Saturday, April 27, 2024
4/27/2024
Project Summary Protein kinases are essential to cellular function and development, and constitutively activated kinases have been widely targeted in the development of cancer therapies. Selectivity is a critical issue in kinase therapeutic discovery and biomedical research, especially in regard to off-target effects. Since the ATP binding site are highly conserved across the kinome, most ATP-competitive kinase inhibitors suffer from specificity issues, and the development of specific kinase inhibitors remains a significant bottleneck in modern drug discovery and biomedical research. Casein kinase 1 (CK1) is involved in various cellular signal transduction pathways including Wnt/ß- catenin, Hedgehog, and Hippo signaling pathways, and its mis-regulation results in various human diseases. There are six CK1 isoforms in humans such as CK1a, CK1¿1, CK1¿2, CK1¿3, CK1d, and CK1e, and each isoform displays individual physiological roles in cellular signal transduction. Currently, CK1 isoform-selective inhibitors are rarely developed, and most of the known CK1-specific agents have been revealed as multi-target or pan-kinase inhibitors upon further investigation. Thus, it will be a compelling future direction to develop isoform selective inhibitors of CK1 for the investigation of specific signaling mechanism of CK1 isoforms in pathophysiological conditions. The objective of this research is to develop isoform selective inhibitors of CK1 via computational analysis, synthetic chemistry, biochemical assays, and structural biology. Molecular dynamics (MD) simulations of CK1 isoforms will be performed to identify distinct features in the ATP-binding pocket of each enzyme for the design of selective inhibitors. In addition, the computational techniques and analysis methods will be applied to explain the mechanisms responsible for selective inhibition of each isoform. X-ray co-crystal structures of CK1 and our new specific inhibitors will be solved to assist further design efforts and validate hypotheses generated by computational analysis and molecular modeling. Multi-step synthesis and characterization of small molecules will be mainly conducted for the optimization of the potency and specificity of our starting agents and designed compounds. ADP-Glo biochemical assays and high-resolution Mass Spectrometry analysis will be used to study the inhibition of kinase activity of CK1 isoforms. A series of cell-based proliferation and functional assays will be performed to assess cellular potency of selective inhibitors such as MTT or CellTiter-Glo cell viability assay and 4E-BP1- and LRP6-phosphorylation analyses.
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