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

Abstract

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.

Chemogenomics for NR1 nuclear hormone receptors

“…To annotate potential activity on critical off-targets, the CG compound candidates were additionally screened by differential scanning fluorimetry (DSF) for binding to a panel of representative kinases and bromodomains that are highly ligandable and/or cause strong phenotypic outcomes when inhibited [30][31][32] . The bromodomain containing proteins BRD4, TRIM24 and BRPF1 represent three diverse bromodomain subfamilies and are the most ligandable proteins within this protein family.…”

“…Liability panel screening by DSF. Recombinantly expressed proteins 32 (produced in-house, for constructs and sequences, refer to Supplementary Table 3) of the liability panel targets were diluted in a buffer containing 10 mM HEPES, pH 7.5 and 500 mM NaCl to a concentration of 2 µM. SYPRO Orange (Invitrogen, #S6650) was added as a 1:1000 dilution, the protein-dye mixtures were transferred to a 384 well plate, and the compounds were added using the Echo 550 liquid handler (Labcyte, San Jose, California, USA, #001-10080) to a final concentration of 20 µM.…”