- ChEMBL_2016537 (CHEMBL4670115) Agonist activity NPY1R (unknown origin) by CRE-luciferase reporter gene assay
- ChEMBL_1281945 (CHEMBL3101343) Agonist activity at human CB2 receptor expressed in CHO CRE-luc cells
- ChEMBL_2016534 (CHEMBL4670112) Agonist activity at NPY5R (unknown origin) by CRE-luciferase reporter gene assay
- ChEMBL_2016536 (CHEMBL4670114) Agonist activity at NPY4R (unknown origin) by CRE-luciferase reporter gene assay
- ChEMBL_2326394 Agonist activity at human TGR5 transfected in HEK293T cells by CRE-driven luciferase reporter assay
- ChEMBL_1837318 (CHEMBL4337451) Agonist activity at mouse H4R expressed in HEK293T cells by CRE-luciferase reporter gene assay
- ChEMBL_595939 (CHEMBL1041788) Agonist activity at TGR5 expressed in CHO cells by CRE-driven luciferase reporter gene assay
- ChEMBL_1855646 (CHEMBL4356375) Agonist activity at human H3R expressed on HEK293T cells assessed as inhibition of forskolin-induced CRE-driven luciferase activity co-incubated with forskolin for 6 hrs and measured after 30 mins by CRE-luciferase reporter gene assay
- ChEMBL_1855652 (CHEMBL4356381) Agonist activity at human H4R expressed on HEK293T cells assessed as inhibition of forskolin-induced CRE-driven luciferase activity co-incubated with forskolin for 6 hrs and measured after 30 mins by CRE-luciferase reporter gene assay
- ChEMBL_1837305 (CHEMBL4337438) Displacement of [3H]-UR-DEBa176 from human H4R expressed in HEK293T-SF-His6-CRE-Luc cells
- ChEMBL_1837306 (CHEMBL4337439) Displacement of [3H]-UR-DEBa176 from mouse H4R expressed in HEK293T-SF-His6-CRE-Luc cells
- ChEMBL_1837307 (CHEMBL4337440) Displacement of [3H]-UR-DEBa176 from rat H4R expressed in HEK293T-SF-His6-CRE-Luc cells
- ChEMBL_1837322 (CHEMBL4337455) Agonist activity at rat H4R expressed in in HEK293T cells by CRE-luciferase reporter gene assay
- ChEMBL_1973790 (CHEMBL4606608) Displacement of UR-DEBa176 from human recombinant H4R expressed in HEK293T-SF-His6-CRE-Luc cells
- ChEMBL_1973792 (CHEMBL4606610) Displacement of UR-DEBa176 from mouse recombinant H4R expressed in HEK293T-SF-His6-CRE-Luc cells
- ChEMBL_2346088 Binding affinity to mouse MC1R expressed in HEK293 cells coexpressing CRE/beta-galactosidase assessed as inhibition constant
- ChEMBL_2346090 Binding affinity to mouse MC5R expressed in HEK293 cells coexpressing CRE/beta-galactosidase assessed as inhibition constant
- ChEMBL_539383 (CHEMBL1027267) Agonist activity at mouse MC1R expressed in HEK293 cells by CRE/beta-galactosidase reporter gene assay
- ChEMBL_539385 (CHEMBL1027269) Agonist activity at mouse MC3R expressed in HEK293 cells by CRE/beta-galactosidase reporter gene assay
- ChEMBL_539391 (CHEMBL1027275) Agonist activity at mouse MC4R expressed in HEK293 cells by CRE/beta-galactosidase reporter gene assay
- ChEMBL_539394 (CHEMBL1027278) Agonist activity at mouse MC5R expressed in HEK293 cells by CRE/beta-galactosidase reporter gene assay
- ChEMBL_1456739 (CHEMBL3371029) Agonist activity at human GPR119 expressed in CHO-K1 cells after 24 hrs by CRE-luciferase assay
- ChEMBL_1837278 (CHEMBL4337411) Binding affinity to human H4R expressed in HEK293T-SF-His6-CRE-Luc cells by saturation binding study
- ChEMBL_1837279 (CHEMBL4337412) Binding affinity to mouse H4R expressed in HEK293T-SF-His6-CRE-Luc cells by saturation binding study
- ChEMBL_1837280 (CHEMBL4337413) Binding affinity to rat H4R expressed in HEK293T-SF-His6-CRE-Luc cells by saturation binding study
- ChEMBL_1837324 (CHEMBL4337457) Binding affinity to human H4R expressed in HEK293T-SF-His6-CRE-Luc cells by kinetic binding study
- ChEMBL_1837325 (CHEMBL4337458) Binding affinity to mouse H4R expressed in HEK293T-SF-His6-CRE-Luc cells by kinetic binding study
- ChEMBL_1837326 (CHEMBL4337459) Binding affinity to rat H4R expressed in HEK293T-SF-His6-CRE-Luc cells by kinetic binding study
- ChEMBL_556999 (CHEMBL960690) Inverse agonist activity at human histamine H4 receptor expressed in HEK293T cells by CRE-beta-galactosidase assay
- ChEMBL_613512 (CHEMBL1072278) Inverse agonist activity at human histamine H4 receptor expressed in HEK293T cells by CRE-beta-galactosidase assay
- ChEMBL_1438565 (CHEMBL3386096) Activity at mouse MC1R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay
- ChEMBL_1438566 (CHEMBL3386097) Activity at mouse MC3R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay
- ChEMBL_1438567 (CHEMBL3386098) Activity at mouse MC4R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay
- ChEMBL_1438568 (CHEMBL3386099) Activity at mouse MC5R expressed in HEK293 cells assessed as cAMP accumulation by CRE/beta-galactosidase reporter gene assay
- ChEBML_1681317 Agonist activity at EYFP-fused human MC3R expressed in HEK293 cells after 16 to 20 hrs by CRE-driven reporter assay
- ChEBML_1681323 Agonist activity at EYFP-fused human MC5R expressed in HEK293 cells after 16 to 20 hrs by CRE-driven reporter assay
- ChEMBL_1334801 (CHEMBL3238908) Agonist activity at human CB2 receptor expressed in human SK-N-MC cells by CRE-beta galactosidase reporter gene assay
- ChEMBL_1334802 (CHEMBL3238909) Agonist activity at human CB1 receptor expressed in human SK-N-MC cells by CRE-beta galactosidase reporter gene assay
- ChEMBL_1350261 (CHEMBL3267244) Agonist activity at GPR119 in mouse GLUTag cells assessed as stimulation of GLP1 secretion by CRE-luciferase reporter gene assay
- ChEMBL_1365356 (CHEMBL3292680) Agonist activity at human TGR5 expressed in HEK293 cells co-expressing CRE after 5.5 hrs by luciferase reporter gene assay
- ChEMBL_1365357 (CHEMBL3292681) Agonist activity at mouse TGR5 expressed in HEK293 cells co-expressing CRE after 5.5 hrs by luciferase reporter gene assay
- ChEMBL_1475559 (CHEMBL3424620) Agonist activity at human TGR5 expressed in HEK293 cells incubated for 5.5 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_1475560 (CHEMBL3424621) Agonist activity at mouse TGR5 expressed in HEK293 cells incubated for 5.5 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_1705548 (CHEMBL4056781) Agonist activity at human GPR119 expressed in CHOK1 cells harboring CRE-luciferase after 6 hrs by luciferase reporter gene assay
- ChEMBL_489015 (CHEMBL986356) Displacement of [3H]N-alpha-methylhistamine from rat histamine H3 receptor expressed in HEK293 cells coexpressed with CRE-beta-lactamase
- ChEMBL_877859 (CHEMBL2187733) Agonist activity at mouse TGR5 expressed in HEK293 cells incubated for 5.5 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_877860 (CHEMBL2187734) Agonist activity at human TGR5 expressed in HEK293 cells incubated for 5.5 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_1543581 (CHEMBL3750699) Displacement of CA200645 from human adenosine A3 receptor expressed in CHO CRE-SPAP cells incubated for 1 hr by fluorescence analysis
- ChEMBL_1681320 (CHEMBL4031597) Agonist activity at EYFP-fused human MC4R expressed in HEK293 cells after 16 to 20 hrs by CRE-driven reporter assay
- ChEMBL_1747052 (CHEMBL4181562) Agonist activity at human GPR119 expressed in CHOK1 cells co-expressing CRE-luciferase after 6 hrs by luciferase reporter gene assay
- ChEMBL_2203902 (CHEMBL5116610) Potentiation of CFTR F508del mutant (unknown origin) expressed in human CFBE41o- cells by CRE- horseradish peroxidase-coupled high throughput screening method
- ChEMBL_538609 (CHEMBL1027399) Agonist activity at human MC1R expressed in HEK293 cells assessed as effect on CRE-driven luminescence by luciferase reporter gene assay
- ChEMBL_538610 (CHEMBL1027400) Agonist activity at human MC3R expressed in HEK293 cells assessed as effect on CRE-driven luminescence by luciferase reporter gene assay
- ChEMBL_538611 (CHEMBL1027401) Agonist activity at human MC4R expressed in HEK293 cells assessed as effect on CRE-driven luminescence by luciferase reporter gene assay
- ChEMBL_697517 (CHEMBL1638849) Agonist activity at human histamine H3 receptor expressed in human SK-N-MC cells by CRE-beta galactosidase reporter gene assay
- ChEMBL_697519 (CHEMBL1638851) Agonist activity at human histamine H4 receptor expressed in human SK-N-MC cells by CRE-beta galactosidase reporter gene assay
- ChEMBL_935595 (CHEMBL2320377) Agonist activity at human GPR119 expressed in HEK293 cells co-expressing CRE beta-lactamase assessed as cAMP accumulation by HTRF assay
- ChEMBL_987703 (CHEMBL2439681) Agonist activity at mouse TGR5 expressed in HEK293 cells assessed CRE-induced luciferase activity after 5.5 hrs by reporter gene assay
- ChEMBL_987708 (CHEMBL2439686) Agonist activity at human TGR5 expressed in HEK293 cells assessed CRE-induced luciferase activity after 5.5 hrs by reporter gene assay
- ChEMBL_1705247 (CHEMBL4056480) Agonist activity at human GPR119 expressed in CHOK1 cells co-expressing CRE-luciferase incubated for 4 hrs by luciferase reporter gene assay
- ChEMBL_1837281 (CHEMBL4337414) Binding affinity to human H4R expressed in HEK293T-SF-His6-CRE-Luc cells assessed as equilibrium dissociation constant by saturation binding study
- ChEMBL_1837282 (CHEMBL4337415) Binding affinity to mouse H4R expressed in HEK293T-SF-His6-CRE-Luc cells assessed as equilibrium dissociation constant by saturation binding study
- ChEMBL_1837283 (CHEMBL4337416) Binding affinity to rat H4R expressed in HEK293T-SF-His6-CRE-Luc cells assessed as equilibrium dissociation constant by saturation binding study
- ChEMBL_2296165 Antagonist activity at mGluR7 (unknown origin) assessed as inhibition of L-AP4 stimulated decrease in forskolin induced cAMP response by CRE-Luc assay
- ChEMBL_341532 (CHEMBL861820) Activity against human H3 receptor as measured by CRE-mediated beta galactosidase reporter gene assay in forskolin-stimulated SK-N-MC cells
- ChEMBL_592751 (CHEMBL1043242) Agonist activity at human TGR5 expressed in CHO cells assessed as increase in CRE-driven gene expression by luciferase reporter gene assay
- ChEMBL_933674 (CHEMBL2320535) Agonist activity at human GPR119 in HEK293 cells assessed as cAMP accumulation after 30 mins by CRE-beta-lactamase reporter gene assay
- ChEMBL_1648545 (CHEMBL3997601) Partial agonist activity at human beta2-AR expressed in CHOK1 cells assessed as inhibition of cimaterol-induced CRE-SPAP production after 5 hrs
- ChEMBL_1766320 (CHEMBL4201567) Agonist activity at human V2 receptor expressed in CHO cells co-expressing CRE-luciferase incubated for 5 hrs by luciferase reporter gene assay
- ChEMBL_1837265 (CHEMBL4337398) Agonist activity at human H4R expressed in HEK293-SF-hH4R-His6-CRE-Luc cells incubated for 5 hrs by luciferase reporter gene assay
- ChEMBL_1837269 (CHEMBL4337402) Agonist activity at mouse H4R expressed in HEK293-SF-mH4R-His6-CRE-Luc cells incubated for 5 hrs by luciferase reporter gene assay
- ChEMBL_1837273 (CHEMBL4337406) Agonist activity at rat H4R expressed in HEK293-SF-rH4R-His6-CRE-Luc cells incubated for 5 hrs by luciferase reporter gene assay
- ChEMBL_1837284 (CHEMBL4337417) Binding affinity to human H4R expressed in HEK293T-SF-His6-CRE-Luc cells assessed as kinetically derived dissociation constant by saturation binding study
- ChEMBL_1837285 (CHEMBL4337418) Binding affinity to mouse H4R expressed in HEK293T-SF-His6-CRE-Luc cells assessed as kinetically derived dissociation constant by saturation binding study
- ChEMBL_1837286 (CHEMBL4337419) Binding affinity to rat H4R expressed in HEK293T-SF-His6-CRE-Luc cells assessed as kinetically derived dissociation constant by saturation binding study
- ChEMBL_1870429 (CHEMBL4371596) Agonist activity at histamine H4 receptor (unknown origin) expressed in HEK293T cells harboring CRE-luciferase after 6 hrs by luciferase reporter gene assay
- ChEMBL_1973730 (CHEMBL4606548) Agonist activity at human H4R expressed in HEK293-SF-hH4R-His6-CRE-Luc cells incubated for 5 hrs by luciferase reporter gene assay
- ChEMBL_1973742 (CHEMBL4606560) Agonist activity at human H3R expressed in HEK293T-SP-FLAG-hH3R-CRE-CBR cells incubated for 5 hrs by luciferase reporter gene assay
- ChEMBL_1973745 (CHEMBL4606563) Agonist activity at mouse H4R expressed in HEK293-SF-mH4R-His6-CRE-Luc cells incubated for 5 hrs by luciferase reporter gene assay
- ChEMBL_505700 (CHEMBL944462) Activity at human PTH/PTH-related peptide receptor expressed in HEK293 cells assessed as induction of luciferase activity by CRE-luciferase reporter assay
- ChEMBL_1935709 (CHEMBL4481468) Agonist activity at mouse MC3R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay
- ChEMBL_1935710 (CHEMBL4481469) Agonist activity at mouse MC4R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay
- ChEMBL_2295723 Allosteric antagonist activity at human FSHR expressed in CHO cells assessed as inhibition of FSH-induced activity incubated for 4 hrs by CRE-luciferase assay
- ChEMBL_407554 (CHEMBL907109) Agonist activity at human histamine H4 receptor in SK-N-MC cells assessed as inhibition of forskolin-induced cAMP-mediated CRE-beta galactosidase activity
- ChEMBL_407556 (CHEMBL907116) Agonist activity at human histamine H3 receptor in SK-N-MC cells assessed as inhibition of forskolin-induced cAMP-mediated CRE-beta galactosidase activity
- ChEMBL_539389 (CHEMBL1027273) Antagonist activity at mouse MC3R expressed in HEK293 cells assessed as effect on MT2 peptide-induced response by CRE/beta-galactosidase reporter gene assay
- ChEMBL_539392 (CHEMBL1027276) Antagonist activity at mouse MC4R expressed in HEK293 cells assessed as effect on MT2 peptide-induced response by CRE/beta-galactosidase reporter gene assay
- ChEMBL_560999 (CHEMBL1015239) Agonist activity at human vasopressin V2 receptor expressed in HEK293 cells assessed as increase in intracellular cAMP level by CRE-luciferase reporter gene assay
- ChEMBL_561010 (CHEMBL1015250) Agonist activity at rat vasopressin V2 receptor expressed in HEK293 cells assessed as increase in intracellular cAMP level by CRE-luciferase reporter gene assay
- ChEMBL_593382 (CHEMBL1040510) Agonist activity at TGR5 expressed in human U2-OS cells assessed as increase in MRE/CRE-driven gene expression by luciferase reporter gene assay
- ChEMBL_702109 (CHEMBL1655339) Agonist activity at human LH receptor expressed in CHO-K1 cells assessed as stimulation of luciferase activity by CRE-driven luciferase reporter gene assay
- ChEMBL_702111 (CHEMBL1655341) Agonist activity at human FSHR receptor expressed in CHO-K1 cells assessed as stimulation of luciferase activity by CRE-driven luciferase reporter gene assay
- ChEMBL_770403 (CHEMBL1833712) Antagonist activity at human beta-3 adrenergic receptor expressed in fenoterol-stimulated CHOK1 cells assessed as CRE-SPAP level by fluorescence correlation spectroscopic analysis
- ChEMBL_816251 (CHEMBL2026776) Antagonist activity at human TRPV1 expressed in CHOluc9aeq cells assessed as inhibition of capsaicin-stimulated response by aequorin and CRE-lucifearase reporter gene assay
- ChEMBL_1935714 (CHEMBL4481473) Full agonist activity at mouse MC1R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay
- ChEMBL_1935715 (CHEMBL4481474) Full agonist activity at mouse MC5R expressed in HEK293 cells co-expressing CRE/beta-galactosidase reporter gene after 6 hrs by beta-galactosidase cAMP assay
- ChEMBL_748631 (CHEMBL1780484) Antagonist activity at rat TRPV1 expressed in CHO cells co-expressing aequorin and CRE-luciferase reporter gene assessed as inhibition of capsaicin-induced calcium flux
- ChEMBL_748632 (CHEMBL1780485) Antagonist activity at human TRPV1 expressed in CHO cells co-expressing aequorin and CRE-luciferase reporter gene assessed as inhibition of capsaicin-induced calcium flux
- ChEMBL_770402 (CHEMBL1833711) Antagonist activity at human beta-2 adrenergic receptor expressed in salbutamol-stimulated CHO-K1 cells assessed as CRE-SPAP level by fluorescence correlation spectroscopic analysis
- ChEMBL_1365585 (CHEMBL3297366) Agonist activity at mouse melanocortin-1 receptor expressed in HEK293 cells co-transfected with CRE/beta-galactosidase reporter gene assessed as stimulatory response after 6 hrs
- ChEMBL_1365586 (CHEMBL3297367) Agonist activity at mouse melanocortin-3 receptor expressed in HEK293 cells co-transfected with CRE/beta-galactosidase reporter gene assessed as stimulatory response after 6 hrs
- ChEMBL_1365587 (CHEMBL3297368) Agonist activity at mouse melanocortin-4 receptor expressed in HEK293 cells co-transfected with CRE/beta-galactosidase reporter gene assessed as stimulatory response after 6 hrs
- ChEMBL_1365588 (CHEMBL3297369) Agonist activity at mouse melanocortin-5 receptor expressed in HEK293 cells co-transfected with CRE/beta-galactosidase reporter gene assessed as stimulatory response after 6 hrs
- ChEMBL_1518307 (CHEMBL3620282) Agonist activity at human GLP1 receptor expressed in BHK cells after 3 hrs by CRE firefly luciferase reporter gene assay in absence of human serum albumin
- ChEMBL_1574207 (CHEMBL3802212) Antagonist activity at human GHS-R1a transfected in mouse LTK cells after 6 hrs by CRE/luciferase reporter gene assay in presence of ghrelin and rolipram
- ChEMBL_1768069 (CHEMBL4220181) Agonist activity at human GLP1 receptor expressed in HEK293 cells assessed as increase in cAMP level after 5 hrs by CRE driven luciferase reporter gene assay
- ChEMBL_1768070 (CHEMBL4220182) Agonist activity at human GCG receptor expressed in HEK293 cells assessed as increase in cAMP level after 5 hrs by CRE driven luciferase reporter gene assay
- ChEMBL_1768071 (CHEMBL4220183) Agonist activity at human GIP receptor expressed in HEK293 cells assessed as increase in cAMP level after 5 hrs by CRE driven luciferase reporter gene assay
- ChEMBL_2106809 (CHEMBL4815484) Displacement of [3H]N-methylspiperone from human D2L receptor expressed in HEK293T cells co-expressing CRE-Luc incubated for 60 mins by microbeta scintillation counting method
- ChEMBL_2106810 (CHEMBL4815485) Displacement of [3H]N-methylspiperone from human D3 receptor expressed in HEK293T cells co-expressing CRE-Luc incubated for 60 mins by microbeta scintillation counting method
- ChEMBL_2171635 (CHEMBL5056769) Activation of human GCCR expressed in human HEK293 cells assessed as luminescence signal incubated for 16 hrs at 37C measured by CRE-driven luciferase reporter assay
- ChEMBL_785118 (CHEMBL1920856) Antagonist activity at full length human H4R expressed in HEK293 cells assessed as reversal of forskolin-induced cAMP production by CRE-beta-lactamase reporter gene assay
- ChEMBL_798257 (CHEMBL1943644) Antagonist activity at human histamine H4 receptor expressed in HEK293 cells assessed as rev inhibition of forskolin-stimulated cAMP accumulation by CRE-betalactamase reporter gene assay
- ChEMBL_2203904 (CHEMBL5116612) Potentiation of CFTR F508del mutant (unknown origin) expressed in human CFBE41o- cells in presence of CFTR corrector C1 by CRE- horseradish peroxidase-coupled high throughput screening method
- ChEMBL_684759 (CHEMBL1285786) Agonist activity at recombinant PTHR1 expressed in HEK293 cells co-transfected with CRE-Luc assessed as increase of adenylyl cyclase activity after 4.5 hrs by luciferase assay
- ChEMBL_770400 (CHEMBL1833709) Antagonist activity at human beta-1 adrenergic receptor site 1 expressed in cimeterol-stimulated CHO-K1 cells assessed as CRE-SPAP level by fluorescence correlation spectroscopic analysis
- ChEMBL_1365574 (CHEMBL3297283) Agonist activity at wild type human melanocortin-4 receptor expressed in HEK293 cells co-transfected with CRE/beta-galactosidase reporter gene assessed as stimulatory response after 6 hrs
- ChEMBL_1499283 (CHEMBL3583165) Antagonist activity at human GSHR1a expressed in mouse LTK cells assessed as inhibition of ghrelin-induced reporter gene expression after 6 hrs by CRE/luciferase reporter gene assay
- ChEMBL_2016538 (CHEMBL4670116) Agonist activity at human NPY2R expressed in HEK293 cells co-transfected with CRE-luciferase incubated for 24 hrs in presence of 10% FBS by One-Glo luciferase assay
- ChEMBL_2091198 (CHEMBL4772461) Agonist activity at mouse TGR5 expressed in HEK293 cells assessed as CRE-driven luciferase reporter incubated for 5.5 hrs and measured by Steady Glow reagent based luminescence assay
- ChEMBL_2115840 (CHEMBL4824781) Activation of GLP1R (unknown origin) CRE-bla expressed in CHO-K1 cells assessed as receptor potency incubated for 1 hrs by time resolved fluorescence resonance energy transfer immunoassay
- ChEMBL_2272412 Agonist activity at human TGR5 expressed in HEK293 cells co-transfected with CRE-Luc reporter plasmid assessed as receptor transactivation incubated for 5.5 hrs by luciferase reporter gene assay
- ChEMBL_608993 (CHEMBL1073135) Agonist activity at human TGR5 receptor expressed in human U2-OS cells assessed as changes in response to cAMP level by MRE/CRE-driven luciferase reporter gene assay
- ChEMBL_770401 (CHEMBL1833710) Antagonist activity at human beta-1 adrenergic receptor site 1 expressed in CGP 12177-stimulated CHO-K1 cells assessed as CRE-SPAP level by fluorescence correlation spectroscopic analysis
- ChEMBL_1775527 (CHEMBL4232519) Agonist activity at human GABA-B1/B2 receptor expressed in HEK293/Cre-luc cells assessed as inhibition of forskolin-stimulated cAMP accumulation after 3 hrs by luciferase reporter gene assay
- ChEMBL_1932395 (CHEMBL4478047) Agonist activity at human NPY Y4 receptor expressed in HEK293 cells co-expressing CRE-Luc gene assessed as inhibition of forskolin stimulated luciferase activity after 4.5 hrs by luminescence assay
- ChEMBL_2091197 (CHEMBL4772460) Agonist activity at human TGR5 expressed in HEK293 cells assessed as CRE-driven luciferase reporter gene activity incubated for 5.5 hrs and measured by Steady Glow reagent based luminescence assay
- ChEMBL_2329026 Agonist activity at human TGR5 expressed in human HEK293 cells co-expressing CRE-driven luciferase reporter gene assessed as luciferase activity incubated for 5.5 hrs by Steady-Glo based luciferase assay
- ChEMBL_2329027 Agonist activity at mouse TGR5 expressed in human HEK293 cells co-expressing CRE-driven luciferase reporter gene assessed as luciferase activity incubated for 5.5 hrs by Steady-Glo based luciferase assay
- ChEMBL_1879060 (CHEMBL4380454) Inhibition of human EP4 transfected in human HEK293 cells co transfected with CRE-luciferase assessed as reduction in PGE2-induced luciferase expression incubated for 24 hrs by luciferase reporter gene Assay
- ChEMBL_2080122 (CHEMBL4735913) Antagonist activity at human histamine H3 receptor expressed in HEK293 cells preincubated for 5 mins followed by forskolin-stimulation and measured after 5 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_2080125 (CHEMBL4735916) Antagonist activity at human histamine H1 receptor expressed in HEK293 cells preincubated for 5 mins followed by forskolin-stimulation and measured after 5 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_2080126 (CHEMBL4735917) Antagonist activity at human histamine H2 receptor expressed in HEK293 cells preincubated for 5 mins followed by forskolin-stimulation and measured after 5 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_2080127 (CHEMBL4735918) Antagonist activity at human histamine H4 receptor expressed in HEK293 cells preincubated for 5 mins followed by forskolin-stimulation and measured after 5 hrs by CRE-driven luciferase reporter gene assay
- 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_1442243 (CHEMBL3376492) Agonist activity at GP-BAR1 (unknown origin) in human HEK293 cells assessed as receptor transactivation at 100 nM to 50 uM incubated for 16 hrs by CRE-driven luciferase reporter gene assay
- ChEMBL_1973743 (CHEMBL4606561) Inverse agonist activity at human H3R expressed in HEK293T-SP-FLAG-hH3R-CRE-CBR cells assessed as reduction in histamine-induced inhibition of forskolin stimulated luciferase activity by luciferase reporter gene assay
- ChEMBL_1973746 (CHEMBL4606564) Inverse agonist activity at human H4R expressed in HEK293-SF-hH4R-His6-CRE-Luc cells assessed as reduction in histamine-induced inhibition of forskolin stimulated luciferase activity by luciferase reporter gene assay
- ChEMBL_1973750 (CHEMBL4606568) Inverse agonist activity at mouse H4R expressed in HEK293-SF-mH4R-His6-CRE-Luc cells assessed as reduction in histamine-induced inhibition of forskolin stimulated luciferase activity by luciferase reporter gene assay
- ChEMBL_2282482 Agonist activity at human EP4 receptor expressed in CHO cells co-transfected with CRE-beta-lactamase reporter gene assessed as increase in intracellular cAMP measured after 3 hrs by TR FRET based assay
- ChEMBL_855309 (CHEMBL2161793) Agonist activity at rat GLP1R expressed in HEK293 cells assessed as stimulation of cAMP levels incubated for 6 hrs by multiple response element/cAMP response element (MRE/CRE)-driven reporter gene assay
- ChEMBL_1577407 (CHEMBL3806541) Agonist activity at human histamine H4 receptor expressed in human SK-N-MC cells assessed as inhibition of forskolin-stimulated cAMP production incubated for 6 hrs by CRE/beta-galactosidase reporter gene assay
- ChEMBL_2031278 (CHEMBL4685436) Agonist activity at human H3 receptor expressed in CHO cells assessed as increase in cAMP accumulation by measuring reduction in forskolin level incubated for 4 hrs by CRE/MRE-luciferase reporter gene assay
- ChEMBL_1929173 (CHEMBL4432349) Activation of glucagon receptor (unknown origin) transfected in HEK293 cells coinfected with luciferase reporter gene linked CRE element assessed as cAMP accumulation after 5 hrs in presence of luclite substrate by liquid scintillation counting
- ChEMBL_1855676 (CHEMBL4356405) Agonist activity at human H3R expressed on SK-N-MC cells assessed as inhibition of forskolin-induced beta galactosidase activity preincubated with forskolin for 6 hrs followed compound addition by CRE-luciferase reporter gene assay
- ChEMBL_1855660 (CHEMBL4356389) Agonist activity at mouse H3R expressed on HEK293T cells assessed as inhibition of forskolin-induced CRE-driven luciferase activity co-incubated with forskolin for 6 hrs and measured after 30 mins by luciferase reporter gene assay
- ChEMBL_1855661 (CHEMBL4356390) Agonist activity at mouse H4R expressed on HEK293T cells assessed as inhibition of forskolin-induced CRE-driven luciferase activity co-incubated with forskolin for 6 hrs and measured after 30 mins by luciferase reporter gene assay
- ChEMBL_2100242 (CHEMBL4808638) Agonist activity at GPR40 (unknown origin) expressed in CHO cells co-expressing luc2P/CRE (Gs) assessed as firefly luciferase activity at 10 uM incubated for 24 hrs by Bright-Glo based serum response element (SRE) luciferase reporter assay
- ChEMBL_2296131 Agonist activity at human mGluR7 expressed in CHO cells expressing CRE-Luc reporter gene assessed as reduction in forskolin-stimulated cAMP production preincubated for 15 mins followed by forskolin stimulation and measured after 5 hrs by luminescence based Steady glo assay
- ChEMBL_2296160 Agonist activity at mouse mGluR7 expressed in CHO cells expressing CRE-Luc reporter gene assessed as reduction in forskolin-stimulated cAMP production preincubated for 15 mins followed by forskolin stimulation and measured after 5 hrs by luminescence based Steady glo assay
- 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
- Counterscreen for S1P2 Antagonists: Dose Response Cell-Based Screen to Identify Antagonists of CRE-BLA Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center Center Affiliation: The Scripps Research Institute (TSRI) Assay Provider: Hugh Rosen, TSRI Network: Molecular Library Screening Center Network (MLSCN) Grant Proposal Number: 1 R03 MH076533-01 Grant Proposal PI: Germana Sanna External Assay ID: CRE _ANT_BLA_384_IC50_Counterscreen_ S1P2_Hits Name: Counterscreen for S1P2 Antagonists: Dose Response Cell-Based Screen to Identify Antagonists of CRE-BLA Description: Sphingosine 1-phosphate (S1P) influences heart rate [1,2], coronary artery caliber, endothelial integrity, lung epithelial integrity [3] and lymphocyte recirculation [2,4] through five related high affinity G-protein coupled receptors [5]. Subtype-selective modulators of S1P receptors will be of broad utility in understanding cell functions in vitro and vascular physiology in vivo, as well as de-convoluting the role of individual subtypes in cellular processes. The S1P receptor 2 (S1P2)
- TGR5 Activity Assay For transient transfection assays to analyze TGR5 activity, Chinese hamster ovary (CHO) cells were transfected with hTGR5 expression plasmid, cAMP-responsive element (CRE)-driven luciferase reporter plasmid (pCRE-Luc), and of pCMV-galactosidase plasmid (pCMVb) in each well using the Lipofectamine 2000 reagent (Invitrogen).
- Evaluation of Agonist Activity Against GLP-1 Receptor This experiment was intended to test the agonist activity of the compound molecules against the GLP-1 receptor and evaluate the in vitro activity of the molecules according to EC50. The experiment adopted a ONE-Glo Luciferase Assay System (Promega, E6110). Under the action of compound molecules, GLP-1R downstream signaling pathways were activated to cause elevated cAMP level. The combination of cAMP and CRE could start the transcription expression of CRE downstream luciferase genes, the luciferase could emit fluorescence when reacting with substrates thereof, and the activity of the compound for agonizing GLP-1 receptors was reflected by measuring fluorescence signals through a ONE-Glo reagent.
- Antagonist Activity Assay Antagonist activity of three test compounds against human TSI at the human TSH receptor was tested in Chinese Hamster Ovary (CHO) cells (cultured 16 h in the absence of serum prior to the assay) stably expressing the human TSH receptor and a CRE-driven firefly luciferase reporter gene. TSI was partially purified from the serum of a GD patient by filtration over a 0.45 mm filter, protein G-Sepharose column chromatography, dialysis against phosphate-buffered saline and subsequent concentrating on a 10K Amicon filter. It was confirmed that the TSI preparation does not activate luciferase activity in control CHO cells lacking the human TSHR. The cAMP phosphodiesterase inhibitor rolipram was included in the assay medium (10 μM) to augment TSHR-induced CRE-luciferase synthesis, which was quantified using a luminescence counter. The cells were incubated with compound A, B or C (0.316 nM-10 μM) together with 3.16 mg/ml TSI (or bovine TSH at a equieffective concentration of 18 nM).
- Counter screen for S1P2 Agonists: Dose Response High Throughput Cell-Based Screen to Identify Activators of CRE-BLA Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center Center Affiliation: The Scripps Research Institute (TSRI) Assay Provider: Hugh Rosen, TSRI Grant Proposal Number: 1 R03 MH076533-01 Grant Proposal PI: Germana Sanna External Assay ID: Counterscreen for S1P2 Agonists: Dose Response High Throughput Cell-Based Screen to Identify Activators of CRE-BLA Name: CRE_AG_BLA_384_EC50_%ACT Description: Sphingosine 1-phosphate (S1P) influences heart rate [1,2], coronary artery caliber, endothelial integrity, lung epithelial integrity [3] and lymphocyte recirculation [2,4] through five related high affinity G-protein coupled receptors [5]. Subtype-selective modulators of S1P receptors will be of broad utility in understanding cell functions in vitro and vascular physiology in vivo, as well as de-convoluting the role of individual subtypes in cellular processes. The S1P receptor 2 (S1P2), also known as endothelial differentiation sphingolipid G-protein-coup
- Counterscreen for S1P2 Agonists: Dose Response High Throughput Cell-Based Screen to Identify Activators of CRE-BLA: S1P2 Purchased Analogues Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center Center Affiliation: The Scripps Research Institute (TSRI) Assay Provider: Hugh Rosen, TSRI Grant Proposal Number: 1 R03 MH076533-01 Grant Proposal PI: Germana Sanna External Assay ID: CRE_AG_BLA_384_EC50_S1P2_Purchased_Analogues Name: Counterscreen for S1P2 Agonists: Dose Response High Throughput Cell-Based Screen to Identify Activators of CRE-BLA: S1P2 Purchased Analogues Description: Sphingosine 1-phosphate (S1P) influences heart rate [1,2], coronary artery caliber, endothelial integrity, lung epithelial integrity [3] and lymphocyte recirculation [2,4] through five related high affinity G-protein coupled receptors [5]. Subtype-selective modulators of S1P receptors will be of broad utility in understanding cell functions in vitro and vascular physiology in vivo, as well as de-convoluting the role of individual subtypes in cellular processes. The S1P receptor 2 (S1P2), also known as endothelial
- Cell-Based Functional Assay (M1) Muscarinic acetylcholine receptor M1 was cloned into an expression vector and transfected in CHO cells along with CRE-Luc reporter system. Isolated colonies were screened with 10,000 nM acetylcholine. Clones exhibiting highest acetylcholine dependent reporter gene induction were utilized in the evaluation of known and novel compounds. Increasing concentrations of the compound were incubated with or without 10,000 nM acetylcholine for antagonist or agonist mode, respectively, for 5 h in 96 well plate. After incubation, cells were harvested, lysed and reporter gene activity was determined.
- Cell-Based Functional Assay (5-HT4) Recombinant CHO cells were established by transfecting an expression vector encoding human 5-HT4a gene and CRE-Luc reporter system. Isolated colonies were screened with 1000 nM 5-HT for the induction of luciferase reporter gene. The clone demonstrating highest 5-HT induced reporter gene was used in the evaluationof the known and novel compounds. Increasing concentrations of the compound were incubated with or without 1000 nM 5-HT for antagonist or agonist mode, respectively, for 5 h in 96 well plate. After incubation, cells were harvested, lysed and reporter gene activity was determined.
- Cell-Based Functional Assay (5-HT7) CHO cells were transfected with an expression vector encoding rat 5-HT7 gene and CRE-Luc reporter system. Colonies were isolated and screened with 1000 nM 5-HTfor the induction of luciferase reporter gene. The clone demonstrating highest 5-HT induced reporter gene was used in the evaluation of the known and novel compounds. Increasing concentrations of the compound were incubated with or without 1000 nM 5-HT for antagonist or agonist mode respectively for 5 h in 96 well plate. After incubation, cells were harvested, lysed and reporter gene activity was measured.
- cAMP Assays To optimize functional activity directed toward Gas coupling, a HEK293/CRE-Luc cell line developed by HDB stably expressing the GLP-1 Receptor was used. 200× concentration of compound working solutions were prepared (Agilent Technologies Bravo) with 1/2 log serial dilution in 384-well Echo LDV plate (Labcyte, Cat #LP-0200). 50 nL/well 200× concentration of compound working solutions were moved to 384-well white low volume plate (Greiner, Cat #784075) using Labcyte ECHO550. 1×105 cells/mL HEK293/GLP1R/CRE-LUC(HD Biosciences) cell suspensions prepared with assay buffer[DPBS containing 0.5 mM IBMX(Sigma, Cat #15879) and 0.1% BSA(GENVIEW, Cat #FA016-100g)], 10 uL cell suspensions were added to each well of previous generated assay plate which already contains 50n1 compound at 200× concentration using ThermoFisher Multidrop Combi(1000 cells/well). Seal the plate and incubate at 37° C. with 5% CO2 for 30 min.After incubation the cAMP assay signal was generated using cAMP dynamic 2 Kit (Cisbio). 5 μL cAMP-d2 working solution was added to each well, followed with 5 μL Anti-cAMP antibody-cryptate working solution added to each well using ThermoFisher Multidrop Combi. Incubate at room temperature for 1 hour protected from light. Read the fluorescence at 665 and 615 nm with Reader PerkinElmer EnVision.% Activity=100%×(mean RLU of test sample−mean RLU of vehicle control)/(mean RLU of MAX control−mean RLU of vehicle control)).
- Cellular In Vitro Assay for Determining Vasopressin Receptor Activity The identification of agonists and antagonists of the V1a and V2 vasopressin receptors from humans, rats and dogs as well as the quantification of the activity of the compounds of the invention is carried out using recombinant cell lines. These cell lines originally derive from a hamster's ovary epithelial cell (Chinese Hamster Ovary, CHO K1, ATCC: American Type Culture Collection, Manassas, Va. 20108, USA). The test cell lines constitutively express the human, rat or dog V1a or V2 receptors. In case of the Gαq-coupled V1a receptors, cells are also stably transfected with a modified form of the calcium-sensitive photoproteins aequorin (human and rat V1a) or obe-lin (dog V1a), which, after reconstitution with the cofactor coelenterazine, emit light when there are increases in free calcium concentrations [Rizzuto R, Simpson A W, Brini M, Pozzan T, Nature 358, 325-327 (1992); Illarionov B A, Bondar V S, Illarionova V A, Vysotski E S, Gene 153 (2), 273-274 (1995)]. The resulting vasopressin receptor cells react to stimulation of the recombinantly expressed Via receptors by intracellular release of calcium ions, which can be quantified by the resulting photoprotein luminescence. The Gs-coupled V2 receptors are stably transfected into cell lines expressing the gene for firefly luciferase under control of a CRE-responsible promoter. Activation of V2 receptors induces the activation of the CRE-responsive promoter via cAMP increase, thereby inducing the expression of firefly luciferase. The light emitted by photoproteins of V1a cell lines as well as the light emitted by firefly luciferase of V2 cell lines corresponds to the activation or inhibition of the respective vasopressin receptor. The bioluminescence of the cell lines is detected using a suitable luminometer [Milligan G, Marshall F, Rees S, Trends in Pharmacological Sciences 17, 235-237 (1996)].
- Luciferase Reporter Assay HEK293-A2AR-luc2p/CRE/Hygro cells were seeded at a density of 5,000 cells/well in DMEM with 1% FBS and 1 U/mL adenosine deaminase (ADA) (Sigma). After 18 h, the cells were treated with 3 nM CGS21680 plus a series dilution of A2AR antagonist, the compounds disclosed herein at the concentration of 0.1 10000 nM, prepared in DMEM with 1% FBS. After 5 h incubation, the luciferase activity in cells were measured using the Bright-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. The luminescence signal was measured using a PHERAstar FS plate reader (BMG Labtech). Luminescence intensity from 10 μM preladenant treatment was set as 0%. Maximal luminescence intensity was determined in the presence of 3 nM CGS21680 and was set as 100%. The IC50 value was calculated from a dose dependent inhibition curve across the range of compound concentrations.
- TGR5/CRE Luciferase Assay In the following tables TGR5 activation by compounds and subsequent increase in intracellular cAMP were evaluated using a luciferase reporter gene assay. Human embryonic kidney (HEK) 293 cells were transiently co-transfected with pCMV tag4b-TGR5 h (to follow hTGR5 activation) or pCMV AC6-TGR5m (to follow mTGR5 activation) expression plasmids and the pCRE TA-Luciferase reporter plasmid using the JET PEI reagent (Polyplus transfection). Transfected cells were seeded in 96-well plates and incubated overnight with the test compounds at increasing concentrations tested in duplicate. Lithocolic acid (LCA) at 10 μM was used as a positive reference compound. The cAMP-dependent luciferase expression was followed using the BrightGlo reagent according to the manufacturer (Promega) instructions. Luminescence was read with a Mithras plate reader (Berthold). Data were expressed as percentage of the 10 μM LCA value and EC50 values were calculated using XL fit 5 software or GraphPad Prism 5. Concentration-response curves were fitted by a nonlinear regression analysis to a 4 parameter logistic equation.
- Cellular In Vitro Assay for Determining Vasopressin V2 Receptor Activity The identification of agonists and antagonists of the V1a and V2 vasopressin receptors from humans, rats and dogs as well as the quantification of the activity of the compounds of the invention is carried out using recombinant cell lines. These cell lines originally derive from a hamster's ovary epithelial cell (Chinese Hamster Ovary, CHO K1, ATCC: American Type Culture Collection, Manassas, Va. 20108, USA). The test cell lines constitutively express the human, rat or dog V1a or V2 receptors. In case of the Gαq-coupled V1a receptors, cells are also stably transfected with a modified form of the calcium-sensitive photoproteins aequorin (human and rat V1a) or obelin (dog V1a), which, after reconstitution with the cofactor coelenterazine, emit light when there are increases in free calcium concentrations [Rizzuto R, Simpson A W, Brini M, Pozzan T, Nature 358, 325-327 (1992); Illarionov B A, Bondar V S, Illarionova V A, Vysotski E S, Gene 153 (2), 273-274 (1995)]. The resulting vasopressin receptor cells react to stimulation of the recombinantly expressed V1a receptors by intracellular release of calcium ions, which can be quantified by the resulting photoprotein luminescence. The Gs-coupled V2 receptors are stably transfected into cell lines expressing the gene for firefly luciferase under control of a CRE-responsible promoter. Activation of V2 receptors induces the activation of the CRE-responsive promoter via cAMP increase, thereby inducing the expression of firefly luciferase. The light emitted by photoproteins of V1a cell lines as well as the light emitted by firefly luciferase of V2 cell lines corresponds to the activation or inhibition of the respective vasopressin receptor. The bioluminescence of the cell lines is detected using a suitable luminometer [Milligan G, Marshall F, Rees S, Trends in Pharmacological Sciences 17, 235-237 (1996)].Vasopressin V1a Receptor Cell Lines:On the day before the assay, the cells are plated out in culture medium (DMEM/F12, 2% FCS, 2 mM glutamine, 10 mM HEPES, 5 μg/ml coelenterazine) in 384-well microtiter plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, test compounds in various concentrations are placed for 10 minutes in the wells of the microtiter plate before the agonist [Arg8]-vasopressin at EC50 concentration is added. The resulting light signal is measured immediately in the luminometer.
- CAMP Assays Activation of GLP-1 receptor is known to stimulate cyclic AMP (cAMP) production in cells which indicates primary coupling to the Gαs subunit of the G protein heterotrimeric complex. Evidence suggests signaling through Gαs induced CAMP stimulation elicits the desired pharmacological response regarding insulin release from pancreatic β-cells. To optimize functional activity directed toward Gαs coupling, a HEK293/CRELuc cell line developed by HDB stably expressing the GLP-1 Receptor was used. 200× concentration of compound working solutions were prepared (Agilent Technologies Bravo) with 1/2 log serial dilution in 384-well Echo LDV plate (Labcyte, Cat #LP-0200). 50 nL/well 200× concentration of compound working solutions were moved to 384-well white low volume plate (Greiner, Cat #784075) using Labcyte ECHO550. 1×105 cells/mL HEK293/GLP1R/CRE-LUC (HD Biosciences) cell suspensions prepared with assay buffer [DPBS containing 0.5 mM IBMX (Sigma,Cat #I5879) and 0.1% BSA (GENVIEW, Cat #FA016-100 g)], 10 uL cell suspensions were added to each well of previous generated assay plate which already contains 50 nl compound at 200× concentration using ThermoFisher Multidrop Combi (1000cells/well). Seal the plate and incubate at 37° C. with 5% CO2 for 30 min.
- Cellular In Vitro Assay for Determining Vasopressin V1a Receptor Activity The identification of agonists and antagonists of the V1a and V2 vasopressin receptors from humans, rats and dogs as well as the quantification of the activity of the compounds of the invention is carried out using recombinant cell lines. These cell lines originally derive from a hamster's ovary epithelial cell (Chinese Hamster Ovary, CHO K1, ATCC: American Type Culture Collection, Manassas, Va. 20108, USA). The test cell lines constitutively express the human, rat or dog V1a or V2 receptors. In case of the Gαq-coupled V1a receptors, cells are also stably transfected with a modified form of the calcium-sensitive photoproteins aequorin (human and rat V1a) or obelin (dog V1a), which, after reconstitution with the cofactor coelenterazine, emit light when there are increases in free calcium concentrations [Rizzuto R, Simpson A W, Brini M, Pozzan T, Nature 358, 325-327 (1992); Illarionov B A, Bondar V S, Illarionova V A, Vysotski E S, Gene 153 (2), 273-274 (1995)]. The resulting vasopressin receptor cells react to stimulation of the recombinantly expressed V1a receptors by intracellular release of calcium ions, which can be quantified by the resulting photoprotein luminescence. The Gs-coupled V2 receptors are stably transfected into cell lines expressing the gene for firefly luciferase under control of a CRE-responsible promoter. Activation of V2 receptors induces the activation of the CRE-responsive promoter via cAMP increase, thereby inducing the expression of firefly luciferase. The light emitted by photoproteins of V1a cell lines as well as the light emitted by firefly luciferase of V2 cell lines corresponds to the activation or inhibition of the respective vasopressin receptor. The bioluminescence of the cell lines is detected using a suitable luminometer [Milligan G, Marshall F, Rees S, Trends in Pharmacological Sciences 17, 235-237 (1996)].Vasopressin V2 Receptor Cell Lines:On the day before the assay, the cells are plated out in culture medium (DMEM/F12, 2% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtiter plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, test compounds in various concentrations and the agonist [Arg8]-vasopressin at EC50 concentration are added together to the wells, and plates are incubated for 3 hours in a cell incubator. Upon addition of the cell lysis reagent Triton and the substrate luciferin, luminescence of firefly luciferase is measured in a luminometer.
- Transactivation Assay In the presence of an activator (agonist), the melanocortin receptor will activate the cAMP pathway which, via the CRE-Luc vector, will result in the synthesis of luciferase. After the addition of a lysis buffer containing a luminescent substrate for luciferase, it will be possible to measure the luminescence proportional to the degree of activation or of inhibition of the receptor. The products are solubilized at 10 mM in DMSO. They are tested in the form of dose-response at a final DMSO concentration of 0.1%. The range comprising 10 points and one zero begins at 10 μM with 4-fold dilutions. For testing agonists, the products are tested alone. For determining the behaviour of antagonists, the products of interest are tested in the presence of 1 nM NDP-MSH (reference agonist). The cells are seeded at a rate of 5000 cells per well (384 well plate) in serum-free DMEM medium, and incubated overnight at 37° C., 5% CO2.The products and the reference ligand (NDP-MSH) are added the following day, and the plates are again incubated for 6 h at 37° C., 5% CO2. After the addition of lysis buffer containing luciferin, the plates are read on a Top-Count instrument. The results are standardized as % activity using the 100% (cells+NDP-MSH at 10 nM) and 0% (cells alone) controls. An EC50 is calculated for each product using the XLFit software. The results are given in nM.
- ULK1 inhibition assay Gamma-32P assays to measure ULK1 kinase activity were performed as previously described. Briefly, Flag ULK1 was transfected into HEK293T cells and 20 hours later treated as indicated. The immmunoprecipitate was washed in IP buffer 3 times, and washed in kinase buffer (25 mM MOPS, pH 7.5, 1 mM EGTA, 0.1 mM Na3VO4, 15 mM MgCl2,). Hot and cold ATP were added at a 100 μM final concentration. As substrates, GST or the recombinant protein GST-Atg101 purified from E. coli were used at 1 μg for each reaction. Reactions were boiled, run out on SDS page gel. The gel was dried, and imaged using PhosphoImager software. For cold assays to asses ULK1, Flag ULK1 which was transiently overexpressed and immunoprecipited from HEK293T cells. Reactions were then run out on SDS page gel, transferred to PVDF membrane and blotted for total levelsFluorescence MicroscopyVps34flox/flox MEFs were reconstituted with Flag-VPS34 and either p40FX or GFP-DFCP1. 48 hours post infection with adenovirus expressing Cre recombinase (MOI of 100), cells were plated on glass coverslips at a density of 3×105 cells per well in 6-well tissue culture plates. 18 h later, cells were fixed in 4% PFA in PBS for 10 minutes and permeabilized in 0.2% Triton in PBS for 10 minutes. The following primary antibodies were used: mouse anti-Myc epitope and LC3B XP antibody (2276 and 3868 respectively, Cell Signaling Technologies). Secondary antibodies were anti-rabbit Alexa488 and anti-mouse Alexa594 (Molecular Probes, 1:1000. Cells were then fixed and counter stained with DAPI. Coverslips were mounted in FluoromountG (SouthernBiothech). Images were acquired on a Zeiss Axioplan2 epifluorescence microscope coupled to the Openlab software. Confocal images of mitotracker were taken on Zeiss LSM 710 laser scanning confocal microscope. 10 random fields per condition were acquired using the 100× objective and representative images shown. Glass coverslips were mounted directly on plate with FluoromountG and images taken on Zeiss Axioplan2 epifluorescence microscope.