Figure 7 |. Optimized phosphonate ENPP1 inhibitors prevent cGAMP degradation in mouse and human plasma at nanomolar concentrations.

(A) In vitro dose-inhibition curves for selected top ENPP1 inhibitors. K_i values were determined using 3 nM ENPP1 and 5 μM cGAMP and (where indicated) in the presence of 40 μM human serum albumin at pH 7.4. Dotted line represents the minimum K_i value (2 nM) measurable with the given enzyme concentration. (B) In mouse and human plasma, IC₅₀ values were determined using 5 μM cGAMP with trace radiolabeled [³²P]cGAMP. In vitro dose-response curves replotted from (A). Chemical structures of inhibitors are displayed below. Dots represent the mean of two independent replicates, and shaded areas around the fitted curves represent the 95% confidence interval of the fit. See also Figure S2. (C) Concentrations of compound 32 in mouse serum, kidney, and liver after one 10 mg/kg dose via intravenous (IV) or subcutaneous (SC) route. IC₉₅ = 40 nM; LOQ = 10 nM. Mean ± SEM is plotted, n = 2 mice for each point except the following, where n = 1: liver SC 4 h (point removed as an outlier using the ROUT method with Q = 1%) and serum 8 h (concentration below LOQ). Data were fit with one-phase decay with 1/Y² weighting to obtain half-life. (D) Concentrations of compound 32 in mouse serum after consecutive once daily dosing at 300 mg/kg via SC route for 6 days. Serum was collected for analysis 24 hours after last dose, immediately before the next dose. (E) Tumor volumes (left; each line represents one mouse) and survival (right) of mice bearing E0771 breast cancer tumors. Mice were treated with vehicle (no treatment, n = 25 mice) or treated with compound 32 (n = 20 mice) dosed once daily at 300 mg/kg via SC route for seven consecutive days after tumors reached 80–120 mm³. For survival, P = 0.0006 (Gehan-Breslow-Wilcoxon test).