Figure 2
From: Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic β-cells
Kinase profiling, molecular docking, and structure-activity relationship analyses reveal selectivity of the TBK1/IKKε inhibitors. (A, B) Chemical structures and kinase profiling of PIAA and iso-PIAA. Dose responses of PIAA and iso-PIAA were generated to determine the potency of the inhibitors (IC50). (C) Ball and stick model of PIAA (grey) and iso-PIAA (red) docked into the binding pocket of TBK1. PIAA adapts a docked pose that has its isoxazole nitrogen and carboxylate moieties engaged in stronger interactions, relative to the same moieties on iso-PIAA, with THR-156 and ASP-157 at the active site of TBK1. (D) Chemical structures of amlexanox and BX795. (E–G) Molecular docking simulations showing interactions of TBK1/IKKε-Is and TBK1. Ball and stick model of PIAA (grey), amlexanox (blue), and BX795 (purple) docked into the binding pocket of TBK1 (E and F). Space filling model of PIAA (grey) and BX795 (purple) docked into the binding pocket of TBK1 (G). The core moieties of PIAA, amlexanox, and BX795 all bound within an unvaried region of the kinase domain of TBK1, while the urea moiety of BX795 extends toward the outer rim of the TBK1 kinase domain and interacts with the carbonyl group of Pro-90. Specifically, the carboxylate moieties of PIAA and amlexanox are placed next to the carboxylate side chain of ASP-157 buried in the TBK1 active site, forming a strong low-barrier H-bonding between these two carboxylate groups. (H) Chemical structures of four PIAA analogs. The moieties that were replaced and different from the original PIAA structure are marked in red (PIAA-1, PIAA-2, and PIAA-4) or blue (PIAA-3). (I) Quantification of the number (mean ± SD) of total regenerated β-cells at 48 hpa treated with DMSO, PIAA-1, PIAA-2, PIAA-3, PIAA-4, and PIAA, respectively (4.8 ± 0.8 (DMSO), 4.2 ± 1.3 (PIAA-1), 6.2 ± 1.3 (PIAA-2), 4.0 ± 1.2 (PIAA-3), 6.0 ± 2.2 (PIAA-4), and 18.6 ± 3.4 (PIAA)). Cells in 20 planes of confocal images from 10 individual larvae were counted per condition. ***P < 0.001. (J) Representative Western blot showing a PIAA dose-dependent decrease of pIRF3 levels in rat INS-1 cells.
