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Details of RareLED ID Assay_1013
Primary information
GENECTSH
NUMBER OF ASSAY792
ASSAY IDs1179619 1179617 1179615 1179613 1179618 1179616 1179614 1179612 1343706 1343705 1125412 1125414 1125413 1125411 1125410 1633 257083 51216 51175 50266 48400 1063742 1063741 273238 273237 273236 360106 273239 48397 620709 1343001 1343000 1343007 1343003 448875 51215 51214 156908 1343163 1125415 723754 723753 1179620 1343002 1342999 1343006 219365 48392 288395 288394 219375 219226 51359 51185 51181 51179 50439 448879 448876 448871 219368 219367 219366 448880 448878 448874 448873 448177 448176 365005 288393 242263 219376 219370 448877 1257963 256977 256976 2214 448872 256992 256978 256975 1344290 307166 307165 51221 50267 51184 47812 51361 51223 51177 50437 448910 448908 448907 448904 448902 448901 448900 448899 448898 448897 448896 448895 448894 448893 448892 448891 448890 448889 448888 448887 448885 448884 448883 448881 448870 448868 448867 448866 448865 448864 4488
Assay Name1. Inhibition of human liver cathepsin H,2. Non-competitive inhibition of goat liver cathepsin H using Leu-betaNA substrate by Lineweaver-Burk plot analysis,3. Competitive inhibition of goat liver cathepsin H using Leu-betaNA substrate by Lineweaver-Burk plot analysis,4. Inhibition of goat liver cathepsin H using Leu-betaNA substrate pre-incubated 30 mins before substrate addition by colorimetry,5. Inhibition of human liver cathepsin B,6. Non-competitive inhibition of goat liver cathepsin B using BANA substrate by Lineweaver-Burk plot analysis,7. Competitive inhibition of goat liver cathepsin B using BANA substrate by Lineweaver-Burk plot analysis,8. Inhibition of goat liver cathepsin B using BANA substrate pre-incubated 30 mins before substrate addition by colorimetry,9. Fluorescent Substrate Kinetic Assay: A substrate peptide is labeled at the N-terminus with tryptophan and at the C-terminus with the fluorophore MR121 (for cathepsin D the 10 amino acid peptide WTSVLMAAPC-MR121 was used; for cathepsin E, MR121-CKLVFFAEDW was used). The fluorescent substrate cathepsin D and cathepsin E kinetic assays were performed at room temperature in 384-well microtiter plates (black with clear flat bottom, non binding surface plates from Corning) in a final volume of 51 ul. The test compounds were serially diluted in DMSO (15 concentrations, 1/3 dilution steps) and 1 ul of diluted compounds were mixed for 10 min with 40 ul of cathepsin D (from human liver, Calbiochem) diluted in assay buffer (100 mM sodium acetate, 0.05% BSA, pH 5.5; final concentration: 200 nM) or with 40 ul of recombinant human cathepsin E (R&D Systems) diluted in assay buffer (100 mM sodium acetate, 0.05% BSA, pH 4.5; final concentration: 0.01 nM). After addition of 10 ul of the cathepsin D subs,10. Fluorescent Substrate Kinetic Assay: A substrate peptide is labeled at the N-terminus with tryptophan and at the C-terminus with the fluorophore MR121 (for cathepsin D the 10 amino acid peptide WTSVLMAAPC-MR121 was used; for cathepsin E, MR121-CKLVFFAEDW was used). The fluorescent substrate cathepsin D and cathepsin E kinetic assays were performed at room temperature in 384-well microtiter plates (black with clear flat bottom, non binding surface plates from Corning) in a final volume of 51 ul. The test compounds were serially diluted in DMSO (15 concentrations, 1/3 dilution steps) and 1 ul of diluted compounds were mixed for 10 min with 40 ul of cathepsin D (from human liver, Calbiochem) diluted in assay buffer (100 mM sodium acetate, 0.05% BSA, pH 5.5; final concentration: 200 nM) or with 40 ul of recombinant human cathepsin E (R&D Systems) diluted in assay buffer (100 mM sodium acetate, 0.05% BSA, pH 4.5; final concentration: 0.01 nM). After addition of 10 ul of the cathepsin D subs,11. Non-competitive inhibition of cathepsin H in goat liver using Leu-betaNA as substrate by colorimetric analysis,12. Inhibition of human liver cathepsin B,13. Competitive inhibition of goat brain cathepsin B,14. Non-competitive inhibition of cathepsin B in goat liver using alpha-N-benzoyl-D,L-arginine-2-naphthylamide as substrate by colorimetric analysis,15. Competitive inhibition of cathepsin B in goat liver using alpha-N-benzoyl-D,L-arginine-2-naphthylamide as substrate by colorimetric analysis,16. Cathepsin L probe dose-response testing,17. Inhibitory activity against humanized rabbit cathepsin K,18. In vitro inhibitory activity against human cathepsin L, using 2 uM of Z -Leu-Arg-AMC as substrate,19. In vitro inhibitory activity against Human Cathepsin K using 2 uM of Z -Leu-Arg-AMC as substrate,20. In vitro inhibitory activity against human cathepsin B, using 2 uM of Z -Leu-Arg-AMC as substrate,21. In vitro inhibitory activity against human cathepsin K, using 2 uM of Z -Leu-Arg-AMC as substrate,22. Inhibition of human Cathepsin H using Arg-aminomethylcoumarin as substrate,23. Inhibition of human Cathepsin H using Arg-aminomethylcoumarin as substrate preincubated for 30 mins followed by substrate addition,24. Inhibition of cathepsin L,25. Inhibition of cathepsin K,26. Inhibition of cathepsin B,27. Inhibition of human recombinant cathepsin H after 10 mins,28. Inhibition of cathepsin S,29. Concentration required to inhibit 50% of cysteine protease cathepsin K of human,30. Reversible inhibition of human recombinant cathepsin B assessed as formation of fluorescent degradation product AMC using Z-Arg-Arg-AMC as substrate by Michaelis-Menten method,31. Inhibition Assay: Assay buffers consist of 20 mM citric acid, 60 mM disodium hydrogen orthophosphate, 1 mM EDTA, 0.1% CHAPS, 4 mM DTT, pH 5.8 for legumain, 50 mM dihydrogen sodium orthophosphate, 1 mM EDTA, 5 mM DTT, pH 6.25 for cathepsin B and cathepsin Land 100 mM Tris, 0.1% CHAPS, 10% sucrose, 10 mM DTT, pH 7.4 for caspase-3. Concentrations of substrates during the measurement were 10 nM (legumain, cathepsin Land caspase-3) and 50 nM (cathepsin B) and concentration of enzymes were 100 nM for cathepsin Land caspase-3, 270 nM for legumain and 360 nM for cathepsin B. Each enzyme was incubated with inhibitor concentrations ranging from 1 nM to 1 mM in the presence of the substrates.,32. Inhibition Assay: Assay buffers consist of 20 mM citric acid, 60 mM disodium hydrogen orthophosphate, 1 mM EDTA, 0.1% CHAPS, 4 mM DTT, pH 5.8 for legumain, 50 mM dihydrogen sodium orthophosphate, 1 mM EDTA, 5 mM DTT, pH 6.25 for cathepsin B and cathepsin Land 100 mM Tris, 0.1% CHAPS, 10% sucrose, 10 mM DTT, pH 7.4 for caspase-3. Concentrations of substrates during the measurement were 10 nM (legumain, cathepsin Land caspase-3) and 50 nM (cathepsin B) and concentration of enzymes were 100 nM for cathepsin Land caspase-3, 270 nM for legumain and 360 nM for cathepsin B. Each enzyme was incubated with inhibitor concentrations ranging from 1 nM to 1 mM in the presence of the substrates.,33. Fluorescence Resonance Energy Transfer (FRET) Assay : The recombinant human BACE1, Cathepsin D, and Cathepsin E enzymes were purchased from R&D Systems (catalog numbers are 931-AS, 1014AS and 1294AS, respectively). The enzymatic inhibition activity assays were determined using the fluorescence resonance energy transfer (FRET) assay. The assays were performed in a 384-well plate format. The recombinant human BACE-1 enzyme (R&D Systems, catalog#931-AS) was diluted to 20 ng/ inverted question markL in assay buffer (100 mM sodium acetate pH 4.0), 10 point 1:3 serial dilutions of compound in DMSO were preincubated with the enzyme for 15 min at room temperature. The concentration of Cathepsin D was 20 ng/ inverted question markL, and 1 ng/ inverted question markL of Cathepsin E was used. CatD and CatE were activated by incubation in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) at room temperature for 30 min. Subsequently, the rhBACE-1 substrate (R&D Systems, catalog# ES004), the Cathepsin ,34. Fluorescence Resonance Energy Transfer (FRET) Assay : The recombinant human BACE1, Cathepsin D, and Cathepsin E enzymes were purchased from R&D Systems (catalog numbers are 931-AS, 1014AS and 1294AS, respectively). The enzymatic inhibition activity assays were determined using the fluorescence resonance energy transfer (FRET) assay. The assays were performed in a 384-well plate format. The recombinant human BACE-1 enzyme (R&D Systems, catalog#931-AS) was diluted to 20 ng/ inverted question markL in assay buffer (100 mM sodium acetate pH 4.0), 10 point 1:3 serial dilutions of compound in DMSO were preincubated with the enzyme for 15 min at room temperature. The concentration of Cathepsin D was 20 ng/ inverted question markL, and 1 ng/ inverted question markL of Cathepsin E was used. CatD and CatE were activated by incubation in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) at room temperature for 30 min. Subsequently, the rhBACE-1 substrate (R&D Systems, catalog# ES004), the Cathepsin ,35. Inhibition of cathepsin H after 10 to 15 mins by fluorescence assay,36. Compound was measured for inhibition of collagenolytic of human Cathepsin L,37. Compound was measured for inhibition of collagenolytic of human Cathepsin L,38. Compound was tested for inhibition of pepsin.,39. Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37 degrees C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.,40. Inhibition of human liver cathepsin H,41. Noncompetitive inhibition of human liver cathepsin H endopeptidase activity using R-AMC substrate assessed as inhibition constant for enzyme-substrate-inhibitor complex,42. Uncompetitive inhibition of human liver cathepsin H endopeptidase activity using R-AMC substrate assessed as inhibition constant for enzyme-substrate-inhibitor complex,43. Competitive inhibition of goat brain cathepsin B,44. Inhibition Assay: Assay buffers consist of 20 mM citric acid, 60 mM disodium hydrogen orthophosphate, 1 mM EDTA, 0.1% CHAPS, 4 mM DTT, pH 5.8 for legumain, 50 mM dihydrogen sodium orthophosphate, 1 mM EDTA, 5 mM DTT, pH 6.25 for cathepsin B and cathepsin Land 100 mM Tris, 0.1% CHAPS, 10% sucrose, 10 mM DTT, pH 7.4 for caspase-3. Concentrations of substrates during the measurement were 10 nM (legumain, cathepsin Land
ACTIVITYSubstance BioActivity: 1 Active, 1 Tested Substance BioActivity: 5 Active, 6 Tested Substance BioActivity: 24 Active, 24 Tested Substance BioActivity: 5 Active, 5 Tested Substance BioActivity: 1 Active, 1 Activity <= 1 µM, 1 Tested Substance BioActivity: 2 Active, 6 Tested Substance BioActivity: 19 Active, 3 Activity <= 1 µM, 24 Tested Substance BioActivity: 5 Active, 3 Activity <= 1 µM, 5 Tested Substance BioActivity: 2 Active, 2 Tested Substance BioActivity: 2 Active, 2 Tested Substance BioActivity: 7 Active, 13 Tested Substance BioActivity: 1 Active, 1 Activity <= 1 µM, 1 Tested Substance BioActivity: 1 Active, 1 Activity <= 1 nM, 1 Activity <= 1 µM, 1 Tested Substance BioActivity: 8 Active, 1 Activity <= 1 µM, 9 Tested Substance BioActivity: 5 Active, 2 Activity <= 1 µM, 5 Tested Substance BioActivity: 1 Active, 1 Activity <= 1 µM, 1 Tested Substance BioActivity: 6 Active, 1 Activity <= 1 nM, 5 Activity <= 1 µM, 6 Tested Substance BioActivity: 22 Active, 5 Activity <= 1 µ