Figure 1 |. ENPP1 rapidly degrades cGAMP but current inhibitors are not potent enough as tools to investigate ENPP1 function.

(A)–(C) cGAMP (5 μM, with trace radiolabeled [³²P]cGAMP) was incubated with C57B6/J mouse plasma (A, B) or human plasma (C) at 37 °C. 50 mM EDTA was included in the indicated reactions. At indicated times, degradation was assessed by separation of cGAMP from degradation products by thin layer chromatography (TLC) and autoradiography. (A) shows a representative experiment for mouse plasma. For (B), data are from 4 independent experiments, mean + SD shown. For (C), data are from 3 donors, each with 2 independent experiments; replicates are plotted individually. (D) Assay for measuring ENPP1 inhibitor K_i values. cGAMP (5μM) was incubated with increasing concentrations of ENPP1 inhibitor (maximum = 10 μM) and ENPP1 (3 nM) in a buffer containing 50 mM Tris, 150 mM NaCl, 1 μM ZnCl₂, and 500 μM CaCl₂ at the indicated pH value. The minimum measurable K_i is 2 nM (determined by enzyme concentration); the maximum is 10 μM. (E) Chemical structures and Ki values of compounds 1 and 2 (mean of at least 2 independent replicates; evaluated in assay described in D).