Through its effects on ULK1 and ULK2, MRT67307 blocks autophagy. MRT67307 prevents the phosphorylation of IRF3 and the production of IFN-β and increases toll-like receptor-induced IL-10 and IL-1ra secretion in macrophages. MRT67307 is a kinase inhibitor that has been shown to inhibit TBK1, MARK1-4, IKKε, and NUAK1 (IC50 values are 19, 27-52, 160, and 230 nM, respectively), the salt-inducible kinases (SIKs; IC50s =250, 67, and 430 nM for SIK1, SIK2, and SIK3, respectively) and ULK1 and ULK2 (IC50s = 45 and 38 nM, respectively).

IKKϵ:160 nM, TBK1:190 nM

在巨噬细胞中，MRT67307 可阻止 IRF3 的磷酸化和 IFNβ 的产生。在野生型 MEFs 中，MRT67307 可增强 IL-1 刺激的 p105 在 Ser933 处的磷酸化以及 RelA 在 Ser468 和 Ser536 处的磷酸化，还可增强 IL-1 刺激的 NF-κB 依赖性基因转录的激活。[1] MRT67307 通过 cAMP 反应元件结合蛋白（CREB）调控转录辅激活因子（CRTC）3 依赖性机制增加 IL-10 的产生并抑制促炎细胞因子的产生。[2] 此外，MRT67307 还能抑制 ULK 并阻断 MEF 细胞的自噬。[3]

Substrates and kinases are diluted in 50?mM Tris/HCl (pH?7.5), 0.1% 2-mercaptoethanol, 0.1?mM EGTA and 10?mM magnesium acetate. Reactions are 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°C 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°C and separation by SDS/PAGE, the phosphorylated proteins are detected by autoradiography.

Cells are rinsed in ice-cold PBS and extracted in lysis buffer (50 mM Tris·HCl at pH 7.4, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium β-glycerol 1-phosphate, 1 mM DTT, 1 mM sodium orthovanadate, 0.27mol/Lsucrose, 1% (vol/vol) Triton X-100, 1 μg/mL aprotinin, 1 μg/ mL leupeptin, and 1 mM phenylmethylsulphonyl fluoride). Cell extracts are clarified by centrifugation at 14,000 × g for 10 min at 4°C, and protein concentrations are determined by using the Bradford assay. FLAG-CRTC3 is purified on anti-FLAG M2 agarose, whereas endogenous CRTC3 is immunoprecipitated from cell extracts by using anti-CRTC3 raised against the peptide CWKEEKHPGFR (S277D bleed 2) and coupled to Protein GSepharose. To detect proteins in cell lysates, 20 μg of protein extract is separated by SDS/PAGE. After transfer to PVDF membranes, proteins are detected by immunoblotting and visualized by treating the blots with ECL followed by autoradiography. The following antibodies are used for immunoblotting: pSer133 CREB, pSer171 CRTC2, total CRTC2, GAPDH, total STAT3, pTyr705 STAT3, FLAG (M2 clone), CRTC3, HA (3F10), and 14-3-3; and antibodies against pSer329 (S256D bleed 2) and pSer370 (S253D bleed 2) of CRTC3 are raised against the phosphopeptides GLQSSRpSNPSIQ and RLFSLpSNPSLST.