In vitro activity: XMU-MP-1 is a potent, selective and ATP-competitive MST1/2 inhibitor. In ELISA-based high-throughput screening assay, XMU-MP-1 at 10 μM inhibited MST2 kinase activity by more than 50% and dose-dependently inhibited the phosphorylation of MOB1 mediated by MST2. In human liver carcinoma (HepG2) cells, XMU-MP-1 (0.1 to 10 μM) dose-dependently reduced the phosphorylation of endogenous MOB1, LATS1/2, and YAP. Also, in a variety of cell lines, including human osteosarcoma (U2OS), human colorectal adenocarcinoma (SW480) and human pleomorphic hepatocellular carcinoma (SNU-423), XMU-MP-1 inhibited H2O2-stimulated MOB1 phosphorylation and MST1/2 autophosphorylation.

Kinase Assay: XMU-MP-1 is dissolved in DMSO (stock concentration, 10 mM). For the in vitro kinase inhibition assays, recombinant GST-tagged MOB1a and various forms of recombinant His-tagged full-length MST1 or MST2 kinase are expressed and purified from Escherichia coli. The assays are performed with the various doses of XMU-MP-1 in the kinase assay buffer for 30 min at 30°C.

Cell Assay: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the expression levels of CTGF and CYR61 in HepG2 cells after XMUMP- 1 treatment for 6 hours is conducted.