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Details of RareLED ID Assay_1005
Primary information
GENEASAH1
NUMBER OF ASSAY29
ASSAY IDs691433 691432 659816 457727 1173818 1344688 736692 734964 736693 659817 767870 676922 457714 691429 1265564 687382 1344687 734978 1281619 1344325 1281626 1281618 1336967 1281616 687383 687387 241756 1192197 1344324
Assay Name1. Inhibition of aCDase expressed in human HL60 cell extract,2. Inhibition of aCDase,3. Inhibition of aCDase in Moh.pAS AcCer10X cells using RBM14C12 as substrate after 1 hr by HPLC-FD analysis,4. Inhibition of NAAA,5. Inhibition of ACDase in human MCF7 cell lysate using [9,10-3H] De-C16-Cer substrate after 1 hr by scintillation counting analysis,6. Acid Ceramidase Activity Assay: A hAC protein preparation (10 inverted question markg) was preincubated with inhibitors (final DMSO concentration 1%) in assay buffer (100 mM sodium phosphate, 0.1% Nonidet P-40, 150 mM NaCl, 3 mM DTT, 100 mM sodium citrate, pH 4.5) for 30 min at 37 degrees C. Reactions were started by the addition of 50 inverted question markM N-lauroyl ceramide (Nu-Chek Prep, Elysian, Minn.) and carried on for 30 min at 37 degrees C. Reactions were stopped by addition of a mixture of chloroform/methanol (2:1, vol/vol) containing 1 nmol 11-lauroleic acid (NuChek Prep). The organic phases were collected, dried under nitrogen and analyzed by UPLC/MS (Acquity, Waters) in the negative-ion mode monitoring the reaction product (lauric acid, m/z=199) using 11-lauroleic acid as internal standard.Lipids were eluted on an Acquity UPLC BEH C18 column (50 mm length, 2.1 mm i.d., 1.7 inverted question markm pore size, Waters) column at 0.5 mL-min inverted question mark1 for 1.5,7. Inhibition of acid ceramidase (unknown origin),8. Inhibition of alkaline ceramidase 2 in human HL-60 cells using 3H]C16-ceramide as substrate after 1 hr by liquid scintillation counting analysis,9. Inhibition of rat recombinant acid ceramidase expressed in human HEK293 cells using N-lauroylceramide as substrate incubated for 30 mins prior to substrate addition measured after 30 mins by LC/MS analysis,10. Inhibition of aCDase using RBM14C12 as substrate after 1 hr by HPLC-FD analysis,11. Inhibition of C-terminal His-6-tagged recombinant human spleen NAAA enzyme expressed in HEK293 cells,12. Inhibition of human recombinant His6-tagged NAAA expressed in HEK293 cells using 10-cis-heptadecenoylethanolamide as substrate after 30 mins by UPLC/MS analysis,13. Inhibition of human recombinant NAAA expressed in human HEK293 cells assessed as conversion of [1,2-14C]palmitoylethanolamine to [1,2-14C]ethanolamine by liquid scintillation counting,14. Inhibition of recombinant human nCDase expressed in Sf9 cells using fluorescently-labeled acyl-NBD-C12-ceramide as substrate after 2 hrs by fluorescence assay,15. Inhibition of recombinant human acid ceramidase expressed in HEK293 cells using N-[(1S,2R)-2-hydroxy-1-(hydroxymethyl)-4-(2-oxochromen-7-yl)-oxybutyl]dodecanamide substrate preincubated for 10 mins,16. Activity of human recombinant acid ceramidase expressed in baculovirus infected Sf9 cells after 150 mins by Lineweaver-Burk plot analysis,17. Enzymatic Assay: r-AC protein samples were pre-incubated with various concentrations of test compounds or vehicle control in 100 mM NaH2PO4/citrate buffer pH 4.5, 0.1% Nonidet P-40, 3 mM DTT for 30 min at 37 degrees C. Samples were incubated with 100 inverted question markM N-lauroyl ceramide (Nu-Chek Prep, Elysian, Minn.) at 37 degrees for 30 min. The reaction was stopped by addition of a mixture of chloroform/methanol (2:1 vol/vol) containing 1 nmol of heptadecanoic acid (HDA; NuChek Prep). The organic phases were collected, dried under nitrogen, and analyzed by LC/MS in the negative-ion mode using heptadecanoic acid (HDA) as internal standard (m/z=199 for lauric acid, m/z=269 for HDA). HDA was eluted on an XDB Eclipse C18 column isocratically at 2.2 mL/min for 1 min with a solvent mixture of 95% methanol and 5% water, both containing 0.25% acetic acid, and 5 mM ammonium acetate. The column temperature was 50 degrees C.,18. Inhibition of human recombinant neutral ceramidase using Acyl-NBD-C12-cer as substrate after 2 hrs by fluorescence assay,19. Inhibition of human acid ceramidase by UPLC/MS analysis,20. Fluorogenic Assay: The assay was run in Optiplate 96-wells black plates, in a total reaction volume of 200 inverted question markL. NAAA protein preparation (4.0 inverted question markg) was pre-incubated for 10 min with various concentrations of test compounds or vehicle control (5% DMSO) in 100 mM citrate/phosphate buffer (pH 4.5) containing 3.0 mM DTT, 0.1% Triton X-100, 0.05% BSA, 150 mM NaCl. N-(4-methyl-2-oxo-chromen-7-yl)-hexadecanamide was used as a substrate (5.0 inverted question markM) and the reaction carried over for 30 min at 37 degrees C. The samples were then read in a Perkin Elmer Envision plate reader using an excitation wavelength of 360 nm and emission 460 nm.,21. Inhibition of human NAAA expressed in HEK293 cells preincubated for 10 mins followed by N-(4-methyl-2-oxo-chromen-7-yl)-hexadecanamide substrate addition for 30 mins by microplate reader analysis,22. Inhibition of human NAAA expressed in HEK293 cells preincubated for 10 mins followed by N-(4-methyl-2-oxo-chromen-7-yl)-hexadecanamide substrate addition for 50 mins by fluorescence assay,23. Inhibition of recombinant human NAAA using PAMCA as substrate preincubated for 30 mins followed by substrate addition measured every 3 mins for 2.5 hrs by fluorescence HTS method,24. Inhibition of recombinant human NAAA expressed in HEK293 cells after 30 mins by UPLC/MS analysis,25. Activity of human recombinant neutral ceramidase after 60 mins by Lineweaver-Burk plot analysis,26. Activity of mouse neutral ceramidase after 30 mins by TLC-based fluorescence imaging,27. Inhibitory concentration against N-palmitoylethanolamine acid amidase,28. Inhibition of human NAAA,29. UPLC/MS Assay: NAAA protein preparation (10 ug) was pre-incubated with various concentrations of test compound or vehicle control in 100 mM NaH2PO4, 100 mM Tri Sodium Citrate Dehydrate, 0.1% Triton-X 100, 3 mM DTT, pH 4.5 for 30 min at 37 degrees C. Duplicate samples were then incubated with 50 uM C17:1 10-cis-heptadecenoylethanolamide (Avanti Polar Lipids, Alabaster, Ala. USA) at 37 degrees C. for 30 minutes. The reaction was terminated by the addition of 0.2 mL of cold methanol containing 1 nmol of heptadecanoic acid (NuChek Prep, Elysian, Minn. USA) as internal standard. Samples were then analyzed by UPLC/MS. Heptadecenoic and heptadecanoic acids were eluted on an Acquity UPLC BEH C18 column (50 mm length, 2.1 mm i.d., 1.7 um pore size, Waters) isocratically at 0.5 mL/min for 1.5 min with a solvent mixture of 95% methanol and 5% water, both containing 0.25% Acetic Acid and 5 mM Ammonium Acetate. The column temperature was 40 degrees C. Electrospray ionization was in the negative
ACTIVITYSubstance BioActivity: 1 Active, 1 Activity <= 1 µM, 1 Tested Substance BioActivity: 1 Active, 1 Tested Substance BioActivity: 3 Tested Substance BioActivity: 4 Active, 5 Tested Substance BioActivity: 1 Tested Substance BioActivity: 4 Active, 4 Activity <= 1 µM, 4 Tested Substance BioActivity: 1 Active, 2 Tested Substance BioActivity: 1 Active, 1 Activity <= 1 µM, 1 Tested Substance BioActivity: 32 Active, 25 Activity <= 1 µM, 37 Tested Substance BioActivity: 3 Tested Substance BioActivity: 5 Active, 5 Activity <= 1 µM, 5 Tested Substance BioActivity: 2 Active, 2 Activity <= 1 µM, 2 Tested Substance BioActivity: 1 Active, 2 Tested Substance BioActivity: 2 Tested Substance BioActivity: 40 Active, 1 Activity <= 1 nM, 40 Activity <= 1 µM, 42 Tested Substance BioActivity: 4 Tested Substance BioActivity: 13 Active, 9 Activity <= 1 µM, 13 Tested Substance BioActivity: 2 Tested Substance BioActivity: 6 Active, 1 Activity <= 1 µM, 6 Tested Substance BioActivity: 15 Active, 15 Activity