A High-throughput Screening Assay For Mst1 And Mst2 Kinase Inhibitors

The Hippo signaling pathway plays an important role in organ size control,tissue regeneration and stem cell self-renewal.The Ste20-like kinases Mstl and 2(Mstl/2),the mammalian Hippo orthologs,are key components of the Hippo signaling pathway along with a scaffolding protein Salvador/WW45(Sav),NDR family kinases NDR1/2 and LATS1/2,and an adaptor protein MOB1.Mst 1/2 phosphorylate and activate LATS 1/2-MOB1,which then phosphorylate their downstream effectors YAP and TAZ.Phospho-YAP/TAZ is either degraded or sequestered in the cytoplasm by 14-3-3 protein.When the Hippo pathway is switched off,YAP/TAZ translocates to the nucleus and forms a functional hybrid transcriptional complex with TEA domain family transcription factors(TEAD1-4)to turn on pro-proliferative and pro-survival genes,thereby enabling cell proliferation.Therefore,Mstl/2 inhibitors would be promising drug candidate for tissue repair and regeneration.In this paper we constructed an ELISA-based high-throughput screening system by using GST-Mobl as the substrate of Mstl/2,and the phosphorylation level of Mob1 as the indicator,aiming at screening out a small compound that specifically inhibits Mstl/2 kinase activity.After screening an in-house compound library(～3000 compounds)designed to target the adenosine triphosphate(ATP)-binding site of the kinases MST1/2,we obtained XMU-MP-1.In vitro kinase assay demonstrated that the inhibition of Mstl/2 by XMU-MP-1 was dose-dependent and ATP competitive.Next,we further verified that XMU-MP-1 can effectively inhibits Mstl/2 in a variety of cell lines.In conclusion,we found a specific Mstl/2 inhibitor XMU-MP-1.By modulating Hippo pathway activity,it shows a promising prospect in clinical application.