In vitro activity: CHMFL-BMX-078 is a highly potent and selective type II irreversible inhibitor of BMX (bone marrow kinase on chromosome X) kinase with an IC50 of 11 nM. CHMFL-BMX-078 exhibits an IC50 of 11 nM by formation of a covalent bond with cysteine 496 residue in the DFG-out inactive conformation of BMX. It displays a high selectivity profile against the 468 kinases/mutants in the KINOMEscan evaluation and achieves at least 40-fold selectivity over BTK kinase (IC50=437 nM). BMX is a member of TEC family nonreceptor tyrosine kinase and is involved in a variety of critical physiological and pathological processes. Given the fact that BMX mediated signaling pathway is still not fully understood, CHMFL-BMX-078 would serve as a useful pharmacological tool to elucidate the detailed mechanism of BMX mediated signaling pathways.

Kinase Assay: CHMFL-BMX-078 is a highly potent and selective type II irreversible inhibitor of BMX (bone marrow kinase on chromosome X) kinase with an IC50 of 11 nM. CHMFL-BMX-078 exhibits an IC50 of 11 nM by formation of a covalent bond with cysteine 496 residue in the DFG-out inactive conformation of BMX. It displays a high selectivity profile against the 468 kinases/mutants in the KINOMEscan evaluation and achieves at least 40-fold selectivity over BTK kinase (IC50=437 nM). The kinase reaction system contains BMX or BTK, 1 μL of serially diluted CHMFL-BMX-078, and substrate Poly peptidewith 100 μM ATP. The reaction in each tube is started immediately by adding ATP and kept going for an hour under 37 °C. After the tube cooled for 5 min at room temperature, 5 μL solvent reactions are carried out in a 384-well plate. Then 5 μL of ADP-Glo reagent is added into each well to stop the reaction and consume the remaining ATP within 40 min. At the end, 10 μL of kinase detection reagent is added into the well and incubated for 30 min to produce a luminescence signal. Luminescence signal is measured with an automated plate reader

Cell Assay: Bone marrow kinase in the X chromosome (BMX, also called ETK) is a nonreceptor tyrosine kinase involved in tumorigenicity, cell motility, adhesion, angiogenesis, proliferation, and differentiation. CHMFL-BMX-078 exhibits an IC50 of 11 nM by formation of a covalent bond with cysteine 496 residue in the DFG-out inactive conformation of BMX. It displays a high selectivity profile against the 468 kinases/mutants in the KINOMEscan evaluation and achieves at least 40-fold selectivity over BTK kinase (IC50=437 nM). For inactive state of BMX kinase, CHMFL-BMX-078 displays a binding Kd of 81 nM, while for the active state of BMX kinase, it exhibits a binding Kd of 10200 nM. CHMFL-BMX-078 exhibits antiproliferative effects against BaF3-TEL-BMX cells (GI50=0.016 μM) and selectivity over parental BaF3 cells. CHMFL-BMX-078 is about 80-fold more potent against BMX wt (EC50=5.8 nM) than C496S mutant (EC50=459 nM) for the inhibition of BMX total tyrosine phosphorylation. CHMFL-BMX-078 would serve as a useful pharmacological tool to elucidate the detailed mechanism of BMX mediated signaling pathways.