Development of selective pyrido[2,3-d]pyrimidin-7(8H)-one-based Mammalian STE20-like (MST3/4) kinase inhibitors

Mammalian STE20-like (MST) kinases 1-4 play key roles in regulating the Hippo and autophagy pathways, and their dysregulation has been implicated in cancer development. In contrast to the well-studied MST1/2, the roles of MST3/4 are less clear, in part due to the lack of potent and selective MST3/4 inhibitors. Here, we re-evaluated literature compounds, and used structure-guided design to optimize the p21-activated kinase (PAK) inhibitor G-5555 ( 8 ) to selectively target MST3/4. These efforts resulted in the development of MR24 ( 24 ) and MR30 ( 27 ) with good kinome-wide selectivity, high potency for MST3/4, and selectivity towards the closely related MST1/2. In combination with the MST1/2 inhibitor PF-06447475 ( 2 ) the two MST3/4 inhibitors can be used to elucidate the multiple roles of MST kinases in cells. We found that MST3/4-selective inhibition caused a cell cycle arrest in the G1 phase, while MST1/2 inhibition resulted in accumulation of cells in the G2/M phase. These data point to distinct functions of these closely related kinases, which can now be addressed with subfamily-selective chemical tool compounds. ### Competing Interest Statement The authors declare the following competing financial interest(s): L.B. is a co-founder and B.-T.B. is a co-founder and the CEO of CELLinib GmbH (Frankfurt am Main, Germany). The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. * AKT : protein kinase B AMPKα : AMP-activated protein kinase ATF : cAMP-dependent transcription factor ATG : autophagy-related gene BECN1 : beclin-1 CGC : chemogenomic compound CK1 : casein kinase 1 DSF : differential scanning fluorimetry EGFR : epidermal growth factor receptor FES : feline sarcoma/Fujinami avian sarcoma oncogene homolog FUCCI : fluorescent ubiquitination-based cell cycle indicator GCK : germinal center kinase GFP : green fluorescent protein HER2 : tyrosine kinase-type cell surface receptor HPLC : high-performance liquid chromatography IQR : interquartile range JAK : Janus kinase LATS : large tumor suppressor homolog LC3B : microtubule-associated proteins 1A/1B light chain 3B LRRK2 : leucine-rich repeat kinase 2 MAP3K5 : mitogen-activated protein kinase kinase kinase 5 MD : molecular dynamics MM/GBSA : molecular mechanics with generalized Born and surface area solvation Mob1A/B : Mps one binder 1 A/B MST : mammalian STE20-like protein kinase NDR : Dbf2-related kinase NEK2 : NimA-related protein kinase 2 NUAK : SNF1/AMP kinase-related kinase PAK : p21-activated kinase RFP : red fluorescent protein Sav1 : Salvador homolog 1 SIK : salt-inducible kinase STE : homologous kinases of the yeast proteins STE20, STE11, and STE7 STK : serine/threonine kinase TAZ : transcriptional coactivator with PDZ-domain motif TEAD : TEA domain family member ULK1 : Unc-51-like kinase 1 YAP : Yes-associated protein YSK1 : Yeast Sps1/Ste20-related kinase 1