| Assay Method Information | |
| | SARM1 Inhibitory Activity |
| Description: | Enzymatic reactions were ran in a 10µL volume consisting of 8nM human SARM1 (aa28-724), 100µM Nicotinamide (NMN) and 30µM Nicotinamide Adenine Dinculeotide (NAD). Assay reagents were prepared in 25mM HEPES pH 7.2, 50mM NaCl, 1mM EDTA and 0.0025% Tween20. To determine compound IC50’s, reactions were incubated for 60minutes at room temperature in the presence of a 12-point concentration response curve of compound (starting concentration 100µM; 1 in 3 dilution between each point; 2% DMSO) and then quenched with 40µL of 0.125% Formic Acid. The peak area of NAD and linear ADPR were measured by a RapidFire High Throughput Mass Spectrometry System (Agilent Technologies, Santa Clara, CA) using an API5000 triple quadrupole mass spectrometer (AB Sciex Framingham, MA). The ratio of linear ADPR to NAD peak area was then plotted against compound concentration to obtain an IC50 as fitted via non-linear regression. |
| Affinity data for this assay | |
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