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Details of RareLED ID Assay_1011
Primary information
GENECTSD
NUMBER OF ASSAY170
ASSAY IDs1343706 1344290 271447 48010 1343003 331709 48033 48029 1186405 1186403 617484 444269 48031 48007 48034 1301525 668998 1137748 254279 240496 219358 48032 48014 48011 48040 48180 1074036 1074035 761331 741674 707181 705011 678315 551757 526843 461698 459623 438584 1344694 1343988 48043 48042 48041 48035 48028 48020 48009 48008 1267558 1200655 290612 48021 1115781 673047 662321 640382 464343 459580 437082 1173618 771312 762872 746789 729136 525776 419439 398171 391464 389515 408675 408453 332043 321131 320514 316808 316776 314038 314033 313980 310772 307638 298146 297412 296065 286640 281967 281500 273398 266640 238412 48027 219361 219359 48045 48039 48026 48015 48012 48030 48019 48006 48017 48016 48186 48185 48025 48018 165449 1230061 1194086 1193714 1190883 1176942 1170259 1161922 1158811 778922 775626 754464 738163 727308 711207 695115 692718 674499 590330 5377
Assay Name1. 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,2. In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCAGKPILFFRLK(Dnp)d-R-NH2 (Enzo Life Sciences, Lorrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium a,3. Inhibition of human cathepsin D,4. Inhibitory activity against human liver Cathepsin D using Cathepsin D assay.,5. 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 ,6. Inhibition of human cathepsin D by FRET assay,7. Inhibitory activity of compound against human Cathepsin D,8. In vitro inhibition of human liver cathepsin D.,9. Inhibition of human liver cathepsin D using MCA-labeled GKPILFFRLK(Dnp)-D-R-NH2 peptide by fluorescence-based assay,10. Inhibition of cathepsin D (unknown origin),11. Inhibition of cathepsin D by FRET assay,12. Inhibition of cathepsin D assessed as reduction in polarization after 110 mins by oregon green based fluorescence polarization assay,13. Compound was tested for inhibitory activity against cathepsin D,14. Compound was tested for inhibitory activity against cathepsin D,15. Inhibitory activity against human Cathepsin D; Range,16. Inhibition of Cathepsin D (unknown origin) by fluorescence assay,17. Inhibition of human recombinant CatD using Mca-GKPILFFRLK(DNP)-dR-NH2 as substrate after 1 hr by fluorescence analysis,18. Inhibition of human cathepsin-D using DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS as substrate preincubated for 30 mins by FRET assay,19. Binding affinity af the compound towards cathepsin D,20. Inhibitory concentration against human Cathepsin D,21. In vitro binding affinity for human cathepsin D.,22. Compound was tested for the inhibitory activity against human liver cathepsin D,23. Inhibitory concentration against cathepsin D,24. Inhibitory concentration against Cathepsin D,25. Inhibition of rabbit liver cathepsin D at pH 4.0,26. Compound was tested for inhibition of cathepsin D,27. Inhibition of human liver cathepsin D measured for 1 hr by FRET assay,28. Inhibition of human spleen cathepsin D measured for 1 hr by FRET assay,29. Inhibition of Cathepsin D (unknown origin) by FRET assay,30. Inhibition of human spleen cathepsin D using 5-FAM/QXL as peptide substrate after 1 hr by FRET assay,31. Inhibition of cathepsin D,32. Inhibition of human cathepsin D,33. Inhibition of human cathepsin D,34. Inhibition of cathepsin D,35. Inhibition of cathepsin D by FRET assay,36. Inhibition of human cathepsin D by FRET assay,37. Inhibition of human liver cathepsin D,38. Inhibition of human cathepsin D by FRET,39. Inhibition Assay: Inhibition assay using Cathepsin D.,40. In Vitro Enzymatic Cathepsin D FRET Assay: Recombinant Cat D was expressed in CHO cells. The assay buffer for CathepsinD is 0.05 M citrate pH 3.5, 10% DMSO final, 5 mM CHAPS. The Cat D enzyme (9 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, is added thereto. The assays are effectively started by the addition of different FRET substrates (20 nM for Cat D) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The Cat D substrate peptide sequence is based on sequence #1 of Table 1 from Gulnik et al. FEBS Letters v413 p 379-384 1997. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (Cat D excitation 500 nm and emission 580 nm).,41. In vitro inhibition of cathepsin D.,42. Compound was evaluated for the inhibition of cathepsin D at concentration of 4.15 ug/mL,43. Compound was evaluated for the inhibition of cathepsin D at an enzyme level of 50 ng/mL,44. Inhibitory activity against human cathepsin D,45. Compound was evaluated for aspartyl protease inhibition selectivity relative to Cathepsin D,46. The compound was tested for its inhibitory activity against human cathepsin D,47. Inhibitory activity against human cathepsin D was determined,48. Inhibition against human cathepsin D was determined,49. Inhibition of cathepsin D (unknown origin),50. Inhibition of CatD (unknown origin) by fluorescence polarization assay,51. Inhibition of cathepsin D,52. The inhibitory concentration value was measured on Human cathepsin D at 31000 nM,53. Inhibition of cathepsin D (unknown origin) using hemoglobin as substrate after 30 min by spectrophotometric analysis,54. Inhibition of cathepsin D by FRET assay,55. Inhibition of cathepsin D,56. Inhibition of human cathepsin D using DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS as substrate by FRET analysis,57. Inhibition of Cathepsin D,58. Inhibition of human cathepsin D,59. Inhibition of human liver cathepsin D,60. Inhibition of cathepsin-D (unknown origin),61. Inhibition of human cathepsin-D,62. Inhibition of cathepsin-D (unknown origin) using C-terminal biotinylated peptide substrate treated 30 mins before addition of peptide substrate measured after 110 mins by fluorescence polarization assay,63. Inhibition of cathepsin-D (unknown origin) after 110 mins by fluorescence polarization assay,64. Inhibition of human recombinant cathepsin-D expressed in baculovirus-infected insect sf9 cells using Ac- E-D(EDANS)-KPILFFRLG-K(Dabcyl)-E-Amid as substrate incubated for 1 hr prior to substrate addition measured after 1 hr by FRET-based enzymatic assay,65. Inhibition of cathepsin-D,66. Inhibition of cathepsin-D,67. Inhibition of cathepsin-D,68. Inhibition of cathepsin D,69. Inhibition of human cathepsin D,70. Inhibition of cathepsin D expressed in CHO cells after 1 hrs by spectrophotometer,71. Inhibition of cathepsin D,72. Inhibition of cathepsin D,73. Inhibition of cathepsin D,74. Inhibition of cathepsin D,75. Inhibition of cathepsin D,76. Binding affinity to human cathepsin D,77. Inhibition of Cathepsin D expressed in CHO cells by FRET assay,78. Inhibition of cathepsin D,79. Inhibition of cathepsin D,80. Inhibition of human recombinant cathepsin D,81. Inhibition of cathepsin D,82. Inhibition human cathepsin D,83. Inhibition of cathepsin D,84. Inhibition of cathepsin D expressed in HEK293 cells,85. Inhibition of human cathepsin D,86. Inhibition of cathepsin D,87. Inhibition of Cathepsin D,88. Inhibition of cathepsin D by FRET based peptide cleavage assay,89. Inhibition of Cathepsin D,90. Inhibitory concentration against the human Cathepsin D,91. Binding affinity towards human cathepsin D.,92. Inhibitory concentration against cathepsin D..,93. Binding affinity against cathepsin D,94. Inhibitory concentration against cathepsin D,95. Inhibitory concentration against Human cathepsin D.,96. Binding affinity against human cathepsin D,97. Inhibitory concentration against human cathepsin D.,98. Inhibitory concentration against Human cathepsin D,99. Compound was tested for inhibition human Cathepsin D,100. The compound was tested for inhibition of Human Cathepsin D,101. Ability to inhibit the human Native cathepsin D aspartic protease enzyme,102. Selectivity for their inhibitory potency against human cathepsin D.,103. Selectivity for their inhibitory potency against human cathepsin D.,104. Inhibitory concentration against cathepsin D,105. Concentration required
ACTIVITYSubstance BioActivity: 2 Active, 2 Tested Substance BioActivity: 42 Active, 1 Activity <= 1 nM, 38 Activity <= 1 µM, 42 Tested Substance BioActivity: 6 Active, 3 Activity <= 1 µM, 14 Tested Substance BioActivity: 36 Active, 1 Activity <= 1 nM, 9 Activity <= 1 µM, 43 Tested Substance BioActivity: 5 Active, 3 Activity <= 1 µM, 8 Tested Substance BioActivity: 3 Active, 2 Activity <= 1 nM, 3 Activity <= 1 µM, 3 Tested Substance BioActivity: 4 Active, 4 Activity <= 1 µM, 7 Tested Substance BioActivity: 18 Active, 17 Activity <= 1 µM, 23 Tested Substance BioActivity: 29 Active, 19 Activity <= 1 µM, 29 Tested Substance BioActivity: 4 Active, 1 Activity <= 1 nM, 4 Activity <= 1 µM, 4 Tested Substance BioActivity: 37 Active, 2 Activity <= 1 µM, 42 Tested Substance BioActivity: 36 Active, 23 Activity <= 1 µM, 41 Tested Substance BioActivity: 3 Active, 3 Tested Substance BioActivity: 1 Tested Substance BioActivity: 1 Tested Substance BioActivity: 6 Active, 5 Activity <= 1 µM, 9 Tested