In vitro activity: FM-381 is a potent covalent reversible inhibitor of JAK3 targeting the unique Cys909 at the gatekeeper (GK) position +7 in JAK3. FM-381 shows an apparent EC50 of 100 nM in a dose dependent BRET assay and blocks IL2-stimulated (JAK3/JAK1 dependent) STAT5 phosphorylation at 100 nM, but not JAK3 independent IL6-stimulated (JAK1/2/TYK dependent) STAT3 signalling in Human CD4+ T cells up to 1 μM.

Kinase Assay: FM381 is screened against a panel of 410 kinases at concentrations of 100 nM and 500 nM. FM381 has no relevant effect on the activity of any tested kinases except JAK3 at a concentration of 100 nM. At 500 nM, FM381 moderately inhibits 11 other kinases besides JAK3 with residual activities below 50%. FM-381 is found to be inactive in a selectivity panel of frequently hit BRDs (BRD4, BRPF, CECR, FALZ, TAF1, BRD9). Strongest hits is 500 nM for TAF1@2. FM381 selectively Inhibit JAK3 Signaling in Human CD4+ T Cells. FM-381 shows an apparent EC50 of 100 nM in a dose dependent BRET assay and blocks IL2 stimulated (JAK3/JAK1 dependent) STAT5 phosphorylation at 100 nM, but not JAK3 independent IL6 (JAK1/2/TYK dependent) stimulated STAT3 signalling in Human CD4+ T cells up to 1 μM.

Cell Assay: CD4+ T Cell cytokine stimulation assay is performed. T cells are purified from peripheral blood mononuclear cells from human donors. Equal numbers of cells are incubated for 1 hr with JAK inhibitors (FM381) (0, 10, 50, 100, 300 nM) or DMSO control and stimulated with cytokines for 30 min. The cells are lysed, and the proteins are separated via PAGE and transferred to a polyvinylidene fluoride membrane. The proteins of interest are blotted with specific antibodies and visualized with an infrared imaging system.