| Assay Method Information | |
| | NAMPT Assay |
| Description: | To test the inhibition activity (IC50) of the compounds of the disclosure against the enzymatic activity of NAMPT, the compounds were dissolved in 100% DMSO to a final concentration of 3 mM. The compounds were then diluted 20 fold in an intermediate dilution to 5% DMSO and water. Finally 10 μL of each compound was added to the assay plate, in duplicate, with highest concentration at 30 μM and the lowest at 0.001 μM in a final volume of 50 μL and 1% DMSO concentration. Each assay also tested, in parallel, two reference compounds, FK866 and GMX1778, with highest concentration at 10 μM and lowest at 0.0003 μM. 200 ng of human NAMPT enzyme (His-hNAMPT obtained from E. coli expression system) diluted in TEST (tris-buffered saline-Polysorbate 20) 1× was added to each well in a volume of 10 μL and the compounds were incubated with the enzyme at room temperature for 20 minutes. After the incubation, 30 μL of master mixture were added to each well. The master mixture contained: 50 mM Tris-HCl, pH 8.0, 12.5 mM MgCl2, 20 μM nicotinamide, 40 μM phosphoribosyl pyrophosphate (PRPP), 20 μM adenosine triphosphate (ATP), 30 μg/mL of alcohol dehydrogenase, 10 μg/mL of NMNAT, 1.5% alcohol, 1 mM dithiothreitol (DTT), 0.02% bovine serum albumin (BSA), 0.01% Tween 20. The reaction was performed at 30° C. for 60 minutes. Fluorescence intensity was measured at an excitation of 340 nm and an emission of 460 nm using a Tecan Infinite M1000 microplate reader. The fluorescence values indicated increase of NADH product. Enzyme activity assay was performed in duplicate at each concentration. The fluorescence data was analyzed and compared. In the absence of the compound, the intensity (Ce) in each data set was defined as 100% activity. In the absence of enzyme, the intensity (C0) in each data set was defined as 0% activity. |
| Affinity data for this assay | |
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