In vitro ENPP1 inhibition assay

CP Congying Pu
HC Hui Cui
HY Huaxing Yu
XC Xin Cheng
MZ Man Zhang
LQ Luoheng Qin
ZN Zhilin Ning
WZ Wen Zhang
SC Shan Chen
YQ Yuhang Qian
FW Feng Wang
LW Ling Wang
XL Xiaoxia Lin
DG David Gennert
FP Frank W Pun
FR Feng Ren
AZ Alex Zhavoronkov
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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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