The inhibitory effect of ISM5939 on ENPP1 enzyme activity at two different pH conditions (6.5 and 7.4) was evaluated using the AMP-Glo luminescence-based assay (Promega). Recombinant human ENPP1 protein (R&D) or purified mouse/dog/monkey ENPP1 protein (Biometas, see below) was incubated with a final concentration of 2.5 µM of either ATP (Promega) or 2’3’-cGAMP (InvivoGen) in assay buffer adjusted to the respective pH. ISM5939 was added at varying concentrations diluted in DMSO, and the reaction was initiated by the addition of AMP-Glo Reagent (Promega) followed by Kinase Detection Reagent (Promega), according to the kit instructions. ENPP-1-IN-1 (WO2019046778A1), the reported ENPP1 inhibitor was used as the positive control. Luminescence signal was measured using a microplate reader (BMG LABTECH), and percentage of inhibition was calculated relative to a control group without the test compound. IC50 values were determined using nonlinear regression analysis in GraphPad Prism 8 software.
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