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(8 H )-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. ### Competing Interest Statement The authors have declared no competing interest. * ABL1 : Abelson murine leukemia viral oncogene homologue 1 AGC : protein kinases A, G and C AKT : protein kinase B AMPK : AMP-activated protein kinase BMPR2 : bone morphogenetic protein receptor type-2 BMX : bone marrow tyrosine kinase gene in chromosome X protein BRAF : serine/threonine protein kinase B-raf CAMK : calmodulin/calcium-dependent kinases CDK2 : cyclin-dependent kinase 2 CEP250 : centrosome-associated protein 250 CK1 : casein kinase 1 CREB : cAMP-response element binding protein CRTC : CREB-regulated transcription coactivators DSF : differential scanning fluorimetry EpHA : ephrin type-A receptor EpHB : ephrin type-B receptor FDA : food and drug administration FGFR1 : fibroblast growth factor receptor 1 GAK : cyclin-G-associated kinase GST : glutathione S-transferase HCC : hepatocellular carcinoma HDAC : histone deacetylase HPLC : high-performance liquid chromatography JNK1 : c-Jun N-terminal kinase 1 LKB1 : liver kinase B1 MST : mammalian STE20-like protein kinase NEK1 : NimA-related protein kinase 1 PAK : p21-activated kinase PI3K : phosphoinositide 3-kinase PKA : protein kinase A PLK1 : polo-like kinase 1 QIK : Qin-induced kinase RSK1 : ribosomal S6 kinase 1 SAR : structure-activity relationship SIK : salt-inducible kinase STE : homologous kinases of the yeast proteins STE20, STE11, and STE7 TK : tyrosine kinases TLK : tyrosine-like kinases ZAK : human leucine zipper-and sterile alpha motif-containing kinase