BDBM323654 US10188667, Dexamethasone
BDBM235670 Dexamethasone sodium phosphate
dexamethasone (tetramethyl-rhodamine conjugated ) BDBM18703
dexamethasone (1R,2S,10S,11S,13R,14R,15S,17S)-1-fluoro-14,17-dihydroxy-14-(2-hydroxyacetyl)-2,13,15-trimethyltetracyclo[8.7.0.0^{2,7}.0^{11,15}]heptadeca-3,6-dien-5-one BDBM18207 US11554172, Compound Dexamethasone dexamethasone (tetramethyl-rhodamine conjugated ) US10869929, Compound Dexamethasone
1-(2-bromo-4-chloro-phenyl)sulfonylpiperidine 1-[(2-bromo-4-chlorophenyl)sulfonyl]piperidine MLS000028544 BDBM44576 DEXAMETHASONE 21-ACETATE MLS000060983 SMR000058327 1-(2-bromo-4-chlorophenyl)sulfonylpiperidine DEXAMETHASONE ACETATE cid_772156 SMR000069748 cid_236702 1-(2-bromanyl-4-chloranyl-phenyl)sulfonylpiperidine
- ChEBML_71399 Displacement of Dexamethasone from human glucocorticoid receptor
- ChEMBL_312086 (CHEMBL833998) Inhibition of glucocorticoid receptor Dexamethasone response
- ChEMBL_71247 (CHEMBL683493) Inhibition of Dexamethasone binding to Glucocorticoid receptor
- ChEMBL_726913 (CHEMBL1686702) Displacement of radiolabeled Dexamethasone from GR
- ChEMBL_728108 (CHEMBL1687050) Displacement of radiolabeled Dexamethasone from GR
- ChEBML_123499 Displacement of [3H]dexamethasone from human mineralocorticoid receptor
- ChEBML_159372 Displacement of [3H]dexamethasone from human progesterone receptor
- ChEBML_36109 Displacement of [3H]dexamethasone from human androgen receptor
- ChEMBL_305896 (CHEMBL874547) Inhibition of [3H]dexamethasone binding to glucocorticoid receptor
- ChEMBL_372312 (CHEMBL869701) Inhibition of fluorescent-labeled Dexamethasone binding to GR
- ChEMBL_437577 (CHEMBL906013) Displacement of radiolabeled Dexamethasone from human GR
- ChEMBL_447642 (CHEMBL896649) Displacement of radiolabeled Dexamethasone from glucocorticoid receptor
- ChEMBL_479540 (CHEMBL931536) Displacement of radiolabeled Dexamethasone from glucocorticoid receptor
- ChEMBL_71405 (CHEMBL680191) Displacement of [3H]- Dexamethasone from glucocorticoid receptor
- ChEMBL_1510389 (CHEMBL3607229) Antagonist activity against glucocorticoid receptor in rat H4-IIE cells assessed as inhibition of dexamethasone-induced receptor transactivation pre-incubated for 1 hr before dexamethasone addition and measured 24 hrs post dexamethasone stimulation by tyrosine aminotransferase enzyme assay
- ChEMBL_305404 (CHEMBL877001) Inhibition of [3H]dexamethasone binding to human glucocorticoid receptor
- ChEMBL_305749 (CHEMBL829520) Inhibition of [3H]dexamethasone binding to human glucocorticoid receptor
- ChEMBL_440116 (CHEMBL890428) Displacement of [3H]dexamethasone from human recombinant GR
- ChEMBL_452405 (CHEMBL902641) Displacement of fluorescent labeled Dexamethasone from glucocorticoid receptor
- ChEMBL_466158 (CHEMBL950162) Displacement of [3H]dexamethasone from human recombinant GR
- ChEMBL_558475 (CHEMBL963881) Displacement of fluorescent labelled Dexamethasone from glucocorticoid receptor
- ChEMBL_71383 (CHEMBL681735) Inhibition of [3H]dexamethasone binding to human Glucocorticoid Receptor
- ChEMBL_71413 (CHEMBL685131) Inhibition of [3H]dexamethasone binding to human Glucocorticoid Receptor
- ChEMBL_716755 (CHEMBL1670637) Displacement of fluorescent-labelled Dexamethasone from glucocorticoid receptor
- ChEMBL_2374956 Displacement of [3H]dexamethasone from human GR by radioligand binding assay
- ChEMBL_306249 (CHEMBL831159) Inhibition of human glucocorticoid receptor alpha by displacement of [3H]dexamethasone
- ChEMBL_312728 (CHEMBL834764) Inhibition of glucocorticoid receptor Dexamethasone response in reporter gene assay
- ChEMBL_423016 (CHEMBL909349) Inhibition of tetramethylrhodamine labeled dexamethasone binding to GR by FP assay
- ChEMBL_423018 (CHEMBL909351) Inhibition of tetramethylrhodamine labeled dexamethasone binding to MR by FP assay
- ChEMBL_447639 (CHEMBL896646) Agonist activity at GR by GRE activation assay relative to Dexamethasone
- ChEMBL_701245 (CHEMBL1648865) Displacement of radiolabeled Dexamethasone from glucocorticoid receptor expressed in baculovirus
- ChEMBL_71101 (CHEMBL679613) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR)
- ChEMBL_739803 (CHEMBL1762863) Displacement of radiolabeled Dexamethasone from glucocorticoid receptor expressed in baculovirus
- ChEBML_71420 Competitive displacement of [3H]dexamethasone from glucocorticoid receptor of rat liver cytosol
- ChEMBL_1559801 (CHEMBL3776014) Displacement of [3H]dexamethasone from glucocorticoid receptor in human IM9 cells
- ChEMBL_1633608 (CHEMBL3876400) Displacement of [3H]dexamethasone from GR receptor in human IM9 cells
- ChEMBL_1735710 (CHEMBL4151246) Antagonist activity at human GR assessed as reduction in dexamethasone-induced transactivation
- ChEMBL_312773 (CHEMBL835185) Inhibition of dexamethasone-induced glucocorticoid receptor mediated alkaline phosphatase activity
- ChEMBL_447640 (CHEMBL896647) Antagonist activity at GR assessed as inhibition of dexamethasone-induced GRE activation
- ChEMBL_448942 (CHEMBL899205) Displacement of TAMRA labeled Dexamethasone at human glucocorticoid receptor in insect cell
- ChEMBL_448943 (CHEMBL899206) Displacement of TAMRA labeled Dexamethasone at human progesterone receptor in insect cell
- ChEMBL_634771 (CHEMBL1119507) Displacement of fluorescent-labeled Dexamethasone from GR by fluorescent polarization assay
- ChEMBL_71395 (CHEMBL681747) Displacement of 10 nM [3H]dexamethasone from human Glucocorticoid receptor
- ChEMBL_71396 (CHEMBL681748) Displacement of 10 nM [3H]dexamethasone from human Glucocorticoid receptor
- ChEMBL_71397 (CHEMBL681749) Binding affinity towards glucocorticoid receptor (GR) by displacing [3H]dexamethasone
- ChEMBL_1700184 (CHEMBL4051166) Antagonist activity at rat glucocorticoid receptor in primary hepatocytes assessed as dexamethasone-induced tyrosine amino transferase activity preincubated for 30 mins followed by dexamethasone addition measured after 20 hrs
- ChEMBL_1700185 (CHEMBL4051167) Antagonist activity at dog glucocorticoid receptor in primary hepatocytes assessed as dexamethasone-induced tyrosine amino transferase activity preincubated for 30 mins followed by dexamethasone addition measured after 20 hrs
- ChEMBL_1700170 (CHEMBL4051152) Antagonist activity at glucocorticoid receptor in human HepG2 cells assessed as inhibition of dexamethasone-induced tyrosine amino transferase activity preincubated for 30 mins followed by dexamethasone addition measured after 20 hrs
- ChEMBL_1700218 (CHEMBL4051200) Antagonist activity at glucocorticoid receptor in human primary hepatocytes assessed as inhibition of dexamethasone-induced tyrosine amino transferase activity preincubated for 30 mins followed by dexamethasone addition measured after 20 hrs
- ChEBML_71100 Binding affinity against human Glucocorticoid receptor (GR) using [3H]dexamethasone as radioligand
- ChEMBL_1292840 (CHEMBL3122504) Displacement of [3H]-dexamethasone from human glucocorticoid receptor expressed in HEK293 cells
- ChEMBL_306508 (CHEMBL828113) Inhibition of dexamethasone-induced glucocorticoid receptor mediated tyrosine aminotransferase in rat hepatocytes
- ChEMBL_457478 (CHEMBL941996) Displacement of [3H]dexamethasone from human glucocorticoid receptor expressed in recombinant baculovirus
- ChEMBL_70937 (CHEMBL683918) Inhibition of Dexamethasone stimulated transcriptional activity in CHO cells expressing glucocorticoid receptor
- ChEMBL_70938 (CHEMBL683919) Inhibition of Dexamethasone stimulated transcriptional activity in CHO cells expressing glucocorticoid receptor
- ChEMBL_839721 (CHEMBL2089548) Antagonist activity at glucocorticoid receptor in rat hepatocytes assessed as inhibition of dexamethasone-induced trypsine aminotransferase expression incubated for 30 mins prior to dexamethasone-induction measured after 4 hrs by reporter gene assay
- ChEMBL_1446326 (CHEMBL3374292) Displacement of tetramethylrhodamine-labeled dexamethasone from PR (unknown origin) by fluorescence polarization assay
- ChEMBL_199711 (CHEMBL802067) Transcriptional repression activity in HEP G2 cells expressing glucocorticoid receptor compared to Dexamethasone
- ChEMBL_306509 (CHEMBL828114) Inhibition of dexamethasone-induced GR-mediated tyrosine amino transferase activity in rat hepatocytes
- ChEMBL_71100 (CHEMBL679612) Binding affinity against human Glucocorticoid receptor (GR) using [3H]dexamethasone as radioligand
- ChEMBL_71400 (CHEMBL681751) Inhibition of Dexamethasone binding to human glucocorticoid receptor expressed in baculovirus SF-12 cells
- ChEMBL_71401 (CHEMBL680187) Displacement of Dexamethasone from human glucocorticoid receptor expressed in baculovirus SF-12 cells
- ChEMBL_726895 (CHEMBL1686601) Displacement of [3H]dexamethasone from Sprague-Dawley rat GR by liquid scintillation counting
- ChEMBL_1338546 (CHEMBL3241423) Displacement of TAMRA-labeled dexamethasone from glucocorticoid receptor (unknown origin) by fluorescence polarization assay
- ChEMBL_1338548 (CHEMBL3241425) Displacement of TAMRA-labeled dexamethasone from mineralocorticoid receptor (unknown origin) by fluorescence polarization assay
- ChEMBL_199710 (CHEMBL802066) Transcriptional repression of IL-1 stimulated IL-6 expression in native cells compared to Dexamethasone
- ChEMBL_466159 (CHEMBL950163) Antagonist activity at GR in SW1353/MMTV5 cells assessed as inhibition of dexamethasone-induced luciferase expression
- ChEMBL_499970 (CHEMBL1019340) Displacement of FITC-dexamethasone from human recombinant glucocorticoid receptor alpha by fluorescence polarization assay
- ChEMBL_637924 (CHEMBL1166110) Displacement of [3H]dexamethasone from human glucocorticoid receptor after 16 hrs by scintillation counting
- ChEMBL_849777 (CHEMBL2150559) Displacement of [3H]-dexamethasone from human full length GR expressed in insect Sf21 cells
- ChEMBL_965461 (CHEMBL2395265) Displacement of [3H]Dexamethasone from glucocorticoid receptor in human HeLaS3 cells after 2 hrs
- ChEBML_71109 Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 150-220 nM
- ChEMBL_1499884 (CHEMBL3583216) Displacement of [3H]-dexamethasone from cytosolic fraction of human recombinant glucocorticoid receptor by scintillation counting analysis
- ChEMBL_1620823 (CHEMBL3863106) Displacement of [3H]dexamethasone from human GR measured after 18 hrs by scintillation counting method
- ChEMBL_1864065 (CHEMBL4365040) Displacement of [3H]dexamethasone from recombinant human GR after 360 mins by scintillation counting analysis
- ChEMBL_2222792 (CHEMBL5136126) Displacement of [3H]-dexamethasone from rat glucocorticoid receptor incubated overnight by liquid scintillation counting analysis
- ChEMBL_873662 (CHEMBL2188778) Displacement of [3H]dexamethasone from human glucocorticoid receptor overexpressed in HEK293 cells by microbeta counting assay
- ChEMBL_977683 (CHEMBL2423841) Displacement of [3H]-Dexamethasone human GR expressed in 293 cells after 16 hrs by scintillation counting
- ChEMBL_1277677 (CHEMBL3096919) Displacement of TAMRA-labeled dexamethasone from glucocorticoid receptor (unknown origin) by fluorescence polarization competitive binding assay
- ChEMBL_1827551 (CHEMBL4327425) Displacement of [3H]dexamethasone from GR in human IM9 cells after 6 hrs by scintillation counting method
- ChEMBL_424794 (CHEMBL909043) Activity at GR expressed in CHO cells assessed as decrease in dexamethasone-stimulated alkaline phosphatase production by GRAF assay
- ChEMBL_71102 (CHEMBL679614) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 10-12 nM
- ChEMBL_71103 (CHEMBL679615) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 106-131 nM
- ChEMBL_71104 (CHEMBL679616) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 1273-2947 nM
- ChEMBL_71105 (CHEMBL679617) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 13-26 nM
- ChEMBL_71106 (CHEMBL679618) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 133-161 nM
- ChEMBL_71107 (CHEMBL679619) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 1382-1049 nM
- ChEMBL_71108 (CHEMBL679620) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 1428-1433 nM
- ChEMBL_71109 (CHEMBL686057) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 150-220 nM
- ChEMBL_71110 (CHEMBL686058) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 159-161 nM
- ChEMBL_71111 (CHEMBL686059) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 17-21 nM
- ChEMBL_71112 (CHEMBL686060) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 2-2 nM
- ChEMBL_71113 (CHEMBL686061) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 215-217 nM
- ChEMBL_71114 (CHEMBL686295) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 23-33 nM
- ChEMBL_71115 (CHEMBL686296) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 26-67 nM
- ChEMBL_71116 (CHEMBL686297) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 28-34 nM
- ChEMBL_71117 (CHEMBL686298) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 294-554 nM
- ChEMBL_71118 (CHEMBL686299) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 3-3 nM
- ChEMBL_71228 (CHEMBL682527) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 3-4 nM
- ChEMBL_71229 (CHEMBL682528) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 366-578 nM
- ChEMBL_71230 (CHEMBL682529) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 378-814 nM
- ChEMBL_71231 (CHEMBL682530) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 379-840 nM
- ChEMBL_71232 (CHEMBL680299) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 39-94 nM
- ChEMBL_71233 (CHEMBL680300) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 43-50 nM
- ChEMBL_71234 (CHEMBL680301) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 44-53 nM
- ChEMBL_71235 (CHEMBL680302) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 5-6 nM
- ChEMBL_71236 (CHEMBL680303) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 536-560 nM
- ChEMBL_71237 (CHEMBL680304) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 559-1423 nM
- ChEMBL_71238 (CHEMBL680305) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 6-7 nM
- ChEMBL_71239 (CHEMBL680306) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 6-8 nM
- ChEMBL_71240 (CHEMBL680307) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 61-63 nM
- ChEMBL_71241 (CHEMBL680308) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 639-673 nM
- ChEMBL_71242 (CHEMBL683338) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 681-974 nM
- ChEMBL_71243 (CHEMBL683339) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 70-92 nM
- ChEMBL_71244 (CHEMBL683340) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 838-988 nM
- ChEMBL_71245 (CHEMBL683341) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 9-12 nM
- ChEMBL_71246 (CHEMBL683342) Displacement of [3H]dexamethasone from human Glucocorticoid receptor (GR); Value ranges from 949-1169 nM
- ChEMBL_772901 (CHEMBL1838304) Displacement of [3H]-dexamethasone from human recombinant glucocorticoid receptor after 18 hrs by liquid scintillation counting
- ChEMBL_372315 (CHEMBL869709) Activity at GR assessed as ability to antagonize dexamethasone-induced MMTV luciferase reporter gene transactivation in human A549 cells
- ChEMBL_372316 (CHEMBL869706) Agonist activity at GR assessed as MMTV-mediated transactivation of renilla luciferase gene in human A549 cells relative to Dexamethasone
- ChEMBL_479545 (CHEMBL931541) Antagonist activity at glucocorticoid receptor assessed as inhibition of dexamethasone-induced glucose response element transcriptional transactivation by luciferase assay
- ChEMBL_71398 (CHEMBL874034) Binding affinity towards human glucocorticoid receptor (GR) was determined using [3H]dexamethasone as radioligand in SF-1 cells
- ChEMBL_728113 (CHEMBL1687055) Agonist activity at human GR expressed in NHDF cells assessed as inhibition of IL-6 production by ELISA relative to Dexamethasone
- ChEMBL_89092 (CHEMBL696546) Repression activity of GR ligand with interleukin-6 receptor in native cell assay using Dexamethasone was determined as maximal potency
- ChEMBL_1438889 (CHEMBL3381814) Displacement of [3H]-Dexamethasone from glucocorticoid receptor (unknown origin) expressed in 293 cells after 16 hrs by scintillation counting
- ChEMBL_2111613 (CHEMBL4820463) Displacement of [3H]dexamethasone from glucocorticoid receptor in human IM-9 cells incubated for 6 hrs by scintillation counting analysis
- ChEMBL_841250 (CHEMBL2089653) Displacement of fluorescent labelled SRC1-4 from dexamethasone-GR complex after 2 to 4 hrs by TR-FRET assay
- ChEMBL_886649 (CHEMBL2216234) Displacement of [3H]dexamethasone from human recombinant glucocorticoid receptor expressed in baculovirus after 18 hrs by scintillation counting analysis
- ChEMBL_936982 (CHEMBL2321589) Displacement of [3H]-dexamethasone human glucocorticoid receptor alpha expressed in 293 MSR cells after 60 mins by scintillation counting
- ChEMBL_1510382 (CHEMBL3607222) Displacement of [3H]-dexamethasone from human glucocorticoid receptor expressed in HEK293 cells by scintillation counting based radioligand competition binding assay
- ChEMBL_1755053 (CHEMBL4189813) Displacement of [3H]dexamethasone from human recombinant glucocorticoid receptor expressed in insect cells after 24 hrs by scintillation proximity assay
- ChEMBL_2172765 (CHEMBL5057899) Antagonist activity at glucocorticoid receptor in human HeLa cells assessed as reduction in dexamethasone-induced luciferase activity by dual-Glo luciferase assay
- ChEMBL_321122 (CHEMBL881382) Effective concentration against inhibition of Dexamethasone induced glucocorticoid receptor transactivation of mouse mammary tumor virus luciferase gene in HeLa cells
- ChEMBL_448641 (CHEMBL897788) Antagonist activity at glucocorticoid receptor in human A549 cells transfected with MMTV luciferase reporter gene assessed as inhibition of dexamethasone-induced activation
- ChEMBL_452410 (CHEMBL902646) Antagonist activity at glucocorticoid receptor in human A549 cells transfected with MMTV luciferase reporter gene assessed as inhibition of dexamethasone-induced activation
- ChEMBL_457479 (CHEMBL941997) Antagonist activity at human glucocorticoid receptor in SW1353 cells assessed as inhibition of dexamethasone-induced luciferase expression by MMTV5 reporter gene assay
- ChEMBL_663368 (CHEMBL1250864) Binding affinity to glucocorticoid receptor expressed in baculovirus-infected insect cells using tetramethylrhodamine labeled Dexamethasone by fluorescence polarization microplate assay
- ChEMBL_663370 (CHEMBL1250866) Binding affinity to mineralocorticoid receptor expressed in baculovirus-infected insect cells using tetramethylrhodamine labeled Dexamethasone by fluorescence polarization microplate assay
- ChEMBL_775646 (CHEMBL1913330) Displacement of tetramethylrhodamine-labeled Dexamethasone from human recombinant glucocorticoid receptor expressed in baculovirus infected insect cells by fluorescence polarization assay
- ChEMBL_775648 (CHEMBL1913332) Displacement of tetramethylrhodamine-labeled Dexamethasone from human recombinant mineralocorticoid receptor expressed in baculovirus infected insect cells by fluorescence polarization assay
- ChEMBL_839719 (CHEMBL2089546) Displacement of [3H]dexamethasone from human glucocorticoid receptor expressed in baculovirus infected Sf9 cells after overnight incubation by scintillation counting
- ChEBML_1685152 Antagonist activity at human GRalpha expressed in HEK293 cells assessed as inhibition of dexamethasone-induced response after 24 hrs by luciferase reporter gene assay
- ChEMBL_1275505 (CHEMBL3090520) Displacement of [3H]dexamethasone from GST-tagged human glucocorticoid receptor ligand binding domain after 4 hrs by liquid scintillation counting
- ChEMBL_1553954 (CHEMBL3767966) Displacement of [3H]-dexamethasone from glucocorticoid receptor (unknown origin) expressed in HEK293 cell lysate incubated overnight by microbeta scintillation counting method
- ChEMBL_1678021 (CHEMBL4028164) Displacement of [3H]-dexamethasone from recombinant human GST-tagged GR ligand binding domain after 4 hrs by liquid scintillation counting
- ChEMBL_1736302 (CHEMBL4151838) Antagonist activity at GR in human PBMC assessed as reduction in dexamethasone-induced GILZ gene expression incubated for 6 hrs by RT-qPCR method
- ChEMBL_1736303 (CHEMBL4151839) Antagonist activity at GR in human PBMC assessed as reduction in dexamethasone-induced FKBP5 gene expression incubated for 6 hrs by RT-qPCR method
- ChEMBL_1736304 (CHEMBL4151840) Antagonist activity at GR in rat PBMC assessed as reduction in dexamethasone-induced GILZ gene expression incubated for 24 hrs by RT-qPCR method
- ChEMBL_1736305 (CHEMBL4151841) Antagonist activity at GR in rat PBMC assessed as reduction in dexamethasone-induced FKBP5 gene expression incubated for 24 hrs by RT-qPCR method
- ChEMBL_1736306 (CHEMBL4151842) Antagonist activity at GR in dog PBMC assessed as reduction in dexamethasone-induced GILZ gene expression incubated for 6 hrs by RT-qPCR method
- ChEMBL_1736307 (CHEMBL4151843) Antagonist activity at GR in dog PBMC assessed as reduction in dexamethasone-induced FKBP5 gene expression incubated for 6 hrs by RT-qPCR method
- ChEMBL_1736312 (CHEMBL4151848) Antagonist activity at GR in human OVCAR5 assessed as reduction in dexamethasone-induced GILZ gene expression incubated for 24 hrs by RT-qPCR method
- ChEMBL_1736313 (CHEMBL4151849) Antagonist activity at GR in human OVCAR5 assessed as reduction in dexamethasone-induced FKBP5 gene expression incubated for 24 hrs by RT-qPCR method
- ChEMBL_474430 (CHEMBL935181) Antagonist activity at human GR ligand binding domain expressed in african green monkey COS7 cells in presence of Dexamethasone by Gal4 hybrid assay
- ChEMBL_839889 (CHEMBL2090090) Displacement of [3H]dexamethasone from human glucocorticoid receptor expressed in baculovirus infected Sf9 cells after 16-18 hrs by beta counting
- ChEMBL_1736310 (CHEMBL4151846) Antagonist activity at GR in mini pig PBMC assessed as reduction in dexamethasone-induced GILZ gene expression incubated for 24 hrs by RT-qPCR method
- ChEMBL_1736311 (CHEMBL4151847) Antagonist activity at GR in mini pig PBMC assessed as reduction in dexamethasone-induced FKBP5 gene expression incubated for 24 hrs by RT-qPCR method
- ChEMBL_702551 (CHEMBL1657195) Modulation of glucocorticoid receptor in human SKGT4 cells assessed as receptor translocation from cytoplasm to nucleus after 4 hrs by Hoechst staining relative to Dexamethasone
- ChEMBL_1736254 (CHEMBL4151790) Antagonist activity at human GR expressed in CHO-K1 cells assessed as reduction in dexamethasone-induced response incubated for 20 hrs by luciferase reporter gene assay
- ChEMBL_802422 (CHEMBL1954865) Antagonist activity at glucocorticoid receptor in human A549 cells assessed as inhibition of dexamethasone-induced transcription at GRE after 24 hrs by luciferase reporter gene assay
- ChEMBL_839891 (CHEMBL2090092) Antagonist activity at glucocorticoid receptor in rat H4IIE cells assessed as inhibition of dexamethasone-induced trypsine aminotransferase expression after 24 hrs by reporter gene assay
- ChEMBL_873649 (CHEMBL2188765) Displacement of TAMRA-labeled Dexamethasone from human mineralocorticoid receptor expressed in baculovirus infected insect cell lysate after 60 mins by fluorescence polarization assay
- ChEMBL_886648 (CHEMBL2216233) Antagonist activity at glucocorticoid receptor in human SW1353 cells assessed as inhibition of dexamethasone-induced luciferase expression after 16 hrs by MMTV5 reporter gene assay
- ChEMBL_1658415 (CHEMBL4008027) Displacement of TAMRA-labeled dexamethasone from full-length human mineralocorticoid receptor expressed in baculovirus infected insect cells after 60 mins by fluorescence polarization assay
- ChEMBL_1735696 (CHEMBL4151232) Antagonist activity at human CMX-GRalpha expressed in HEK293 cells assessed as reduction in dexamethasone-induced transactivation activity after 24 hrs by luciferase reporter gene assay
- ChEMBL_2222793 (CHEMBL5136127) Displacement of [3H]-dexamethasone from recombinant full length human glucocorticoid receptor expressed in baculovirus infected insect cells incubated overnight by liquid scintillation counting analysis
- ChEMBL_2278368 Antagonist activity at glucocorticoid receptor (unknown origin) transfected in human HeLa cells assessed as inhibition of dexamethasone-induced transactivation of receptor incubated for 18 hrs by firefly luciferase assay
- ChEMBL_839720 (CHEMBL2089547) Antagonist activity at human glucocorticoid receptor expressed in GRAF cells assessed as inhibition of dexamethasone-induced alkaline phosphatase expression after 48 hrs by reporter gene assay
- ChEMBL_839890 (CHEMBL2090091) Antagonist activity at human glucocorticoid receptor expressed in CHOK1 cells assessed as inhibition of dexamethasone-induced alkaline phosphatase expression after 46 hrs by reporter gene assay
- ChEMBL_1295697 (CHEMBL3132584) Competitive binding affinity to mineralocorticoid receptor (unknown origin) expressed in baculovirus-infected cell system after 1 hr by fluorescence polarization assay in presence of tetramethylrhodamine-labeled dexamethasone
- ChEMBL_1295699 (CHEMBL3129511) Competitive binding affinity to glucocorticoid receptor (unknown origin) expressed in baculovirus-infected cell system after 1 hr by fluorescence polarization assay in presence of tetramethylrhodamine-labeled dexamethasone
- ChEMBL_1499886 (CHEMBL3583218) Antagonist activity at human glucocorticoid receptor transfected in CHOK1 cells assessed as inhibition of dexamethasone-induced receptor transcriptional activity after 6 hrs by luciferase reporter gene assay
- ChEMBL_634775 (CHEMBL1119511) Antagonist activity at GR in human A549 cells transfected with luciferase gene linked to MMTV promoter assessed as inhibition of dexamethasone-induced luciferase transactivation activity after 24 hrs
- ChEMBL_728109 (CHEMBL1687051) Agonist activity at GR expressed in african green monkey CV1 cells transfected with luciferase gene linked to MMTV promoter assessed as induction of luciferase transactivation activity relative to Dexamethasone
- ChEMBL_1759556 (CHEMBL4194564) Antagonist activity at glucocorticoid receptor (unknown origin) expressed in human ChaGoK1 cells assessed as inhibition of dexamethasone induced transactivation incubated for 24 hrs by beta-galactosidase reporter gene assay
- ChEMBL_1904911 (CHEMBL4407269) Antagonist activity at human glucocorticoid receptor expressed in CHO-K1 cells assessed as inhibition of glucocorticoid receptor-dexamethasone coactivator protein-protein interaction incubated for 24 hrs by bioluminescence assay
- ChEMBL_631316 (CHEMBL1105653) Inhibition of dexamethasone-induced human NR1-1a/NR2A receptor-mediated excitotoxicity in (S)-glutamate/glycine-stimulated mouse L12-G10 cells assessed as LDH release after 30 mins
- ChEMBL_631318 (CHEMBL1105655) Inhibition of dexamethasone-induced human NR1-1a/NR2B receptor-mediated excitotoxicity in (S)-glutamate/glycine-stimulated mouse L13-E6 cells assessed as LDH release after 30 mins
- ChEMBL_728111 (CHEMBL1687053) Agonist activity at GR expressed in IL-1beta- and TNFalpha-stimulated HepG2 cells assessed as inhibition of NFKB- or AP-1 mediated E-selectin transcription by luciferase reporter gene assay relative to Dexamethasone
- ChEMBL_2207452 (CHEMBL5120160) Displacement of [3H]ifenprodil from recombinant human GluN2B NMDA receptor (unknown origin) expressed in dexamethasone-induced mouse L-M(TK-) cell after 120 mins by microbeta scintillation counting method
- GR Binding Assay and GRAF Assay For compounds able to displace the 3(H)-dexamethasone from the receptor an IC50 value (the concentration required to inhibit 50% of the binding of 3(H)-dex) was determined by a nonlinear four parameter logistic model. The EC50s are the concentrations of compounds required to inhibit 50% of dexamethasone-induced alkaline phosphatase (ALP) expression in GRAF cell line, which is produced by CHO-K1 cells stably transfected with GR and a reporter construct containing a glucocorticoid response element driving expression of ALP.
- GR Binding Assay and Human IL-6 Assay The IC50s were determined by incubating the receptors with radiolabeled dexamethasone in the presence of full log scale concentrations (10-11 M to 10-6 M) of cold dexamethasone or the ligands. The IC50s were calculated assuming a single binding site sigmoidal dose-response model. The EC50s were obtained from transcriptional repression of IL-6-release using the human A549 lung epithelial cell line and freshly isolated mouse thioglycolate-elicited peritoneal exudate cells.
- ChEMBL_1497288 (CHEMBL3579001) Antagonist activity at human glucocorticoid receptor expressed in CHOK1 cells assessed as inhibition of dexamethasone-induced protein interaction with steroid receptor co-activator peptide after overnight incubation by beta-galactosidase reporter gene assay
- Receptor Binding Assay Binding Assay (I):In order to assess the affinity of test compounds for the human glucocorticoid receptor, a commercially available kit was used (Glucocorticoid Receptor Competitor Assay Kit, Invitrogen Part #2893). Briefly, purified human recombinant full-length glucocorticoid receptor (2 nM) was mixed with fluorescently labeled glucocorticoid (1 nM Fluormone GS Red) in the presence or absence of test compound. After two hour incubation at room temperature in the dark, the fluorescence polarization (FP) of the samples was measured. The FP of a mixture of receptor, fluorescent probe (i.e., Fluormone GS Red) and 5 μM dexamethasone represented background fluorescence or 100% inhibition, whereas, the FP of the mixture without dexamethasone (but in the presence of vehicle) was taken to be 100% binding. The percentage inhibition of test compounds were then compared to the sample with 5 μM dexamethasone and expressed as % relative binding activity with dexamethasone being 100% and no inhibition is 0%. Test compounds were analyzed in the concentration range from 8.5E-05 μM to 5 μM. Binding Assay (II):In order to measure the binding of compounds on the glucocorticoid receptor a commercially available kit was used (Glucocorticoid receptor competitor assay kit, PanVera Co., Madison, Wis., P2816). Briefly, a cell lysate containing recombinantly expressed human full-length glucocorticoid receptor was mixed with a fluorescently labeled glucocorticoid (1 nM Fluormone GS1) in the presence or absence of test compound. After one hour at room temperature, the fluorescence polarization (FP) of the samples were measured. The FP of a mixture of receptor, fluorescent probe (i.e., Fluormone GS1) and 1 mM dexamethasone represented background fluorescence or 100% inhibition, whereas, the FP of the mixture without dexamethasone was taken to be 100% binding. The percentage inhibition of test molecules were then compared to the sample with 1 mM dexamethasone and expressed as % relative binding activity with dexamethasone being 100% and no inhibition is 0%. Test molecules were analyzed in the concentration range from 2.4 nM to 40 μM.Site I binding assays for any NHR (Nuclear Hormone Receptor) are conducted similarly to the above. An appropriate cell lysate or purified NHR is used as the source of the NHR. The fluorescent probe and unlabeled competitor are appropriate for the specific NHR, i.e., are ligands for the specific NHR.
- ChEMBL_1658413 (CHEMBL4008025) Antagonist activity at GAL4-fused human GR LBD (443 to 777 residues) expressed in CHO-K1 cells assessed as inhibition of dexamethasone-induced transactivation activity after 5 to 6 hrs by luciferase reporter gene assay
- Binding Assay In order to measure the binding of compounds on the glucocorticoid receptor a commercially available kit was used (Glucocorticoid receptor competitor assay kit, PanVera Co., Madison, Wis., P2816). Briefly, a cell lysate containing recombinantly expressed human full-length glucocorticoid receptor was mixed with a fluorescently labeled glucocorticoid (1 nM Fluormone GS1) in the presence or absence of test compound. After one hour at room temperature, the fluorescence polarization (FP) of the samples were measured. The FP of a mixture of receptor, fluorescent probe (i.e., Fluormone GS1) and 1 mM dexamethasone represented background fluorescence or 100% inhibition, whereas, the FP of the mixture without dexamethasone was taken to be 100% binding. The percentage inhibition of test molecules were then compared to the sample with 1 mM dexamethasone and expressed as % relative binding activity with dexamethasone being 100% and no inhibition is 0%. Test molecules were analyzed in the concentration range from 2.4 nM to 40 μM.Site I binding assays for any NHR (Nuclear Hormone Receptor) are conducted similarly to the above. An appropriate cell lysate or purified NHR is used as the source of the NHR. The fluorescent probe and unlabeled competitor are appropriate for the specific NHR, i.e., are ligands for the specific NHR.
- GR-Mediated Antagonist Activity Assay and IL-6 Repression Assay GR-Mediated Antagonist Activity Assay- GR-mediated antagonist activity is measured in the presence of a concentration of dexamethasone empirically determined to give half-maximal response. The antagonist activity of compounds, their activity in inhibiting dexamethasone response to basal level, is reported as % absolute efficacy. Potencies of compounds are reported as the concentration at which they reach half their antagonist (IC50) activity. IL-6 Repression Assay- The GR-mediated repression of IL-6 production in a human fibroblast (NHDFneo) cell line is measured with a modified ELISA assay.
- Competitive Ligand Binding Assay, GR-Mediated Antagonist Activity Assay, and IL-6 Repression Assay Competitive Ligand Binding Assay- The Ki values were determined by the application of the Cheng-Prusoff equation: Ki=IC50/(1+[L]/Kd) where [L] is the concentration of labeled ligand and Kd is the dissociation constant of the labeled ligand determined in the saturation analysis. GR-Mediated Antagonist Activity Assay- GR-mediated antagonist activity is measured in the presence of a concentration of dexamethasone empirically determined to give half-maximal response. The antagonist activity of compounds, their activity in inhibiting dexamethasone response to basal level, is reported as % absolute efficacy. Potencies of compounds are reported as the concentration at which they reach half their antagonist (IC50) activity. IL-6 Repression Assay- The GR-mediated repression of IL-6 production in a human fibroblast (NHDFneo) cell line is measured with a modified ELISA assay.
- GR Binding Assay To assess GR binding, test compound (top dose 1 μM, 4 fold serial dilution, 8 point dose response) and control (dexamethasone) were transferred to the assay plate. Cytosol from IM-9 cells was added to the plate, followed by addition of radiolabeled 3H-Dexamethasone at a final concentration of 1.5 nM. The plate was sealed, and the reaction was incubated at 300 rpm at 4° C. for 24 hrs. Radioligand absorption buffer (10 mM Tris-HCl, pH 7.4; 1.5 mM EDTA; 1 mM DTT; 0.25% charcoal; 0.0025% dextran) was then added to the plate, mixed, and incubated at 4° C. for 15 minutes. The plate was then centrifuged at 3000 pm for 30 minutes at 4° C. The supernatant was transferred to the scint-tube and Tri-carb was used for scintillation counting. The data was analyzed using GraphPadPrism v5.0 and binding IC50 was determined as the concentration where 50% inhibition of radioligand binding was observed.
- GR-Mediated Agonist Activity Assay and IL-6 Repression Assay GR-Mediated Agonist Activity Assay- GR-mediated activation by compounds is measured against maximal activation of dexamethasone in the same experiment and reported as % relative efficacy. Potencies of compounds are reported as the concentration at which they reach half their agonist (EC50). IL-6 Repression Assay- The GR-mediated repression of IL-6 production in a human fibroblast (NHDFneo) cell line is measured with a modified ELISA assay.
- Competitive Molecular Binding Assay The MR competitive binding assay is based on the binding and displacement of a TAMRA-labeled Dexamethasone probe with fluorescence polarization (FP) detection. The assay is performed in 384-well, low volume NBS black plates (Corning #3676) in assay buffer consisting of 10 mM TES, pH 7.4, 50 mM KCl, 20 mM Sodium molybdate, 1.5 mM EDTA, 0.04% CHAPS, 10% Glycerol and 1 mM DTT. Full Length human mineralocorticoid receptor (hMR) present in a baculovirus infected insect cell lysate is diluted 2 fold in assay buffer and 10 μL of this dilution is added to the assay plate. Blank wells receive 10 μL of the diluted MR lysate containing 3 μM Dexamethasone. 2 μL diluted test compound is transferred to the assay plate for a final starting top concentration of 10 μM in 1% DMSO. The reaction is started by adding 3 μL of 25 nM probe in assay buffer for a final assay concentration of 5 nM.
- Competitive Ligand Binding Assay, GR-Mediated Agonist Activity Assay, and IL-6 Repression Assay Competitive Ligand Binding Assay- The Ki values were determined by the application of the Cheng-Prusoff equation: Ki=IC50/(1+[L]/Kd) where [L] is the concentration of labeled ligand and Kd is the dissociation constant of the labeled ligand determined in the saturation analysis. GR-Mediated Agonist Activity Assay- GR-mediated activation by compounds is measured against maximal activation of dexamethasone in the same experiment and reported as % relative efficacy. Potencies of compounds are reported as the concentration at which they reach half their agonist (EC50). IL-6 Repression Assay- The GR-mediated repression of IL-6 production in a human fibroblast (NHDFneo) cell line is measured with a modified ELISA assay.
- GRE Antagonist Assay A reporter cell line (ChagoK1 18:7:2 s4/GRE) was established by stable transfection of the human bronchogenic carcinoma celline, ChaGo K1 (ATCC: HTB 168) with a MMTV-GRE-LacZ reporter construct. The generated cell line allows for identification of compounds showing antagonist activity at the human glucocorticoid receptor (GR) via reduction of LacZ gene expression. Dexamethasone-activated GR binds to the Glucocorticoid Response Element (GRE) in the promoter of the LacZ gene and transcription is initiated. Antagonistic properties of compounds are assessed as beta-galactosidase intensity reduction from pre-stimulation with dexamethasone through a colour reaction (change in absorbance).Cryo-preserved ChagoK1 18:7:2 s4/GRE cells were suspended in RPMI medium with 10% FBS, 1% NEAA and 1% sodium pyruvate, and seeded as 50000 cells/200 ul/well in 96-well plates and cultured at 37° C. with 5% CO2 and 95% humidity for 24 hr. Cells were pre-stimulated with 2 μl dexamethasone (70 nM final conc) for 4-5 hr, before addition of 1 μl compound at different concentrations and incubation for an additional 24 hr. Cells were washed once in PBS and lysed with 50 μl of 0.1% Triton-X for 10 min at room temperature. 40 μl of reaction mixture (2.5 mM MgCl2, 0.1 M β-mercapto ethanol, 1.7 mg/ml ONPG and 42.5 mM sodium phosphate, pH 7.5), was added to each well and kept at 37° C. for 60 min. The reaction was then terminated by addition of 100 μl stop solution (300 mM glycine, 15 mM EDTA, pH 11.3, adjusted with NaOH). The plates were measured at 420 nm for absorbance in a SpectraMax reader (Molecular Device).The relative efficacy (% effect) of a compound is calculated based on the full antagonist effect of the reference compound Mifepristone (RU486): % Effect=((Sample abs−min abs)/(max abs−min abs))×100To calculate IC50, max, min and slope factor for each compound, a concentration response curve is fitted by plotting % Effect versus compound concentration using the 4 parameter logistic equation: y=A+(B−A)/(1+((10C)/x)D)Where A=min Y, B=max Y, C=log IC50 and D=Slope factor.
- Glucocorticoid Receptor Transactivation Potencies for Cortisol and 17-ester Derivatives Glucocorticoid receptor (GR) activation potency was assessed using a HeLa cell line containing the MMTV-bla reporter (MMTV-bla HeLa CELLSENSOR , Invitrogen Corp., Carlsbad, Calif.). This cell line was stably transfected with an expression construct containing β-lactamase cDNA under control of the MMTV response element previously identified as a glucocorticoid receptor response element.Results from one experiment performed in duplicate for 9 compounds and the control compound, dexamethasone, are summarized in Table 3. All assays were performed as 10-point dose responses using a half log-fold dilution series starting with a maximum compound concentration of 100 nM. The compounds were incubated for 5 hours. The activation of endogenous GR leads to expression of the reporter β-lactamase which is detected by the conversion of a FRET substrate in a ratiometric assay format. This functional assay allows for measurement of receptor agonism by compounds and can be used to determine compound potency and selectivity.
- GR Antagonist Assay COS-7 cells (ATCC, Manassas, Va.) were plated in 24 well plates in DME+5% csFBS without phenol red at 70,000 cells/well. Once the cells attached to the plates (typically after overnight incubation after plating), they were transfected in OPTIMEM medium (Life Technologies) using lipofectamine reagent (Life Technologies) with 0.25 μg GRE-LUC, 25 ng pCR3.1 GR, and 10 ng CMV-renilla LUC per well. Twenty-four hours after transfection, the cells were fed with DME+5% csFBS without phenol red (Fisher Scientific, Waltham, Mass.) and treated with the test compounds (1 pM to 10 μM dose range) in the presence of 0.1 nM dexamethasone (Sigma, St. Louis, Mo.). Sixteen to twenty-four hours after treatment, a luciferase assay was performed using the Dual Luciferase assay kit (Promega, Madison, Wis.). Firefly luciferase values were normalized to Renilla luciferase numbers.
- Radioligand Binding Assay In the GR radioligand binding assay, test compounds were serially diluted in semi-log steps (10 concentrations) with a final concentration of 10 uM. Test compounds (1 uL) and controls (1 uL) in 100% DMSO were added to 96 Greiner V-bottom polypropylene plates. 0% control was 6.7% DMSO (final concentration in assay) and 100% control was 6.7 uM Dexamethasone.The full length GR was diluted to a final concentration of 3.3% (0.495 mg/ml) in assay buffer (20 mM Tris-HCl, 1 mM EDTA, 10% (w/v) Glycerol, 20 mM Sodium molbydate, pH 7.4). 45 uL of GR was added to each well and the plates were incubated for 15 min at room temperature.3H-dexamethasone solution was diluted to a concentration of 70 nM in assay buffer (7 nM final assay concentration) and 5 uL was added to each well. The samples were mixed for 5 min using a plate shaker at 700 rpm, before incubation for 2 h at room temperature,50 uL ice-cold charcoal solution.
- Radioligand Binding of Compounds to AR, GR and ER ReceptorsGR (human) (agonist radioligand) IM-9 cells (cytosol)[3H]dexamethasone 1.5 nM 1.5 nM triamcinolone (10 μM) 6 h 4° C. Scintillation counting (Clark, A. F et al. (1996) Invest. Ophtalmol. Vis. Sci., 37: 805-813).ER (nonselective) (human) (agonist radioligand) MCF-7 cells (cytosol)[3H]estradiol 0.4 nM 0.2 nM 17(3-estradiol (6 μM) 20 h 4° C. Scintillation counting(Parker, G. J et al. (2000) J. Biomol. Screen., 5: 77-88).AR (human) (agonist radioligand) LNCaP cells (cytosol)[3H]methyltrienolone 1 nM 0.8 nM mibolerone (1 μM) 24 h 4° C. Scintillation counting.Zava, D. T et al. (1979) Endocrinology, 104: 1007-1012.The results are expressed as a percent of control specific binding measured specific binding*100 control specific binding and as a percent inhibition of control specific binding 100-(measured specific binding*100) control specific binding obtained in the presence of compound.
- Radioligand Binding Assay In the GR radioligand binding assay, test compounds were serially diluted in semi-log steps (10 concentrations) with a final concentration of 10 μM. Test compounds (1 μL) and controls (1 μL) in 100% DMSO were added to 96 Greiner V-bottom polypropylene plates. 0% control was 6.7% DMSO (final concentration in assay) and 100% control was 6.7 μM Dexamethasone.The full length GR was diluted to a final concentration of 3.3% (0.495 mg/ml) in assay buffer (20 mM Tris-HCl, 1 mM EDTA, 10% (w/v) Glycerol, 20 mM Sodium molybdate, pH 7.4). 45 μL of GR was added to each well and the plates were incubated for 15 min at room temperature. 3H-dexamethasone solution was diluted to a concentration of 70 nM in assay buffer (7 nM final assay concentration) and 5 μL was added to each well. The samples were mixed for 5 min using a plate shaker at 700 rpm, before incubation for 2 h at room temperature.50 μL ice-cold charcoal solution (pH 7.4: 2% Charcoal, 0.2% Dextran T70 in 20 mM Tris-HCl, 1 mM EDTA and 20 mM Sodium molybdate) was added to each well and the samples were mixed on plate shaker for 5 minutes.
- In Vitro GR Luciferase Reporter Assay Cell Line: CHO-K1-GR-MMTV-Luc reporter cellsCulture Media: DMEM (with phenol red)+10% FBSAssay Media: DMEM (without phenol red)+10% CSSCulture CHO-K1-GR-MMTV-Luc reporter cells in 15 cm plates in Culture Media at conditions less than 90% confluence.Prepare 200×DMSO 1:5 serial dilutions of control and test compounds in 96-well non-sterile V bottom plate in DMSO, 8 serial dilutions for each compound.Prepare 5× Assay Media diluted compound serial dilutions in 96-well non-sterile V bottom plate: Add 97.5 uL/well of Assay Media into 96-well then add 2.5 ul of 200× concentration of compounds and mix well.Seed cells for Antagonist Assay: 1.5×106 CHO-K1-GR-MMTV-Luc reporter cells were seeded in a Corning 3707 flat clear bottom 384-well white TC plate in 20 ul of Assay Media containing 12.5 nM Dexamethasone (final concentration=10 nM).Add compounds: 5 ul of assay media diluted compounds were added to appropriate wells and followed a quick spin (1000 rpm, 10 sec) to bring media and cells to the bottom of plate. The plates were covered with SealMate film to avoid evaporation and placed in 37° C. incubator for approximately 18-24 hours.Read plates: Equilibrate appropriate amount of Promega OneGlo luciferase reagent to room temperature. Remove the plates from incubator and add 25 uL of OneGlo reagent/well by multiple channel pipette and read the plates with Tecan F500 luminometer within 3 minutes.The ability of the compounds disclosed herein to inhibit GR activity was quantified and the respective IC50 value was determined.
- Inhibition Assay The CYP3A4 inhibition assays were conducted using Human Liver Microsomes purchased from Invitrogen and designed to screen potential inhibitors of Cytochrome P450 in physiological condition. Initially the following reagents/mixtures were prepared: (i) Assay buffer: 0.1 M Phosphate buffer pH 7.4 (ii) Cofactor: 15 mM stock was prepared in assay buffer. Final concentration in assay 1.5 mM (iii) Substrate 50 mM DMSO stock was prepared for testosterone. From this a 10 mM sub-stock was prepared in MeCN. Further, a working stock solution of 700 μM was prepared in assay buffer. Final concentration in assay 70 μM (iv) Enzyme: 20 mg/mL stock was provided by manufacturer. Final concentration in assay was 0.5 mg/mL. At the start of the experiment, various concentrations of compound (7 different concs.) or positive control (Ketoconazole at a single concentration) were prepared in assay buffer. For 100 μL of final reaction system, 2.5 μL of HLM (20 mg/ml), 50 μL of test compound/reference compound from each concentration was added. Subsequently, 10 μL of substrate (testosterone 700 μM) and 10 μL of Cofactor (NADPH; 15 mM) were added. The volume was increased to 100 μL by adding assay buffer. DMSO concentration was kept as 0.5% uniform across all the reactions. The reaction was then allowed to incubate for 45 min at 37° C. After completion of the incubation period, the reaction was terminated by addition of 200 μL of chilled MeCN containing internal standard (Dexamethasone). The samples were than centrifuged and supernatants were analyzed using LCMS/MS.
- GRE Agonist Assay A reporter cell line (ChagoK1 18:7:2 s4/GRE) was established by stable transfection of the human bronchogenic carcinoma cell line, ChaGo K1 (ATCC: HTB 168) with a MMTV-GRE-LacZ reporter construct. The generated cell line allows for identification of compounds showing agonist activity at the human glucocorticoid receptor (GR) via induction of LacZ gene expression. Ligand-activated GR binds to the Glucocorticoid Response Element (GRE) in the promoter of the LacZ gene and transcription is initiated. The resulting beta-galactosidase activity is measured through a colour reaction (change in absorbance).Cryo-preserved ChagoK1 18:7:2 s4/GRE cells were suspended in RPMI medium with 10% FBS, 1% NEAA and 1% sodium pyruvate, and seeded as 50000 cells/200 ul/well in 96-well plates and cultured at 37° C. with 5% CO2 and 95% humidity for 24 hours. 1 μl compound was added at different concentrations to the cells and incubated for another 24 hours. Cells were washed once in PBS and lysed with 50 μl of 0.1% Triton-X for 10 min at room temperature. 40 μl of reaction mixture (2.5 mM MgCl2, 0.1 M β-mercapto ethanol, 1.7 mg/ml ONPG and 42.5 mM sodium phosphate, pH 7.5), was added to each well and kept at 37° C. for 60 min. The reaction was then terminated by addition of 100 μl stop solution (300 mM glycine, 15 mM EDTA, pH 11.3, adjusted with NaOH). The plates were measured at 420 nm for absorbance in a SpectraMax reader (Molecular Device).The relative efficacy (% effect) of a compound is calculated based on the full agonist effect of dexamethasone: % Effect=((Sample abs−min abs)/(max abs−min abs))×100To calculate EC50, max, min and slope factor for each compound, a concentration response curve is fitted by plotting % Effect versus compound concentration using the 4 parameter logistic equation: y=A+(B−A)/(1+((10C)/x)D)Where A=min Y, B=max Y, C=log EC50 and D=Slope factor.
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 oC water bath and then transferred to 20 mL of PHH thawing medium (Sigma , InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells.50 μl of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFLO384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 oC water bath and then transferred to 20 mL of PHH thawing medium (Sigma, InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells, and then 50 µl of cells were transferred to each well 384-well collagen I coated plate withappropriate liquid handling equipment, e.g. Integra VI AFL0384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol.
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 oC water bath and then transferred to 20 mL of PHH thawing medium (Sigma , InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells.50 μl of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFLO384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufactures protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 °C water bath and then transferred to 20 mL of PHH thawing medium (Sigma, InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells, and then 50 mΐ of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFL0384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902- 1 OOmg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72 -hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 °C water bath and then transferred to 20 mL of PHH thawing medium (Sigma, InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma, InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells, and then 50 μL of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFL0384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco,Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco,Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwichculture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).