Inhibition of Caspase 5 (unknown origin) using Ac-LEHD-AMC substrate assessed as residual activity at 10 uM incubated for 15 mins prior to substrate addition measured for 2 hrs
Fluorometric Enzymatic Assay: The inhibition of the activity of Caspase-1 to -10 by four different compounds (#53, #111, #123 & Z-DEVD-FMK used as a prodrug: z-D(OMe)E(OMe)VD(OMe)FMK (comparison control compound, SM Biochemicals LLC, Cat# SMFMK003)) and their respective IC50 values were determined using a fluorometric enzymatic assay (Active Recombinant Caspase Set IV (BioVision, Cat# K233-10-25) and Caspase Fluorometric Substrate Set II Plus (BioVision, Cat# K137-9-25)) on a Flexstation 3x (Molecular Devices). In brief, each compound was serially diluted for a final test concentration range of 0.1 nM to 3.3 uM (10-point titration). Each active recombinant Caspase (Active Recombinant Caspase Set IV (BioVision, Cat# K233-10-25)) was added to the respective wells of the 96-well High Efficiency plates (Molecular Devices, Cat#75-000-0005), followed by the addition of the diluted test compounds. DMSO (Final=1%) or Z-VAD-FMK (Caspase-Family Inhibitor, Z-VAD-FMK (BioVision, Cat#1010-100).
Fluorometric Enzymatic Assay: Selectivity of compound 55, compound 63, compound 48, compound 57, compound 88 toward caspase-1 (pro-inflammatory group), caspase-5 (group I), caspase-9 (group II) and caspase-3 and caspase-7 (group III) was evaluated by using fluorometric methods using the Caspase-1, -3, -5, -7, -9 Inhibitor Drug Screening Kit (Catalog #: K151-100, K153-100, K155-100/K157-100, K159-100 respectively, BioVision). Briefly, using instructions of the manufacturer, a wide range of different concentrations of the compound: 3333, 1000, 333, 100, 33, 10, 3, and 1 nM (final concentration) was added directly to the reaction mixtures containing the substrate and the enzyme in a final volume of 10 ul. After a 30-minute incubation at 37 C., the liberation of AFC was measured as an endpoint assay using the Flexstation3 (Molecular Devices) with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The level of inhibition of caspase-1, -3, -5, -7, -9 activity was determined by comparison.
Inhibition of human recombinant DTT-activated caspase-5 expressed in Escherichia coli using Ac-WEHD-AMC as substrate preincubated for 30 mins followed by substrate addition by fluorescence assay
Inhibition of human recombinant DTT-activated caspase-5 expressed in Escherichia coli using Ac-WEHD-AMC as substrate preincubated for 15 mins followed by substrate addition by fluorescence assay
Inhibition of human recombinant DTT-activated caspase-5 expressed in Escherichia coli using Ac-WEHD-AMC as substrate preincubated for 45 mins followed by substrate addition by fluorescence assay
Ratio of IC50 for human recombinant DTT-activated caspase-5 expressed in Escherichia coli preincubated for 15 mins followed by Ac-WEHD-AMC substrate addition to IC50 for human recombinant DTT-activated caspase-5 expressed in Escherichia coli preincubated for 30 mins followed by Ac-WEHD-AMC substrate addition
Ratio of IC50 for human recombinant DTT-activated caspase-5 expressed in Escherichia coli preincubated for 15 mins followed by Ac-WEHD-AMC substrate addition to IC50 for human recombinant DTT-activated caspase-5 expressed in Escherichia coli preincubated for 45 mins followed by Ac-WEHD-AMC substrate addition