In vitro activity: In macrophages, MRT67307 prevents the phosphorylation of IRF3 and the production of IFNβ. In wild-type MEFs, MRT67307 enhances the IL-1-stimulated phosphorylation of p105 at Ser933 and RelA at both Ser468 and Ser536, and also enhances IL-1-stimulated activation of NF-κB-dependent gene transcription. MRT67307 increases IL-10 production and suppresses proinflammatory cytokine production via a cAMP response element-Binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3 Dependent Mechanism. In addition, MRT67307 inhibit ULK and block autophagy in MEF cells.
Kinase Assay: Enzyme assay: Substrates and kinases were diluted in 50 mM Tris/HCl (pH 7.5), 0.1% 2-mercaptoethanol, 0.1 mM EGTA and 10 mM magnesium acetate. Reactions were initiated with [γ-32P]ATP (2500 c.p.m./pmol) to a final concentration of 0.1 mM and terminated after 15 min at 30 ℃ by the addition of SDS and EDTA (pH 7.0) to final concentrations of 1.0% (w/v) and 20 mM respectively. After heating for 5 min at 100 ℃ and separation by SDS/PAGE, the phosphorylated proteins were detected by autoradiography.
Cell Assay: In macrophages, MRT67307 prevented the production of IFNβ and the phosphorylation of IRF3 without suppressing the activation of NF-κB, which showed that MRT67307 blocked the induction of Pellino 1 through inhibiting TBK1/IKKε kinase activity [1] [3]. Also, MRT67307 completely blocked the TBK1- or IKKε-induced decrease in the mobility of Pellino 1. | 
