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 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 and high cellular potency. The distinct cellular functions of closely related MST kinases can now be elucidated with subfamily-selective chemical tool compounds using a combination of the MST1/2 inhibitor PF-06447475 (2) and the two MST3/4 inhibitors developed. We found that MST3/4-selective inhibition caused a cell-cycle arrest in the G1 phase, whereas MST1/2 inhibition resulted in accumulation of cells in the G2/M phase.

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 – 32 . The bromodomain containing proteins BRD4, TRIM24 and BRPF1 represent three diverse bromodomain subfamilies and are the most ligandable proteins within this protein family.…”

“…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.…”