BX-795

N-(3-(5-iodo-4-(3-(thiophene-2-carboxamido)propylamino)pyrimidin-2-ylamino)phenyl)pyrrolidine-1-carboxamide

BX795 is a potent and specific TBK1, IKKepsilon and PDK-1 inhibitor with IC50 of 6 nM, 41 nM and 111 nM, respectively.

BX795 also suppresses MARK1, MARK2, MARK4, NUAK1, VEGFR, MLK1, MLK2, MLK3 with IC50 of 55, 53, 19, 5, 157, 50, 46 and 42 nM, respectively. BX795 does not prevent the following protein-tyrosine kinases at 1 uM: ephrin receptors A2 and B3, Syk, Bruton's tyrosine kinase, and fibroblast growth factor receptor 1. However, the vascular endothelial growth factor receptor is inhibited, albeit much less potently than TBK1. The ability of BX795 to inhibit the TBK1-catalyzed phosphorylation of IRF3 at Ser-396 declined as the ATP concentration is increased, indicating that BX795 is an ATP competitive inhibitor of TBK1 as is the case for PDK1. BX795 blocks TBK1- and IKKepsilon-mediated activation of IRF3 and production of IFN-beta. IRF3 accumulated in the nucleus on a similar time scale after poly(I:C) treatment, which is blocked by BX795. BX795 inhibits IRF3-dependent gene transcription. BX795 blocks the secretion of IFN-beta from macrophages whether stimulated by LPS, a TLR4 agonist. BX795 has no effect on the LPS-stimulated phosphorylation of p70 ribosomal S6 kinase 1 at Thr-229, the site that is targeted by PDK1. BX795 does not affect activation of the IKKalpha/beta complex or NFkappaB-dependent gene transcription by LPS, poly(I:C), IL-1alpha, or TNFalpha. BX795 strongly suppresses the LPS- or poly(I:C)-stimulated phosphorylation of the activating Thr-Pro-Tyr motif on JNK1/2 and partially inhibits the phosphorylation of the activating Thr-Gly-Tyr motif on p38alpha MAP kinase. BX795 also suppresses the phosphorylation of JNK1/2 and p38alpha MAPK in MEFs stimulated with IL-1alpha or TNFalpha. The effect of BX795 on the activation of JNK1/2 and p38alpha MAPK does not result from the inhibition of TBK1/IKKepsilon.[1]

Protein kinase assays, Assays are initiated by adding [gamma-32P]ATP (1000 cpm/pmol) to a final concentration of 0.1 mM. When GST-IRF3 is used as the substrate, the inhibitory reactions of BX795 are terminated after 30 min at 30 C by the addition of SDS containing 40 mM EDTA, pH 7.0, heated for 5 min at 100 C and separated by SDS-PAGE, and phosphorylated proteins are detected by autoradiography. Quantification is performed by phosphorimaging analysis. For assays with the IkappaBalpha substrate peptide, reactions are terminated after 10 min at 30 C by spotting an aliquot of the reaction on to a 2 x 2-cm 2 piece of phosphocellulose P81 paper followed by immersion in 75 mM phosphoric acid. After washing six times in phosphoric acid and once in acetone, the papers are dried and counted. One unit of TBK1 activity is defined as that amount of enzyme catalyzing the incorporation of 1 nmol of phosphate into substrate in 1 min.

591, 47

DMSO 118 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL