor in CHO cell membranes using [3H]NECA
- ChEMBL_325471 (CHEMBL860042) Displacement of [3H]NECA from cloned human adenosine A2A receptor expressed in CHO cells
- ChEMBL_337185 (CHEMBL862545) Inhibition of NECA-stimulated cAMP accumulation in CHO cells expressing human adenosine A2B receptor
- ChEMBL_428387 (CHEMBL917987) Displacement of [3H]NECA from human recombinant adenosine A2A receptor expressed in CHO cells
- ChEMBL_428388 (CHEMBL917988) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells
- ChEMBL_436395 (CHEMBL904703) Inhibition of NECA-induced IL6 production in human fibroblast after 24 hrs by ELISA
- ChEMBL_461290 (CHEMBL927307) Displacement of [3H]NECA from human recombinant adenosine A2A receptor expressed in CHO cells
- ChEMBL_461291 (CHEMBL927310) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells
- ChEMBL_529843 (CHEMBL967546) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in HEK293T cells
- ChEMBL_535232 (CHEMBL991729) Displacement of [3H]NECA from Sprague-Dawley rat adenosine A2A receptor by scintillation counting
- ChEMBL_556458 (CHEMBL955675) Displacement of [125I]NECA from human cloned adenosine A2A receptor expressed in CHO cells
- ChEMBL_562703 (CHEMBL1012738) Displacement of [3H]NECA from human cloned adenosine A2A receptor expressed in CHO cells
- ChEMBL_562992 (CHEMBL1015329) Displacement of [3H]NECA from human recombinant adenosine A2A receptor expressed in CHO cells
- ChEMBL_588679 (CHEMBL1056089) Displacement of [3H]NECA from human recombinant adenosine A2A receptor expressed in CHO cells
- ChEMBL_588680 (CHEMBL1056090) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells
- ChEMBL_623792 (CHEMBL1117075) Displacement of [3H]NECA from human cloned adenosine A2a receptor expressed in CHO cells
- ChEMBL_623793 (CHEMBL1117076) Displacement of [3H]NECA from human cloned adenosine A3 receptor expressed in CHO cells
- ChEMBL_818428 (CHEMBL2034748) Displacement of [3H]NECA from human A2A adenosine receptor expressed in CHO cell membrane
- ChEMBL_878513 (CHEMBL2188178) Displacement of [3H]NECA from human adenosine A3 receptor expressed in CHO cell membrane
- ChEMBL_878514 (CHEMBL2188179) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cell membrane
- ChEMBL_937677 (CHEMBL2317191) Displacement of [3H]NECA from human adenosine A3 receptor expressed in human HeLa cells
- ChEMBL_629514 (CHEMBL1120803) Antagonist activity human recombinant adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-mediated [3H]cAMP accumulation treated 15 mins before NECA challenge measured after 30 mins by liquid scintillation spectrometry
- ChEMBL_1278934 (CHEMBL3097234) Displacement of [3H]NECA from adenosine A3 receptor in human HeLa cells after 180 mins
- ChEMBL_153751 (CHEMBL761206) Tested for inhibition of rat PC12 adenylate cyclase stimulation by using NECA as a radioligand
- ChEMBL_1743109 (CHEMBL4158859) Displacement of [3H]NECA from GRP94 in porcine pancreas rough microsomes by liquid scintillation spectrometry
- ChEMBL_30302 (CHEMBL639471) Inhibitory activity against NECA stimulated cAMP accumulation in CHO cells expressing human adenosine A2B receptor
- ChEMBL_30534 (CHEMBL643181) Concentration required for 50% inhibition of [3H]NECA binding on rat brain adenosine A2 receptor
- ChEMBL_30693 (CHEMBL646321) Binding affinity against Adenosine A2 receptor from rat striatal membrane using [3H]NECA as radioligand
- ChEMBL_30711 (CHEMBL646478) Binding affinity for adenosine A2 receptor in rat striatal membranes using [3H]NECA as radioligand
- ChEMBL_30717 (CHEMBL646484) Binding affinity towards adenosine A2 receptor on rat striatal membrane using [3H]NECA as radioligand
- ChEMBL_30854 (CHEMBL643571) Tested for inhibition of rat PC12 adenylate cyclase stimulation by using NECA as a radioligand
- ChEMBL_367228 (CHEMBL865365) Inhibition of NECA-stimulated cAMP level in CHO cells transfected with human adenosine A2B receptor
- ChEMBL_436396 (CHEMBL904704) Inhibition of NECA-induced IL6 production in Swiss mouse fibroblasts after 24 hrs by ELISA
- ChEMBL_538318 (CHEMBL1036752) Displacement of [3H]NECA from human cloned adenosine A3 receptor expressed in human HeLa cells
- ChEMBL_1645470 (CHEMBL3994399) Antagonist activity at human adenosine A2B receptor expressed in HEK293 cells assessed as reduction in NECA-induced increase in cAMP level preincubated for 15 mins followed by NECA addition measured after 15 mins by HTRF assay
- ChEMBL_1439619 (CHEMBL3383137) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells after 120 mins
- ChEMBL_1569188 (CHEMBL3791371) Displacement of [3H]NECA from human Adenosine A3 receptor expressed in HeLa cells after 30 mins
- ChEMBL_27927 (CHEMBL642335) Inhibition of NECA binding to adenosine A2 receptor mediates reduced adenylate cyclase activity in human platelets
- ChEMBL_30564 (CHEMBL649876) Binding affinity against Adenosine A2 receptor in rat brain membrane, using [3H]-NECA as the radioligand.
- ChEMBL_30726 (CHEMBL648805) Binding affinity against adenosine A2 receptor in rat striatum by using [3H]NECA as a radioligand
- ChEMBL_312875 (CHEMBL874953) Inhibition of 100 nM NECA-stimulated cAMP production in CHO cells expressing human adenosine A2B receptor
- ChEMBL_32143 (CHEMBL646353) Ability to inhibit binding of [3H]NECA to Adenosine A2A receptor in rat brain striatal membranes
- ChEMBL_613658 (CHEMBL1067001) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in human HeLa cell membrane
- ChEMBL_685061 (CHEMBL1286677) Displacement of [3H]NECA from human adenosine A3 receptor expressed in HeLa cells after 180 mins
- ChEMBL_1575935 (CHEMBL3802558) Antagonist activity at human adenosine A2B receptor expressed in CHO cell membranes assessed as inhibition of NECA-stimulated cAMP level preincubated for 10 mins followed by NECA stimulation by scintillation counting method in presence of [3H]-cyclic AMP
- ChEMBL_1575939 (CHEMBL3802562) Antagonist activity at human adenosine A2A receptor expressed in CHO cell membranes assessed as inhibition of NECA-stimulated cAMP level preincubated for 10 mins followed by NECA stimulation by scintillation counting method in presence of [3H]-cyclic AMP
- ChEMBL_1663193 (CHEMBL4012874) Antagonist activity at human adenosine receptor A2B expressed in CHO cell membranes assessed as inhibition of NECA-induced increase of cAMP accumulation preincubated for 10 mins followed by NECA addition measured after 10 mins by GloSensor cAMP assay
- ChEMBL_1663194 (CHEMBL4012875) Antagonist activity at human adenosine receptor A2A expressed in CHO cell membranes assessed as inhibition of NECA-induced increase of cAMP accumulation preincubated for 10 mins followed by NECA addition measured after 10 mins by GloSensor cAMP assay
- ChEMBL_1904963 (CHEMBL4407321) Antagonist activity at recombinant human A2A receptor expressed in HEK293T cells co-expressing Gs assessed as inhibition of NECA-stimulated cAMP accumulation preincubated for 15 mins followed by NECA addition and measured after 15 mins by Glosensor chemiluminescence assay
- ChEMBL_629515 (CHEMBL1120804) Antagonist activity human recombinant adenosine A3 receptor expressed in CHO cells assessed as blockade of NECA-mediated inhibition of forskolin-stimulated [3H]cAMP production treated 15 mins before NECA challenge measured after 30 mins by liquid scintillation spectrometry
- ChEBML_1690747 Displacement of [3H]NECA from human adenosine A3 receptor expressed in human HeLa cell membranes after 180 mins
- ChEMBL_1467363 (CHEMBL3411600) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells by Top Count analysis
- ChEMBL_1842420 (CHEMBL4342847) Displacement of [3H]-NECA from human A2A receptor expressed in CHO cells by radioligand competitive binding assay
- ChEMBL_1991285 (CHEMBL4625020) Displacement of [3H]NECA from Sprague-Dawley rat striatal membrane A2A adenosine receptor by scintillation counting analysis
- ChEMBL_2019976 (CHEMBL4673789) Displacement of [3H]-NECA from human A2A receptor expressed in CHO cell membrane by radioligand binding assay
- ChEMBL_27613 (CHEMBL643986) Binding affinity against A2 adenosine receptors from rat striatal membranes with 50 nM CPA using [3H]NECA
- ChEMBL_27614 (CHEMBL643987) Binding affinity against Adenosine A2 receptor from rat striatal membrane with 50 nM CPA using [3H]NECA
- ChEMBL_27759 (CHEMBL643364) Binding affinity against Adenosine A2 receptor from rat striatal membranes with 50 nM CPA using [3H]-NECA
- ChEMBL_27784 (CHEMBL645920) Binding affinity for adenosine A2 receptor using [3H]- NECA antagonism of adenylate cyclase activation in human platelets
- ChEMBL_30303 (CHEMBL639472) Inhibitory activity on NECA-induced cyclic-AMP accumulation in CHO-K1 cells expressing human Adenosine A2B receptor
- ChEMBL_306686 (CHEMBL830020) Inhibition of 100 nM-NECA stimulation of cAMP levels in human adenosine A2b receptor expressing CHO cells
- ChEMBL_30702 (CHEMBL644777) Binding affinity against adenosine A2 receptor from rat striatal membranes with 50 nM CPA using [3H]-NECA
- ChEMBL_30927 (CHEMBL647736) In vitro binding affinity against Adenosine A2a receptor in rat forebrain membranes by [3H]NECA (+CPA) displacement.
- ChEMBL_578989 (CHEMBL1061438) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in HEK293T cells by scintillation counting
- ChEMBL_772800 (CHEMBL1837539) Displacement of [3H]NECA from human recombinant adenosine 2A receptor expressed in CHO cells after 3 hrs
- ChEMBL_772802 (CHEMBL1837541) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells after 3 hrs
- ChEMBL_988047 (CHEMBL2438815) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cell membrane after 90 mins
- ChEMBL_956569 (CHEMBL2380019) Antagonist activity at human adenosine A2a receptor expressed in CHOK1 cells assessed as inhibition of NECA/forskolin-induced cAMP accumulation incubated for 15 mins prior to NECA/forskolin challenge measured after 5.5 to 6 hrs by beta-galactosidase reporter gene assay
- ChEMBL_151406 (CHEMBL754752) Binding activity of P3 purinoceptor-like protein (P3LP) using radioligand 40 nM [3H]NECA from rat brain membranes
- ChEMBL_1677774 (CHEMBL4027917) Displacement of [3H]NECA from recombinant human adenosine A2A receptor expressed in CHO cell membranes after 3 hrs
- ChEMBL_1700311 (CHEMBL4051293) Displacement of [3H]NECA from human adenosine A3 receptor expressed in human Hela cell membranes after 180 mins
- ChEMBL_1822663 (CHEMBL4322427) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cell membranes by radioligand competition assay
- ChEMBL_1822676 (CHEMBL4322440) Displacement of [3H]NECA from rat adenosine A2A receptor expressed in CHO cell membranes by radioligand competition assay
- ChEMBL_1878367 (CHEMBL4379761) Displacement of [3H]NECA from human adenosine receptor A2A expressed in CHO cell membranes by radioligand competition assay
- ChEMBL_1920493 (CHEMBL4423338) Displacement of [3H] NECA from human adrenergic alpha 2A receptor expressed in MDCK cells by radioligand binding assay
- ChEMBL_2213744 (CHEMBL5126876) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells measured by radioligand competition assay
- ChEMBL_2213748 (CHEMBL5126880) Displacement of [3H]NECA from human adenosine A3 receptor expressed in HeLa cells measured by scintillation counting assay
- ChEMBL_27918 (CHEMBL642326) In vitro binding affinity to Adenosine A2 receptor of human platelets by inhibition of NECA-stimulated adenylate cyclase
- ChEMBL_30565 (CHEMBL649877) Binding affinity against Adenosine A2 receptor in rat striatum by displacement of [3H]N-ethyladenosine-5''-uronamide(NECA)
- ChEMBL_30724 (CHEMBL648803) Binding affinity against Adenosine A2 receptor using [3H]NECA with 50 nM CPA in rat striatal brain membranes
- ChEMBL_31355 (CHEMBL644647) Binding affinity against Human recombinant Adenosine A2A receptor stably transfected in CHO cells using [3H]NECA as radioligand
- ChEMBL_31705 (CHEMBL644949) Binding affinity against Human recombinant Adenosine A3 receptor stably transfected in CHO cells using [3H]NECA as radioligand
- ChEMBL_497307 (CHEMBL995869) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells by liquid scintillation counting
- ChEMBL_499870 (CHEMBL976202) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in HeLa cells by beta scintillation counter
- ChEMBL_535309 (CHEMBL982634) Antagonist activity at NECA-stimulated human adenosine A3 receptor expressed in CHO cells by [35S]GTPgammaS binding assay
- ChEMBL_542426 (CHEMBL1010663) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells by liquid scintillation counting
- ChEMBL_575806 (CHEMBL1024507) Displacement of [3H]NECA from human recombinant adenosine A1 receptor expressed in CHO cells by liquid scintillation counting
- ChEMBL_715074 (CHEMBL1664180) Displacement of [3H]NECA from human adenosine A3 receptor expressed in human HeLa cells by liquid scintillation counting
- ChEMBL_818429 (CHEMBL2034749) Antagonist activity at human A2B receptor expressed in CHO cells assessed as inhibition of NECA-mediated cAMP accumulation
- ChEMBL_878512 (CHEMBL2188177) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as NECA-induced adenylate cyclase activity
- ChEMBL_1543585 (CHEMBL3750703) Competitive antagonist activity at YFP linked human adenosine A3 receptor expressed in CHO CRE-SPAP cells assessed as inhibition of NECA-induced receptor internalization preincubated for 30 mins followed by addition of NECA measured after 60 mins by H33342 staining-based fluorescence analysis
- ChEMBL_1590519 (CHEMBL3831101) Antagonist activity at human adenosine receptor A2b expressed in CHO cell membranes assessed as inhibition of NECA-induced cAMP accumulation measured as NECA EC50 at 2 uM incubated for 15 mins in presence of [3H]cAMP by liquid scintillation counting (Rvb = 73 nM)
- ChEMBL_1590524 (CHEMBL3831106) Antagonist activity at human adenosine receptor A2a expressed in CHO cell membranes assessed as inhibition of NECA-induced cAMP accumulation measured as NECA EC50 at 5 uM incubated for 15 mins in presence of [3H]cAMP by liquid scintillation counting (Rvb = 68 nM)
- ChEMBL_1590529 (CHEMBL3831111) Antagonist activity at human adenosine receptor A2a expressed in CHO cell membranes assessed as inhibition of NECA-induced cAMP accumulation measured as NECA EC50 at 7 uM incubated for 15 mins in presence of [3H]cAMP by liquid scintillation counting (Rvb = 77 nM)
- ChEMBL_1707355 (CHEMBL4058588) Displacement of [3H]-NECA from A2A receptor in rat striatal membrane in presence of A1 receptor agonist N6-cyclopentyladenosine
- ChEMBL_2053442 (CHEMBL4708443) Displacement of [3H]-NECA from human A2A receptor stably expressed in CHO cell membranes by radioligand competitive binding assay
- ChEMBL_2213731 (CHEMBL5126863) Antagonist activity at human adenosine A2A receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP production
- ChEMBL_2224409 (CHEMBL5137922) Displacement of [3H]NECA from A2AAR in rat striatal membrane assessed as inhibition constant by radioligand competitive binding assay
- ChEMBL_30293 (CHEMBL639463) Receptor-stimulated activity in CHO cells stably transfected with Human recombinant Adenosine A2B receptor using [3H]NECA as radioligand
- ChEMBL_30570 (CHEMBL649882) Binding affinity against adenosine A2 receptor in rat striatum by the displacement of [3H]N-ethyladenosine-5''-uronamide(NECA).
- ChEMBL_31206 (CHEMBL640303) Antagonist activity as inhibition of cAMP generation after agonist-modulation of human Adenosine A2A receptor with 100 nM NECA
- ChEMBL_445201 (CHEMBL894349) Antagonist activity at human adenosine A3 receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP accumulation
- ChEMBL_450738 (CHEMBL901021) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP accumulation
- ChEMBL_473541 (CHEMBL939195) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP levels
- ChEMBL_556464 (CHEMBL955681) Antagonist activity at human adenosine A3 cells expressed in CHO cells assessed as inhibition of NECA-induced cAMP accumulation
- ChEMBL_609496 (CHEMBL1071768) Displacement of [3H]NECA from human recombinant adenosine A2A receptor expressed in Sf21 cells co-expressing GalphaS2, beta4, gamma2
- ChEMBL_834864 (CHEMBL2073181) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP production
- ChEMBL_2326235 Antagonist activity at human A1R expressed in Flp-In-CHO cells assessed as NECA-mediated reduction in forskolin-stimulated cAMP accumulation by measuring equilibrium dissociation constant incubated for 30 to 60 mins followed by forskolin/NECA addition measured after 30 mins by LANCE cAMP assay
- ChEMBL_988045 (CHEMBL2438813) Antagonist activity at human recombinant adenosine A1 receptor expressed in CHO cells assessed as inhibition of NECA-mediated reduction of intracellular [3H]-cAMP accumulation incubated for 15 mins prior to NECA induction measured after 30 mins by liquid scintillation spectrometric analysis in presence of forskolin
- ChEMBL_1274848 (CHEMBL3090422) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEMBL_1278721 (CHEMBL3096095) Displacement of [3H]NECA from human adenosine A3 receptor expressed in CHO cells after 60 mins by scintillation counting analysis
- ChEMBL_1452898 (CHEMBL3366214) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEMBL_1467364 (CHEMBL3411601) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced adenylyl cyclase activity
- ChEMBL_1548614 (CHEMBL3757433) Displacement of [3H]NECA from adenosine A2A receptor in Sprague-Dawley rat striatal membrane after 1 hr by scintillation counting
- ChEMBL_1713851 (CHEMBL4123900) Displacement of [3H]NECA from A3 receptor in human HeLa cell membranes after 180 mins by beta-scintillation counting method
- ChEMBL_1833776 (CHEMBL4333784) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cells after 3 hrs by scintillation counting method
- ChEMBL_1905176 (CHEMBL4407534) Antagonist activity at human A2BR expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP production by alphascreen assay
- ChEMBL_1986040 (CHEMBL4619446) Displacement [3H]NECA from adenosine A3 receptor in human HeLa cell membranes incubated for 180 mins by scintillation counting method
- ChEMBL_1995266 (CHEMBL4629161) Antagonist activity against human A2B adenosine receptor expressed in CHO cells assessed as inhibition of NECA-induced adenylyl cylase activity
- ChEMBL_2046324 (CHEMBL4701023) Antagonist activity at human adenosine 2A receptor expressed in HEK293 cells assessed as inhibition of NECA-induced cAMP by immunoassay
- ChEMBL_27936 (CHEMBL649117) Binding affinity against Adenosine A2 receptor in rat striatal membranes using [3H]5'-(N-ethylcarboxamido)-adenosine (NECA) as the ligand
- ChEMBL_30535 (CHEMBL643182) Evaluated for the binding affinity towards the Adenosine A2 receptor in corpora striata of rats using [3H]NECA as radioligand.
- ChEMBL_436392 (CHEMBL904700) Activity at human A2B receptor expressed in HEK293 cells assessed as inhibition of NECA-induced intracellular cAMP levels by ELISA
- ChEMBL_436393 (CHEMBL904701) Activity at mouse A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced intracellular cAMP levels by ELISA
- ChEMBL_559971 (CHEMBL1014570) Antagonist activity at human cloned adenosine A2B cells expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP production
- ChEMBL_562993 (CHEMBL1015330) Agonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEMBL_566531 (CHEMBL955117) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEMBL_587705 (CHEMBL1037719) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced adenylyl cyclase activity
- ChEMBL_611641 (CHEMBL1071011) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in human HeLa cells by microplate beta scintillation counter
- ChEMBL_623794 (CHEMBL1117077) Antagonist activity at human cloned adenosine A2b receptor expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP accumulation
- ChEMBL_627568 (CHEMBL1113574) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEMBL_719013 (CHEMBL1679075) Displacement of [3H]NECA from human adenosine A3 receptor expressed in HeLa cells after 3 hrs by filtration binding assay
- ChEMBL_772801 (CHEMBL1837540) Antagonist activity human recombinant adenosine 2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEMBL_805338 (CHEMBL1955231) Displacement of [3H]NECA from A2A adenosine receptor expressed in rat striatal membranes after 30 mins by liquid scintillation counting
- ChEMBL_964094 (CHEMBL2394447) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEBML_1663191 Displacement of [3H]NECA from human adenosine receptor A2A expressed in CHO cell membranes after 3 hrs by microbeta scintillation counting method
- ChEBML_27615 Binding affinity against Adenosine A2 receptor from rhesus moneky striatum with N-[3H] ethyladenosin-5''-uronamide (NECA) and 30 nM (R)-PIA
- ChEMBL_1291662 (CHEMBL3119409) Displacement of [3H]-NECA from human adenosine A2A receptor expressed in CHO cells after 3 hrs by topcount scintillation counting analysis
- ChEMBL_1539019 (CHEMBL3738124) Antagonist activity at human wild-type A2A receptor expressed in CHO-K1 cells assessed as NECA-induced cAMP by HTRF assay
- ChEMBL_1539073 (CHEMBL3738289) Displacement of [3H]NECA from adenosine A2A receptor in Sprague-Dawley rat striatal membranes after 1 hr by scintillation counting analysis
- ChEMBL_1549261 (CHEMBL3757839) Antagonist activity at human A2B adenosine receptor expressed in CHO cell membranes assessed as inhibition of NECA stimulated adenylyl cyclase activity
- ChEMBL_1626998 (CHEMBL3869519) Displacement of [3H]NECA from adenosine receptor A2A in Sprague-Dawley rat striatal membranes after 60 mins by liquid scintillation counting
- ChEMBL_1714787 (CHEMBL4124836) Antagonist activity at human A2B adenosine receptor assessed as inhibition of NECA-stimulated cAMP level after 30 mins by Alphascreen assay
- ChEMBL_31395 (CHEMBL643653) Effective concentration for inhibition of NECA-stimulated [35S]GTP-gamma-S, binding at human adenosine A3 receptor expressed in CHO cells
- ChEMBL_590632 (CHEMBL1052236) Displacement of [3H]NECA from human recombinant adenosine A1 receptor expressed in CHO cells after 60 mins by liquid scintillation counting
- ChEMBL_616081 (CHEMBL1102044) Displacement of [3H]NECA from human adenosine A3 receptor expressed in human HeLa cells after 3 hrs by filtration binding assay
- ChEMBL_616403 (CHEMBL1100689) Displacement of [3H]NECA from human adenosine A3 receptor expressed in human HeLa cells after 3 hrs by filtration binding assay
- ChEMBL_629909 (CHEMBL1108373) Displacement of [3H]NECA from human adenosine A3 receptor expressed in human HeLa cells after 180 mins by beta scintillation counting
- ChEMBL_794134 (CHEMBL1931872) Antagonist activity at human recombinant adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity
- ChEMBL_1754719 (CHEMBL4189479) Antagonist activity at recombinant human adenosine A2B receptor expressed in CHO cell membranes assessed as inhibition of NECA-induced cAMP accumulation preincubated for 1 hr followed by NECA induction measured after 5 mins in presence of [3H]cAMP and bovine cAMP-binding protein by liquid scintillation counting method
- ChEBML_1591032 Displacement of [3H]NECA from recombinant human adenosine A2A receptor expressed in CHO cell membrane after 3 hrs by microbeta scintillation counting method
- ChEMBL_1292708 (CHEMBL3123575) Displacement of [3H]-NECA from human recombinant adenosine A2A receptor expressed in CHO cell membranes after 3 hrs by microbeta counting analysis
- ChEMBL_1550084 (CHEMBL3756030) Antagonist activity at human A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP production by scintillation counting method
- ChEMBL_1663191 (CHEMBL4012872) Displacement of [3H]NECA from human adenosine receptor A2A expressed in CHO cell membranes after 3 hrs by microbeta scintillation counting method
- ChEMBL_1701162 (CHEMBL4052144) Displacement of [3H]NECA from recombinant rat adenosine A3 receptor expressed in CHO cell membranes after 3 hrs by microbeta scintillation counting
- ChEMBL_1701166 (CHEMBL4052148) Displacement of [3H]NECA from recombinant human adenosine A2A receptor expressed in CHO cell membranes after 3 hrs by microbeta scintillation counting
- ChEMBL_2028575 (CHEMBL4682733) Displacement of [3H]NECA from rat adenosine receptor A3 expressed in CHO cell membranes incubated for 60 mins by radioligand competition assay
- ChEMBL_2094406 (CHEMBL4775669) Displacement of [3H]NECA from adenosine A3 receptor expressed in human HeLa cell membranes incubated for 180 mins by scintillation counting method
- ChEMBL_2157931 (CHEMBL5042681) Binding affinity to NanoLuc human A1 adenosine receptor expressed in HEK293-A cells in prescence of NECA by NanoBRET competitive binding assay
- ChEMBL_2160174 (CHEMBL5044924) Displacement of [3H]NECA from human A3AR expressed in HeLa cell membrane incubated for 180 mins by microbeta trilux scintillation counter analysis
- ChEMBL_2161287 (CHEMBL5046037) Displacement of [3H] NECA from human recombinant A3 receptor expressed in HeLa cells incubated for 180 mins by radioligand binding competition assay
- ChEMBL_2213739 (CHEMBL5126871) Antagonist activity at human adenosine A2B receptor expressed in mouse NIH/3T3 cells assessed as inhibition of NECA-induced IL-6 release
- ChEMBL_934961 (CHEMBL2320330) Antagonist activity at human adenosine A2b receptor transfected in CHO cells assessed as inhibition of NECA-induced cAMP accumulation by scintillation counting
- ChEMBL_940035 (CHEMBL2327844) Displacement of [3H]NECA from human recombinant adenosine A3 receptor expressed in CHO cells after 180 mins by liquid scintillation counting analysis
- ChEMBL_1447963 (CHEMBL3379789) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP level by scintillation counting analysis
- ChEMBL_1447967 (CHEMBL3379793) Antagonist activity at human adenosine A2A receptor expressed in CHO cells assessed as inhibition of NECA-stimulated cAMP level by scintillation counting analysis
- ChEMBL_1591032 (CHEMBL3831117) Displacement of [3H]NECA from recombinant human adenosine A2A receptor expressed in CHO cell membrane after 3 hrs by microbeta scintillation counting method
- ChEMBL_1736931 (CHEMBL4152681) Displacement of [3H]NECA from recombinant human adenosine A2A receptor transfected in CHO cell membranes after 3 hrs by microbeta scintillation counting analysis
- ChEMBL_1736933 (CHEMBL4152683) Displacement of [3H]NECA from recombinant human adenosine A3 receptor transfected in CHO cell membranes after 3 hrs by microbeta scintillation counting analysis
- ChEMBL_1802979 (CHEMBL4275271) Displacement of [3H]NECA from human adenosine A2A receptor expressed in CHO cell membranes after 3 hrs by micro beta scintillation counting method
- ChEMBL_1846323 (CHEMBL4346864) Displacement of [3H]NECA from rat A2A adenosine receptor expressed in CHO cell membranes measured after 60 mins by liquid scintillation counting method
- ChEMBL_1913508 (CHEMBL4416091) Displacement of [3H]NECA from human A3 receptor expressed in human HeLa cell membranes incubated for 180 mins by radioligand binding competition assay
- ChEMBL_2046311 (CHEMBL4701010) Antagonist activity at adenosine 2A receptor in human blood assessed as reversal of NECA/LPS-stimulated TNF-alpha release incubated for 5 hrs
- ChEMBL_2046330 (CHEMBL4701029) Antagonist activity at adenosine 2A receptor in human peripheral blood lymphocytes assessed as reduction in NECA-stimulated CREB phosphorylation by flow cytometric analysis
- ChEMBL_2053448 (CHEMBL4708449) Antagonist activity at human A2B receptor expressed in CHO cell membrane assessed as reduction in NECA-stimulated AMP level by GloSensor CAMP assay
- ChEMBL_2053449 (CHEMBL4708450) Antagonist activity at human A2A receptor expressed in CHO cell membrane assessed as reduction in NECA-stimulated AMP level by GloSensor CAMP assay
- ChEMBL_2151812 (CHEMBL5036274) Displacement of [3H]NECA from human adenosine A3 receptor expressed in human HeLa cells incubated for 30 mins by fluorescence polarization binding assay
- ChEMBL_2224406 (CHEMBL5137919) Displacement of [3H]NECA from A2AAR in rat striata assessed as inhibition constant in presence of N6-cyclopentyladenosine by radioligand competitive binding assay
- ChEMBL_562709 (CHEMBL1012744) Antagonist activity at human adenosine A3 receptor expressed in CHO cells assessed as blockade of NECA-mediated inhibition of forskolin-stimulated cAMP accumulation
- ChEMBL_589472 (CHEMBL1053620) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated [3H]cAMP production by scintillation counting
- ChEMBL_742838 (CHEMBL1769841) Antagonist activity at human cloned adenosine A3 receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP accumulation after 15 mins
- ChEMBL_958098 (CHEMBL2384072) Antagonist activity at recombinant human adenosine A2A receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP production after 60 mins
- ChEBML_31409 Inhibition of [35S]GTP-gamma-S, binding stimulated by 20 uM 5''-N-ethyluronamidoadenosine (NECA) in membranes of HEK293 cells expressing human Adenosine A3 receptor
- ChEMBL_1846317 (CHEMBL4346858) Displacement of [3H]NECA from recombinant mouse A3 adenosine receptor expressed in CHO cell membranes measured after 180 mins by liquid scintillation counting method
- ChEMBL_2046307 (CHEMBL4701006) Antagonist activity at adenosine 2A receptor in human PBMC assessed as reversal of NECA/CD3/CD28-stimulated IL-2 release incubated for 24 hrs
- ChEMBL_2213725 (CHEMBL5126857) Antagonist activity at human adenosine A2A receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP production measured by scintillation counter assay
- ChEMBL_2216614 (CHEMBL5129746) Antagonist activity at human adenosine A2A receptor expressed in HEK293T cells assessed as inhibition of NECA-induced Gs short protein activation by TRUPATH assay
- ChEMBL_2311476 Displacement of [3H]NECA from human A3AR expressed in human HeLa cell membrane assessed as inhibition constant incubated for 180 mins by competitive binding assay
- ChEMBL_794928 (CHEMBL1936207) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP production by competition protein binding assay
- ChEMBL_1292705 (CHEMBL3123572) Binding affinity to human recombinant adenosine A2B receptor expressed in CHO cell membranes assessed as inhibition of NECA-stimulated adenylyl cyclase activity after 20 mins
- ChEMBL_1471051 (CHEMBL3420496) Antagonist activity at human recombinant adenosine A3 receptor transfected in CHO cells assessed as reversal of NECA-induced inhibition of forskolin-induced adenylyl cyclase activity
- ChEMBL_1822665 (CHEMBL4322429) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity by Glo-sensor cAMP assay
- ChEMBL_1822666 (CHEMBL4322430) Antagonist activity at human adenosine A2A receptor expressed in CHO cells assessed as inhibition of NECA-stimulated adenylyl cyclase activity by Glo-sensor cAMP assay
- ChEMBL_2019975 (CHEMBL4673788) Antagonist activity at human A2B receptor expressed in CHO cell membrane assessed as reduction in NECA-stimulated adenylyl cyclase activity in presence of [alpha32P]ATP
- ChEMBL_2094590 (CHEMBL4775853) Inhibition of A2AR (unknown origin) expressed in CHO cells assessed as reduction in NECA-stimulated cellular cAMP levels incubated for 90 mins by FRET assay
- ChEMBL_31409 (CHEMBL644552) Inhibition of [35S]GTP-gamma-S, binding stimulated by 20 uM 5''-N-ethyluronamidoadenosine (NECA) in membranes of HEK293 cells expressing human Adenosine A3 receptor
- ChEMBL_31410 (CHEMBL644553) Inhibition of [35S]GTP-gamma-S, binding stimulated by 5 uM 5''-N-ethyluronamidoadenosine (NECA) in membranes of HEK293 cells expressing human Adenosine A3 receptor
- ChEMBL_687329 (CHEMBL1292005) Antagonist activity at adenosien A2B receptor in human HMC-1 cells assessed as inhibition of NECA-induced IL-8 release after 6 hr by ELISA
- ChEMBL_757184 (CHEMBL1803408) Antagonist activity human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-mediated [3H]cAMP accumulation after 150 mins by scintillation counter
- ChEMBL_817486 (CHEMBL2027709) Antagonist activity at human adenosine A1 receptor expressed in CHO cells assessed as inhibition of NECA-induced increase of intracellular cAMP level after 5 hrs
- ChEMBL_1438093 (CHEMBL3387894) Allosteric enhancer activity at human adenosine A1 receptor expressed in CHO cell membranes assessed as [3H]NECA affinity constant at 1 uM (Kd = 0.87 +/- 0.04 nM)
- ChEMBL_1461353 (CHEMBL3396203) Inhibition of human adenosine A2A receptor expressed in FlpIn-CHO cells assessed as inhibition of NECA-induced cAMP accumulation incubated for 30 mins by fluorescence assay
- ChEMBL_1878369 (CHEMBL4379763) Antagonist activity at human adenosine receptor A3 expressed in CHO cells assessed as suppression of NECA-induced decrease of cAMP accumulation by Glo-sensor cAMP assay
- ChEMBL_1878370 (CHEMBL4379764) Antagonist activity at human adenosine receptor A2B expressed in CHO cells assessed as suppression of NECA-induced decrease of cAMP accumulation by Glo-sensor cAMP assay
- ChEMBL_1885444 (CHEMBL4387026) Displacement of [3H]NECA from human A3 receptor expressed in HeLa-A3 cells incubated for 180 mins by scintillation counting method based radioligand competitive binding assay
- ChEMBL_2046326 (CHEMBL4701025) Antagonist activity at human adenosine 2A receptor expressed in HEK293 cells assessed as reduction in NECA-induced cAMP level incubated for 15 mins by HTRF assay
- ChEMBL_2491587 Displacement of [3H]NECA from human adenosine A3 receptor extracted from human HeLa cell membrane assessed as inhibition constant incubated for 180 mins by microplate scintillation counter
- ChEMBL_1439622 (CHEMBL3383140) Antagonist activity at human recombinant adenosine A1 receptor expressed in CHO cells assessed as inhibition of NECA-induced cAMP accumulation after 15 mins by liquid scintillation spectrometry
- ChEMBL_1802980 (CHEMBL4275272) Antagonist activity at human adenosine A2B receptor expressed in CHO cell membranes assessed as inhibition of NECA-stimulated adenylyl cyclase activity by [alpha-32P]ATP based assay
- ChEMBL_2119773 (CHEMBL4828920) Displacement of [3H]NECA from human adenosine A3 receptor expressed in HeLa cell membrane incubated for 180 mins by microplate beta scintillation counting based radioligand inhibition assay
- ChEMBL_2199482 (CHEMBL5111998) Antagonist activity at wild type A1R (unknown origin) expressed in CHO-K1 cells assessed as inhibition of NECA reduced forskolin stimulated cAMP accumulation incubated for 30 mins
- ChEMBL_718297 (CHEMBL1679601) Antagonist activity at human adenosine A2B receptor expressed in HEK293 cells assessed as inhibition of NECA-induced increase of intracellular cAMP level after 30 mins by ELISA
- ChEMBL_718298 (CHEMBL1679602) Antagonist activity at mouse adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced increase of intracellular cAMP level after 30 mins by ELISA
- ChEMBL_806471 (CHEMBL1959510) Antagonist activity at human recombinant adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced [3H]cAMP production after 150 mins by scintillation counting
- ChEMBL_941716 (CHEMBL2330939) Inhibition of human A2B adenosine receptor transfected in CHO cells assessed as inhibition of NECA-induced cyclic AMP production after 10 mins by competitive protein binding assay
- ChEMBL_2199478 (CHEMBL5111994) Antagonist activity at wild type A3R (unknown origin) expressed in Flp-In-CHO cells assessed as inhibition of NECA reduced forskolin stimulated cAMP accumulation incubated for 30 mins
- ChEMBL_30696 (CHEMBL644771) Binding affinity to A2 adenosine receptor in rat striatal membranes by [3H]NECA (1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide) displacement.
- ChEMBL_1291661 (CHEMBL3119408) Antagonist activity at human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced adenylyl cyclase activity after 20 mins in presence of [alpha-32P]ATP
- ChEMBL_1736932 (CHEMBL4152682) Antagonist activity at recombinant human adenosine A2B receptor expressed in CHO cells assessed as inhibition of NECA-induced adenylyl cyclase activity after 20 mins by [alpha-32P]ATP assay
- ChEMBL_1846297 (CHEMBL4346838) Displacement of [3H]NECA from recombinant mouse A3 adenosine receptor expressed in CHO cell membranes measured after 180 mins in presence of adenosine deaminase by liquid scintillation counting method
- ChEMBL_2028580 (CHEMBL4682738) Antagonist activity at rat A3AR expressed in CHO cells assessed as reduction in NECA-induced inhibition of forskolin-stimulated cAMP accumulation incubated for 2 hrs by fluorescence based assay
- ChEMBL_303806 (CHEMBL829027) Inhibition of luciferase production elicited by NECA by compound in CHO cells transfected with human adenosine A2b receptor and a luciferase expressing reporter plasmid in a reporter gene assay
- ChEMBL_1915028 (CHEMBL4417611) Antagonist activity at human A1AR expressed in CHO-K1 cells assessed as reduction in NECA-induced inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by fluorescence based assay
- ChEMBL_520192 (CHEMBL951835) Antagonist activity at adenosine A2B receptor in human Jurkat T cells assessed as inhibition of NECA-induced increase in intracellular calcium concentration by fluorimetric measurement in presence of MSX-2
- ChEMBL_1591035 (CHEMBL3831120) Antagonist activity at recombinant human adenosine A2B receptor expressed in CHOK1 cell membrane assessed as inhibition of NECA-stimulated adenylyl cyclase activity incubated for 20 mins in presence of [alpha32P]ATP
- ChEMBL_817485 (CHEMBL2027708) Antagonist activity at human adenosine A3 receptor expressed in forskolin-stimulated CHO cells assessed as inhibition of NECA-induced CRE-SPAP gene transcription by measuring intracellular cAMP level after 5 hrs
- ChEMBL_1701172 (CHEMBL4052154) Antagonist activity at recombinant human adenosine A3 receptor expressed in CHO cell membranes assessed as reversal of NECA-mediated inhibition of adenylyl cyclase activity after 10 mins by [alpha-32P]ATP assay
- ChEMBL_2028581 (CHEMBL4682739) Antagonist activity at rat A3AR expressed in CHO cells assessed as reduction in NECA-induced inhibition of forskolin-stimulated cAMP accumulation at 3 uM incubated for 2 hrs by fluorescence based assay
- ChEMBL_2028582 (CHEMBL4682740) Antagonist activity at rat A3AR expressed in CHO cells assessed as reduction in NECA-induced inhibition of forskolin-stimulated cAMP accumulation at 10 uM incubated for 2 hrs by fluorescence based assay
- ChEMBL_2106114 (CHEMBL4814789) Antagonist activity at human A2A expressed in human HEK293 cells assessed as reduction in cAMP production preincubated for 10 mins followed by NECA stimulation and measured after 30 mins by micro plate reader method
- ChEMBL_1671316 (CHEMBL4021345) Antagonist activity at human adenosine A2B receptor expressed in HEK293 cells assessed as inhibition of NECA-induced increase in cAMP accumulation incubated for 15 mins followed by agonist treatment for 15 mins by HTRF assay
- ChEMBL_2046345 (CHEMBL4701044) Antagonist activity at adenosine 2A receptor (unknown origin) expressed in CHO cells assessed as reduction in intracellular cAMP level preincubated for 15 mins followed by NECA addition and measured after 15 mins by immunoassay method
- ChEMBL_2152684 (CHEMBL5037231) Antagonist activity at human adenosine A1 receptor expressed in Flp-In-CHO cell membrane assessed as inhibition of NECA-induced cAMP accumulation in presence of forskolin measured after 40 mins by alpha screen LANCE assay
- ChEMBL_2152685 (CHEMBL5037232) Antagonist activity at human adenosine A2A receptor expressed in Flp-In-CHO cell membrane assessed as inhibition of NECA-induced cAMP accumulation in presence of forskolin measured after 40 mins by alpha screen LANCE assay
- ChEMBL_2152686 (CHEMBL5037233) Antagonist activity at human adenosine A2B receptor expressed in Flp-In-CHO cell membrane assessed as inhibition of NECA-induced cAMP accumulation in presence of forskolin measured after 40 mins by alpha screen LANCE assay
- ChEMBL_2152687 (CHEMBL5037234) Antagonist activity at human adenosine A3 receptor expressed in Flp-In-CHO cell membrane assessed as inhibition of NECA-induced cAMP accumulation in presence of forskolin measured after 40 mins by alpha screen LANCE assay
- ChEMBL_590641 (CHEMBL1053707) Agonist activity at human recombinant adenosine A3 receptor expressed in CHO cells assessed as inhibition of forskolin-stimulated cAMP production treated 45 mins before forskolin challenge measured after 15 mins by radioimmunoassay relative to NECA
- ChEMBL_1671317 (CHEMBL4021346) Antagonist activity at human adenosine A2B receptor expressed in mouse NIH/3T3 cells assessed as inhibition of NECA-induced IL-6 release incubated for 15 mins followed by agonist treatment for 15 mins by HTRF assay
- ChEMBL_2046310 (CHEMBL4701009) Antagonist activity at adenosine 2A receptor in human whole blood assessed as reduction in phosphorylated CREB level preincubated for 20 mins followed by adenosine receptor agonist NECA addition and measured after 30 mins by flow cytometric analysis
- ChEMBL_2234772 (CHEMBL5148544) Antagonist activity at A2AAR in human T -cells assessed as increase in IL-2 production preincubated for 1 hr followed by CD3/CD28 monoclonal antibody addition and measured after 24 hrs in presence of NECA by ELISA
- A2A Adenosine Receptor Binding Assay Aliquots of cell membranes (30 ug) were incubated at 25 °C for 90 min in 500 uL of binding buffer (50 mM Tris-HCl, 2 mm MgCl2, 2 units/mL ADA, pH 7.4) in the presence of 30 nM of [3H]NECA and six different concentrations of the tested compounds. Non-specific binding was determined in the presence of 100 uM R-PIA. The dissociation constant (Kd) of [3H]NECA in human A2A AR CHO cell membranes was 30 nM.
- ChEMBL_1654448 (CHEMBL4003814) Antagonist activity at human adenosine A2B receptor expressed in HEK293 cell membranes assessed as decrease in NECA/forskolin-induced cAMP level pretreated for 15 mins followed by agonist addition for 15 mins and subsequent forskolin stimulation measured after 30 mins
- ChEMBL_2046327 (CHEMBL4701026) Antagonist activity at human adenosine 2A receptor expressed in HEK293 cells assessed as reduction in cAMP production preincubated for 10 mins followed by 3 nM A2A agonist NECA addition and measured after 30 mins by EU-cAMP tracer based TR-FRET assay
- ChEMBL_2046328 (CHEMBL4701027) Antagonist activity at human adenosine 2A receptor expressed in HEK293 cells assessed as reduction in cAMP production preincubated for 10 mins followed by 5 nM A2A agonist NECA addition and measured after 30 mins by EU-cAMP tracer based TR-FRET assay
- ChEMBL_2152682 (CHEMBL5037229) Antagonist activity at human adenosine A1 receptor expressed in Flp-In-CHO cell membrane assessed as inhibition of forskolin-stimulated cAMP accumulation preincubated for 10 min followed by forskolin stimulation and further incubated for 30 mins in presence of NECA by alphascreen LANCE assay
- Radioligand Binding Assay The radioligands used were 1 nM (2R,3R,4S,5R)-2-(2-chloro-6-cyclopentylamino-purin-9-yl)-5-hydroxymethyl-tetrahydro-3,4-diol ([3H]CCPA) for hA1, 10 nM (1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuronamide) ([3H]NECA) for hA2a, and 1 nM 2-(1-hexynyl)-N6-methyladenosine [3H] ([3H]HEMADO) for hA3 receptors.
- A3 Adenosine Receptor Binding Assay Aliquots of cell membranes (90 ug) were incubated at 25 °C for 180 min in 500 uL of binding buffer (50 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, 2 units/mL ADA, pH 7.4) in the presence of 30 nm [3H]NECA and six different concentrations of the tested compounds. Non-specific binding was determined in the presence of 100 uM R-PIA.
- Inhibitory Activity on A2A and A2B Receptors Assay The screening procedure was as follows:1. adjusting the cell density to 6×105 cells/mL with serum-free medium;2. adding 5 mL of cytosol, 2.5 mL of NECA (Sigma, 119140-10MG), and 2.5 mL of compound solution sequentially to each well of a 384-well plate (Greiner Bio-One, 784075), in which the final concentration of NECA is 1 μM (CHO-K1/ADORA2A) or 0.1 μM (CHO-K1/ADORA2B), and the final concentration of the compound starts from 3 μM to triple dilutions less than 3 μM;3. placing the 384-well plate in 37° C. incubator for 30 min;4. adding 5 μL of cAMP-d2 and 5 μL of cAMP-ab (Cisbio, 62AM4PEB) sequentially;5. leaving the 384-well plate at room temperature for 1 h, protected from light; and6. reading the plate (Victor X5, PerkinElmer) and analyzing the data by XLfit nonlinear regression to calculate the IC50 of the compound.
- Radioligand Binding Assay Dissociation constants at A1, A2A and A3 receptors (Ki-values) were determined in radioligand competition experiments as reported earlier [19,20]. All binding experiments were done in a microplate format utilizing a 96-well microplate filtration system (Millipore Multiscreen MAFC). As radioligands the agonists [3H]CCPA (1 nM) for A1, [3H]NECA (10 nM) for A2A, and [3H]HEMADO (1 nM) for A3 receptors were used. Samples were incubated with 10 µg of membrane protein for 3 h at 25 °C, filtered through the built-in filter at the bottom of the wells and washed three times with 200 ml of ice-cold binding buffer. After addition of 20 µl of scintillator to the dried filter plates samples were counted in a Wallac Micro-Beta counter. Nonspecific binding was determined in the presence of 1 mM theophylline ([3H]CCPA) or 100 mM R-PIA (N6-phenylisopropyladenosine; [3H]NECA and [3H] HEMADO).
- Assay for Binding Affinity for Adenosine Receptor A2 HEK-293 cell membrane homogenates (40 μg protein), in which A2a adenosine receptors were expressed, are incubated for 120 min at 22° C. with 6 nM [3H]CGS 21680 in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 10 μM NECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is NECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
- Radiological Binding Assay For A1 adenosine receptors, 40 mg of protein were incubated for 60 min at 25°C with [3H]DPCPX 0.5 nM (Kd = 0.4 nM) and increasing concentrations of the compounds, in a final volume of 0.4 mL of Tris-HCl buffer. Binding reaction was terminated by dilution with ice-cold 50 mM Tris-HCl buffer, pH 7.4.For A2A and A3 adenosine receptors, 40 mg of protein were incubated with [3H]ZM241385 6 nM (Kd = 4 nM) in the experiments involving A2A and with [3H]NECA 15 nM (Kd = 150 nM) involving A3 and the compounds to be assayed, at fixed concentration (10 µM) or at increasing concentrations of the compounds in duplicate, in a final volume of 0.4 mL of Tris-HCl buffer for 120 min at 25°C. Non-specific binding was measured in the presence of 100 µM NECA in the case of A2A and 100 µM R-PIA in the case of A3 binding assay. Bound radioactivity of all samples was measured in a liquid scintillation counter (1600 TR Packard) after the addition of 4 mL of scintillation liquid (Emulsifier-Safe; Hewlett-Packa
- Evaluation of A2a Receptor Binding Affinity A radioligand binding test was conducted by using [3H]-NECA (5′-N-[adenine-2,8-3H]-ethylcarboxamidoadenosine) and an adenosine A2a membrane. As the adenosine A2a membrane, a cell membrane prepared from HEK-293 cells transfected with human adenosine A2a receptor was used. The membrane used for the test was prepared by incubating with a radioligand until equilibrium was reached. In order to separate the radioligand-bound membrane, the unbound radioligand was separated by using Packard filtermate Harverster and glass filter plates.10 μL of test compound dissolved in binding buffer (50 mM Tris, 10 mM MgCl2, 1 mM EDTA pH 7.4) and 20 μL of [3H]-NECA (final concentration of 37 nM) or reference inhibitor was mixed with 20 μL of A2a membrane in an unbound 96-well plate and incubated at room temperature for one hour. Prior to filtration, a 96-well harvest filter plate was coated with 0.33% polyethylenimine for 30 minutes and then washed with assay buffer. The binding reaction was transferred to the filter plate and washed three times with wash buffer. The dish was then dried, scintillant was added, and radioactivity was counted in a scintillation counter (Topcount NXT, Packard).
- Competition Radioligand Binding Assay A2A Adenosine Receptor: Competitive assays were carried out by incubation of membranes from human A2A receptors transfected to HeLa cells, [3H]-ZM241385, buffer (Tris-HCl 50 mM (pH=7.4), 10 mM MgCl2, EDTA 1 mM and 2 units/ml adenosine deaminase), and unlabelled ligand in a total volume of 0.2 ml for 60 min at 25° C. NECA was used to determine nonspecific binding. It was filtered over Schleicher & Schuell GF/52 (presoaked with 0.5% polyethyleneimine) in a Brandel cell harvester. The unbound radioligand was removed with 3×250 μl of 20 mM Tris-HCl 50 mM (pH=7.4), 10 mM MgCl2, EDTA 1 mM.
- Competition Radioligand Binding Assay A3 Adenosine Receptor:Competitive assays were carried out by incubation of membranes from human A3 receptors transfected to CHO cells, [3H]-NECA, buffer (20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 2 units/ml adenosine deaminase), and unlabelled ligand in a total volume of 0.2 ml for 60 min at 25° C. R-PIA was used to determine nonspecific binding. It was filtered over Schleicher & Schuell GF/52 (presoaked with 0.5% polyethyleneimine) in a Brandel cell harvester. The unbound radioligand was removed with 3×250 μl of 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2.
- cAMP Production Assay hese assays were performed at adenosine receptors transfected using a cAMP enzymeimmunoassay kit (Amersham Biosciences). CHO-A2A cells were seeded (10000 cells/well) in 96-well culture plates and incubated at 37 C. in an atmosphere with 5% CO2 in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 (DMEM F-12), containing 10% Foetal Calf Serum (FCS) and 1% L-Glutamine. Cells were washed 3x with 200 ul assay medium (DMEM-F12 and 25 mM HEPES pH=7.4) and pre-incubated with assay medium containing 30 uM rolipram and test compounds at 37 C. for 15 min. 1 uM NECA was incubated for 15 min at 37 C. (total incubation time 30 min). Reaction was stopped with lysis buffer supplied in the kit and the enzymeimmunoassay was carried out for detection of intracellular cAMP at 450 nm in an Ultra Evolution detector (Tecan).
- Competition Radioligand Binding Assay Competition assays were carried out by incubation of membranes from hA1 receptors transfected to CHO cells, [3H]-DPCPX as radioligand, buffer (HEPES 20 mM (pH=7.4), 10 mM MgCl2, 100 mM NaCl, 2 units/ml adenosine deaminase), and unlabelled ligand in a total volume of 0.2 ml for 60 min at 25 C. R-PIA was used to determinate non-specific binding. Filter over Schleicher&Schuell GF/52 filters (pre-soaked 0.5% polyethylenimine) in a Brandel cell harvester. Unbound radioligand was removed with (3x 250 ul) HEPES 20 mM (pH=7.4), 100 mM NaCl and 10 mM MgCl2. Competition assays were carried out by incubation of membranes from hA2a receptors transfected to HeLa cells, [3H]ZM241385 as radioligand, buffer (50 mM Tris-HCl (pH=7.4), 10 mM MgCl2, 1 mM EDTA, 2 units/ml adenosine deaminase), and unlabelled ligand in a total volume of 0.2 ml for 30 min at 25 C. NECA was used to determinate non-specific binding.
- Radioligand Binding Assay Radioligand binding assays at A1, A2A, and A3ARs were performed according to the procedures described previously. Each tube in the binding assay contained 100 uL of membrane suspension (20 ug of protein), 50 pt of a stock solution of agonist radioligand, and 50 uL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2. Nonspecific binding was determined using a final concentration of 10 uM NECA, a non-specific agonist, diluted with the buffer.The mixtures were incubated at 25° C. for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under a reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburg, Md.). Filters were washed three times with 5 ml of 50 mM ice-cold Tris-HCl buffer (pH 7.5). The radioactive agonists [3H]R-PIA and [3H]CGS21680 were used for the A1 and A2AAR assays, respectively, while [125I]AB-MECA was used for the A3AR assay.
- Radioligand Binding Assays For the A2a receptor binding study, the incubation contained 10 mg of the original tissue weight of the striatal membranes, 4 nM [3H]NECA, 50 nM CPA, 10 mM MgCl2, 0.2 units/mL adenosine deaminase, the test compound and 1% DMSO. For the A1 receptor binding study, the incubations contained 5 mg of the original tissue weight of the whole brain membranes, 0.1 nM [3H]DPCPX, 0.2 units/mL adenosine deaminase, the test compound and 1% DMSO. The incubations were vortexed and incubated for 60 min at 25 °C in a shaking waterbath. Half an hour after incubation was started, the incubations were vortexed again. The incubations were terminated via filtration. The tubes were washed twice with 4 mL ice-cold Tris buffer and the filters were washed once more with 4mL ice-cold Tris buffer. The damp filters were place in scintillation vials and 4 mL of scintillation fluid was added. The vials were shaken and incubated for two hours before being counted.
- Adenosine Receptor Binding Assay Binding affinity of test compounds to four human adenosine receptors, A1, A2A, A2B and A3 was determined in radioligand competitive binding assay (conducted by Cerep, France) using following protocols. For A1 receptor (A1R), membrane homogenates from CHO cells transfected with A1R were incubated for 60 min at 22° C. with 1 nM [3H]DPCPX in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA/Tris and 2 UI/mL ADA. For A2AR, membrane homogenates from HEK293 cells transfected with A2AR were incubated for 120 min at 22° C. with 6 nM [3H]CGS21680 in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 2 UI/mL ADA. For A2B receptor (A2BR), membrane homogenates from HEK293 cells transfected with A2BR were incubated for 60 min at 22° C. with 5 nM [3H]CPX in the absence or presence of the test compound in a buffer containing 10 mM Hepes/Tris (pH 7), 1 mM MgCl2, and 1 mM EDTA. For A3 receptor (A3R), membrane homogenates from HEK293 cells transfected with A3R were incubated for 120 min at 22° C. with 0.15 nM [125I] AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/mL ADA. Nonspecific binding was determined in the presence of unlabeled 1 μM DPCPX, 10 μM NECA, 100 μM NECA, and 1 μM IB-MECA in A1R, A2AR, A2BR, A3R binding assays, respectively. Following incubation, the samples are rapidly filtered and washed with ice-cold 50 mM Tris-HCl. Then filters are dried and counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). Duplicate experiments were performed for each assay. The results are expressed as a percent inhibition of control radioligand specific binding.Mouse BBB Assay
- Adenosine A2B Receptor Cyclic AMP GS Assay Stably transfected HEK-293 cells expressing the human adenosine A2B receptor (Perkin Elmer) were maintained in MEM culture medium with 10% FBS and 100 μg/mL Geneticin (Life Technologies). 18 to 24 hours prior to assay, geneticin was removed from culture. The cisbio cAMP-GS Dynamic kit utilizing the FRET (Fluorescence Resonance Energy Transfer) technology was used to measure cAMP accumulation in the cells. Compounds of the present disclosure at an appropriate concentration were mixed with 10000 cells/well in white 96 well half area plates (Perkin Elmer) for 30 min at room temperature gently shaking. Agonist, NECA (R&D Technologies) at 12 nM was added to each well for 60 min at room temperature gently shaking. Detection reagents, d2-labeled cAMP (acceptor) and anti-cAMP cryptate (donor) were added to each well for 60 min at RT gently shaking. Plates were read on Pherastar (BMG Labtech), fluorescence ratio 665/620 was calculated and EC50 determination was performed by fitting the curve of percent of control versus the log of the compound concentration using GraphPad Prism.
- Radioligand Binding Assay In all radioligand binding experiments, the samples were incubated in a final volume of 0.1 ml of binding buffer for 60 minutes at 25° C. and then filtered over Unifilter plates (Perkin Elmer) pre-treated for 2 hours to limit tracer non-specific binding. Filters were washed five times with 0.5 ml of ice-cold washing buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl, 1 mM EDTA), and 50 μL of Microscint 20 (Perkin Elmer) were added to each filter. The plates were incubated 15 min at room temperature on an orbital shaker and then counted with a TopCount™ for 1 min/well.For A1 receptor, the assay was performed with [3H]-DPCPX and membranes from CHO-Kl cells transfected with human A1 receptor (Euroscreen FAST-001B).For A2A receptor, the assay was performed with [3H-NECA and membranes from HEK293 cells transfected with human A2A receptor (Euroscreen FAST-002B).For A2B receptor, the assay was carried with [3H]-DPCPX and membranes prepared from HEK293 cells transfected with human A2B receptor (Euroscreen FAST-003B).For A3 receptor, the assay was carried out with [125IJ-MECA and membranes from CHO-Kl cells transfected with human A3 receptor (Euroscreen FAST-004B).
- Human Adenosine A2A Receptor Binding Assay Test compound is weighed, dissolved in DMSO to make a stock solution of 10 mM, diluted with DMSO to prepare working solutions, then 100-fold diluted to the indicated concentrations. Human recombinant adenosine A2A receptors expressed in HEK-293 cells were used to prepare membranes in incubation buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 2 U/ml Adenosine Deaminase). Test compound or vehicle (2.2 μL) was added in 200 μL of membranes and incubated with 20 μL of 50 nM [3H] CGS-21680 for 90 minutes at 25 C. Non-specific binding was estimated in the presence of 50 μM NECA (5′-N-ethylcarboxamidoadenosine). The incubation was stopped by vacuum filtration onto 0.3% PEI (polyethylenimine) presoaked GF/B filters using a harvester followed by four washes with ice-cold 50 mM Tris-HCl, pH 7.4, and the radioactivity on GF/B filtermats counted in a scintillation counter (PerkinElmer TOPCOUNT ) to determine [3H] CGS-21680 specifically bound. Final concentration of vehicle DMSO is 1%.Data is fitted using the non-linear curve fitting routines in Meth-IQ software (ID Business Solutions Ltd., UK). IC50 is converted to Ki by Cheng-Prusoff equation: Ki=IC50/(1+[L]/KD) where [L] is concentration of radiolabeled ligand used in the assay.
- Binding Assays [3H]R N6-Phenylisopropyladenosine (40, [3H]R-PIA, 63 Ci/mmol), [3H](2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine) (41, [3H]CGS21680, 40.5 Ci/mmol) and [25I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (42, [125I]I-AB-MECA, 2200 Ci/mmol) were purchased from Perkin-Elmer Life and Analytical Science (Boston, Mass.). Test compounds were prepared as 5 mM stock solutions in DMSO and stored frozen. Pharmacological standards 1b (A3AR agonist), adenosine-5′-N-ethylcarboxamide (43, NECA, nonselective AR agonist) and 2-chloro-N6-cyclopentyladenosine (44, CCPA, A1AR agonist) were purchased from Tocris R&D Systems (Minneapolis, Minn.).Cell Culture and Membrane Preparation CHO cells stably expressing the recombinant hA1 and hA3ARs and HEK293 cells stably expressing the hA2AAR were cultured in Dulbecco's modified Eagle medium (DMEM) and F12 (1:1) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 μmol/mL glutamine. In addition, 800 μg/mL geneticin was added to the A2A media, while 500 μg/mL hygromycin was added to the A1 and A3 media. After harvesting, cells were homogenized and suspended in PBS. Cells were then centrifuged at 240 g for 5 min, and the pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2. The suspension was homogenized and was then ultra-centrifuged at 14,330 g for 30 min at 4° C. The resultant pellets were resuspended in Tris buffer, incubated with adenosine deaminase (3 units/mL) for 30 min at 37° C. The suspension was homogenized with an electric homogenizer for 10 sec, pipetted into 1 mL vials and then stored at −80° C. until the binding experiments. The protein concentration was measured using the BCA Protein Assay Kit from Pierce Biotechnology, Inc. (Rockford, Ill.).27Binding Assays:Into each tube in the binding assay was added 50 μL of increasing concentrations of the test ligand in Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2, 50 μL of the appropriate agonist radioligand, and finally 100 μL of membrane suspension. For the A1AR (22 μg of protein/tube) the radioligand used was [3H]40 (final concentration of 3.5 nM). For the A2AAR (20 μg/tube) the radioligand used was [3H]41 (10 nM). For the A3AR (21 μg/tube) the radioligand used was [125I]42 (0.34 nM). Nonspecific binding was determined using a final concentration of 10 μM 43 diluted with the buffer. The mixtures were incubated at 25° C. for 60 min in a shaking water bath. Binding reactions were terminated by filtration through Brandel GF/B filters under a reduced pressure using a M-24 cell harvester (Brandel, Gaithersburg, Md.). Filters were washed three times with 3 mL of 50 mM ice-cold Tris-HCl buffer (pH 7.5). Filters for A1 and A2AAR binding were placed in scintillation vials containing 5 mL of Hydrofluor scintillation buffer and counted using a Perkin Elmer Liquid Scintillation Analyzer (Tri-Carb 2810TR). Filters for A3AR binding were counted using a Packard Cobra II γ-counter. The Ki values were determined using GraphPad Prism for all assays.Similar competition binding assays were conducted using HEK293 cell membranes expressing mARs using [125I]42 to label A1 or A3ARs and [3H]41 to label A2AARs. IC50 values were converted to Ki values as described.28 Nonspecific binding was determined in the presence of 100 μM 43.