- Paulsen, ES; Villadsen, J; Tenori, E; Liu, H; Bonde, DF; Lie, MA; Bublitz, M; Olesen, C; Autzen, HE; Dach, I; Sehgal, P; Nissen, P; Møller, JV; Schiøtt, B; Christensen, SB Water-mediated interactions influence the binding of thapsigargin to sarco/endoplasmic reticulum calcium adenosinetriphosphatase. J Med Chem 56: 3609-19 (2013)
- Skytte, DM; Møller, JV; Liu, H; Nielsen, HØ; Svenningsen, LE; Jensen, CM; Olsen, CE; Christensen, SB Elucidation of the topography of the thapsigargin binding site in the sarco-endoplasmic calcium ATPase. Bioorg Med Chem 18: 5634-46 (2010)
- ChEMBL_2124957 (CHEMBL4834190) Inhibition of IL2 in thapsigargin stimulated human whole blood
- ChEMBL_2124958 (CHEMBL4834191) Inhibition of TNF-alpha in thapsigargin stimulated human whole blood
- ChEMBL_990824 (CHEMBL2444391) Inhibition of thapsigargin-induced autophosphorylation of PERK in human A549 cells preincubated for 1 hr followed by thapsigargin-induction measured after 1 hr by Western blotting analysis
- ChEMBL_1467749 (CHEMBL3412175) Inhibition of PERK in human HT1080 cells assessed as inhibition of thapsigargin-induced CHoP mRNA expression preincubated for 1 hr followed by thapsigargin-induction measured after 2 hrs by bDNA assay
- ChEMBL_1513934 (CHEMBL3611616) Antagonist activity at Kv1.3 in human whole blood assessed as inhibition of thapsigargin-induced IL-2 secretion incubated for 30 prior to thapsigargin induction measured after 48 hrs by electrochemilluminescent immunoassay
- ChEMBL_1513935 (CHEMBL3611617) Antagonist activity at Kv1.3 in human whole blood assessed as inhibition of thapsigargin-induced IFN-gamma secretion incubated for 30 prior to thapsigargin induction measured after 48 hrs by electrochemilluminescent immunoassay
- ChEMBL_1661706 (CHEMBL4011318) Inhibition of thapsigargin-stimulated 15-LOX in human primary polymorphonuclear leukocytes using arachidonic acid as substrate preincubated for 5 mins followed by thapsigargin stimulation for 5 mins by RP-HPLC method
- ChEMBL_2156967 (CHEMBL5041627) Inhibition of 5-LO in thapsigargin stimulated human PMNL cells assessed as reduction in 5-LO product level preincubated for 5 mins followed by thapsigargin addition and measured after 15 mins by RP-HPLC analysis
- ChEMBL_2288536 Inhibition of thapsigargin-induced XBP1-luciferase activation in human HeLa cells incubated for 24 hrs by luciferase assay
- ChEMBL_862852 (CHEMBL2172954) Inhibition of thapsigargin-induced autophosphorylation of PERK in human A549 cells after 2 hrs by Western blotting
- ChEMBL_1467748 (CHEMBL3412174) Inhibition of PERK-mediated protein synthesis in human U2OS cells harboring thapsigargin-induced ER stress assessed as incorporation of L-azidohomoalanine into protein preincubated for 1 hr followed by thapsigargin induction measured over 30 mins by AlexaFluor 488 alkyne dye-based HCS assay
- ChEMBL_1875709 (CHEMBL4377103) Inhibition of human TRPC6 expressed in thapsigargin pretreated HEK293 cells assessed as reduction in GPCR-induced calcium entry by fluo-4/AM dye based assay
- ChEMBL_1875685 (CHEMBL4377079) Inhibition of mouse TRPC5 expressed in thapsigargin treated HEK293/TREx cells assessed as reduction in intracellular calcium level in presence of GPCR agonist by fluorescence method
- ChEMBL_1875687 (CHEMBL4377081) Inhibition of YFP-tagged human TRPC3 expressed in thapsigargin treated HEK293 cells assessed as reduction in amix-induced calcium level by fluo-4 dye based fluorescence assay
- ChEMBL_1875688 (CHEMBL4377082) Inhibition of YFP-tagged human TRPC6 expressed in thapsigargin treated HEK293 cells assessed as reduction in amix-induced calcium level by fluo-4 dye based fluorescence assay
- ChEMBL_1875689 (CHEMBL4377083) Inhibition of YFP-tagged mouse TRPC7 expressed in thapsigargin treated HEK293 cells assessed as reduction in amix-induced calcium level by fluo-4 dye based fluorescence assay
- ChEMBL_1899752 (CHEMBL4401867) Inhibition of thapsigargin-induced IRE1alpha (unknown origin) expressed in HEK293 cells assessed as reduction in IRE1alpha-dependent splicing of XBP1u-luciferase mRNA by luciferase reporter gene assay
- ChEMBL_1875707 (CHEMBL4377101) Inhibition of ORAI/STIM1 in thapsigargin treated rat RBL2H3 cells assessed as reduction in store-operated calcium entry after 5 mins by fura-2-AM dye based assay
- ChEMBL_2437021 Inhibition of IRE1alpha RNase activity (unknown origin) using XBP1 as substrate preincubated for 1 hr followed by thapsigargin stimulation for 5 hrs by One-Glo luciferase reagent based luminescence assay
- ChEMBL_2437042 Inhibition of IRE1alpha in human KMS-11 cells assessed as reduction in XBP1 splicing preincubated for 24 hrs followed by thapsigargin addition and measured after 30 mins by luminescence assay
- ChEMBL_1912162 (CHEMBL4414745) Inhibition of 5-LO in human PMNL cells assessed as reduction in leukotriene formation preincubated for 5 mins followed by thapsigargin stimulation measured after 15 mins by RP-HPLC analysis
- ChEMBL_2019488 (CHEMBL4673066) Inhibition of IRE1alpha (unknown origin) assessed as XBP-1 slicing luciferase activity incubated for 1 hr followed by stimulation with thapsigargin measured after 5 hrs by luciferase reporter gene assay
- ChEMBL_1875686 (CHEMBL4377080) Inhibition of YFP-tagged mouse TRPC4 beta expressed in thapsigargin treated HEK293 cells co-expressing M3 receptor assessed as reduction in carbachol-induced calcium level by fluo-4 dye based fluorescence assay
- ChEMBL_2437043 Inhibition of IRE1alpha in human KMS-11 cells assessed as reduction in XBP1 splicing preincubated for 24 hrs followed by thapsigargin addition and measured after 30 mins under FCS medium by luminescence assay
- ChEMBL_2437044 Inhibition of IRE1alpha RNase activity (unknown origin) using XBP1 as substrate preincubated for 1 hr followed by thapsigargin stimulation for 5 hrs under FCS medium by One-Glo luciferase reagent based luminescence assay
- NFAT Transcriptional Activity HEK 293 cells were stably transfected with a NFAT-Luc reporter gene. 30,000-80,000 cells were seeded per well. Test compounds from this invention were added to the cells at different concentrations. Thapsigargin (TG) was added after 10 mins and the cells were incubated for 4-8 h. NFAT transcriptional activity was measured using BrightGlo reagent (Promega USA). Luminescence observed in cells treated with thapsigargin was considered 100% maximal signal and the reduced fluorescent signal observed in the presence of test compounds was expressed as percent inhibition of the maximal signal. The data was analyzed using 4-parametric sigmoidal dose response (variable slope) curve-fit.
- In-Vitro CRAC Channel Inhibition Assay in Jurkat Cells Inhibition of CRAC channels was determined following thapsigargin (Sigma, Cat #T9033) induced endoplasmic calcium release in Jurkat cells, (see Yasurio Yonetoky et. al Bio. & Med Chem. 14 (2006) 4750-4760). Cells were centrifuged and re-suspended in equal volumes ° f Ca2+ and Mg2+ free Hanks buffer and Fluo-8 NW dye (ABD Bioquest, Inc., Sunnyvale, Calif.) loading solution at 2×105 cells/100 μl/well in 96-well black plate. Plate is incubated at 37° C./5% CO2 for 30 min followed by further 15 min incubation at room temperature. Test compounds (DMSO stocks diluted in Ca2+ and Mg2+ free Hanks buffer) at desired concentrations were added to the wells and incubated for 15 min. Thapsigargin (1 μM final concentration) was added to the wells and incubated for 15 min to inhibit the Sarco-endoplasmic reticulum Ca2+ ATPase pump thereby depleting endoplasmic calcium and raising cytosolic calcium concentrations. Store-operated calcium entry was initiated by adding extracellular Ca2+ to a final concentration of 1.8 mM. Fluorescence was monitored over 5 min on a plate reader (BMG Labtech., Germany) with excitation at 485 nm and; an emission wavelength at 520 nm. Data were analyzed using GraphPad Prism. IC50 for each compound was determined based on the percent inhibition of thapsigargin-induced calcium influx into cells.
- In-Vitro Inhibition Assay Inhibition of CRAC channels was determined following thapsigargin (Sigma, Cat # T9033) induced endoplasmic calcium release in Jurkat cells. (see Yasurio Yonetoky et. al Bio. & Med. Chem. 14 (2006) 4750-4760). Cells were centrifuged and re-suspended in equal volumes f Ca2+ and Mg2+ free Hanks buffer and Fluo-8 NW dye (ABD Bioquest, Inc., Sunnyvale, Calif.) loading solution at 2x105 cells/100 ul/well in 96-well black plate. Plate is incubated at 37 C., 5% CO2 for 30 min followed by further 15 min incubation at room temperature. Test compounds (DMSO stocks diluted in Ca2+ and Mg2+ free Hanks buffer) at desired concentrations were added to the wells and incubated for 15 mM Thapsigargin (1 uM final concentration) was added to the wells and incubated for 15 min to inhibit the Sarco-endoplasmic reticulum Ca2+ ATPase pump thereby depleting endoplasmic calcium and raising cytosolic calcium concentrations.
- Inhibition Assay Inhibition of CRAC channels was determined following thapsigargin (Sigma, Cat # T9033) induced endoplasmic calcium release in Jurkat cells. (see Yasurio Yonetoky et. al Bio. & Med. Chem. 14 (2006) 4750-4760). Cells were centrifuged and resuspended in equal volumes f Ca2+ and Mg2+ free Hanks buffer and Fluo-8 NW dye (ABD Bioquest, Inc., Sunnyvale, Calif.) loading solution at 2x105 cells/100 ul/well in 96-well black plate. Plate is incubated at 37 C./5% CO2 for 30 mM followed by further 15 mM incubation at room temperature. Test compounds (DMSO stocks diluted in Ca2+ and Mg2+ free Hanks buffer) at desired concentrations were added to the wells and incubated for 15 mM. Thapsigargin (1 uM final concentration) was added to the wells and incubated for 15 min to inhibit the Sarco-endoplasmic reticulum Ca2+ ATPase pump thereby depleting endoplasmic calcium and raising cytosolic calcium concentrations. Store-operated calcium entry was initiated by adding extracellular Ca2+.
- SOCE inhibition Jurkat E6.1 cells were seeded at a density of 1-2×105 cells per well in calcium-4 dye prepared in calcium free HBSS (Sigma, USA). Test compounds from this invention were added to the cells at different concentrations. This was followed by the addition of thapsigargin (TG), a SERCA inhibitor, to empty the stores of calcium. Calcium chloride was added to the cells after 10-30 min to induce calcium influx and the fluorescence was measured for 10 min using the FLIPR-Tetra detection system. Fluorescence was also measured using a plate reader at 485 nm excitation and 520 nm emission (Synergy2, Biotek, USA) after 30-90 minutes of calcium addition. Fluorescence observed in cells treated with Thapsigargin and calcium chloride solution was considered 100% maximal signal and the reduced fluorescent signal observed in the presence of test compounds was expressed as percent inhibition of the maximal signal. The dose response data was analyzed using 4-parametric sigmoidal dose response (variable slope) curve-fit.
- Cell-Based Assay Initial cell-based XBP-1 mRNA splicing assays confirmed IRE-1α inhibition with several potent 5-bromo and 6 bromo o-vanillins. HEK293 cells were incubated with compound either overnight or for 2 hours prior to IRE-1α activation with the UPR inducing reagent thapsigargin. IRE-1α mediated XBP-1 splicing was measured by RT-PCR using XBP-1 specific primers flanking the 26 bp intron excised by IRE-1α. The results are shown in FIG. 1. It can be observed that at the higher concentrations, there is relatively more of the unspliced XBP-1 (upper band: substrate) compared to the spliced form (lower band: product).