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 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.

Shifting the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one inhibitors towards the salt-inducible kinase (SIK) subfamily.

Salt-inducible kinases 1-3 (SIK1-3) are key regulators of the LKB1-AMPK pathway and play an important role in cellular homeostasis. Dysregulation of any of the three isoforms has been associated with tumorigenesis in liver, breast, and ovarian cancers. We have recently developed the dual pan-SIK/group I p21-activated kinase (PAK) chemical probe MRIA9. However, inhibition of p21-activated kinases has been associated with cardiotoxicity in vivo, which complicates the use of MRIA9 as a tool compound. Here, we present a structure-based approach involving the back-pocket and gatekeeper residues, for narrowing the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one-based inhibitors towards SIK kinases, eliminating PAK activity. Optimization was guided by high-resolution crystal structure analysis and computational methods, resulting in a pan-SIK inhibitor, MR22, which no longer exhibited activity on STE group kinases and displayed excellent selectivity in a representative kinase panel. MR22-dependent SIK inhibition led to centrosome dissociation and subsequent cell-cycle arrest in ovarian cancer cells, as observed with MRIA9, conclusively linking these phenotypic effects to SIK inhibition. Taken together, MR22 represents a valuable tool compound for studying SIK kinase function in cells.

The Small-Molecule Inhibitor MRIA9 Reveals Novel Insights into the Cell Cycle Roles of SIK2 in Ovarian Cancer Cells.

The activity of the Salt inducible kinase 2 (SIK2), a member of the AMP-activated protein kinase (AMPK)-related kinase family, has been linked to several biological processes that maintain cellular and energetic homeostasis. SIK2 is overexpressed in several cancers, including ovarian cancer, where it promotes the proliferation of metastases. Furthermore, as a centrosome kinase, SIK2 has been shown to regulate the G2/M transition, and its depletion sensitizes ovarian cancer to paclitaxel-based chemotherapy. Here, we report the consequences of SIK2 inhibition on mitosis and synergies with paclitaxel in ovarian cancer using a novel and selective inhibitor, MRIA9. We show that MRIA9-induced inhibition of SIK2 blocks the centrosome disjunction, impairs the centrosome alignment, and causes spindle mispositioning during mitosis. Furthermore, the inhibition of SIK2 using MRIA9 increases chromosomal instability, revealing the role of SIK2 in maintaining genomic stability. Finally, MRIA9 treatment enhances the sensitivity to paclitaxel in 3D-spheroids derived from ovarian cancer cell lines and ovarian cancer patients. Our study suggests selective targeting of SIK2 in ovarian cancer as a therapeutic strategy for overcoming paclitaxel resistance.

Structure-Based Design of Selective Salt-Inducible Kinase Inhibitors.

Salt-inducible kinases (SIKs) are key metabolic regulators. The imbalance in SIK function is associated with the development of diverse cancers, including breast, gastric, and ovarian cancers. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based p21-activated kinase (PAK) inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets, MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.