Serine-threonine protein kinases of the DYRK and CLK families regulate a variety of vital cellular functions. In particular, these enzymes phosphorylate proteins involved in pre-mRNA splicing. Targeting splicing with pharmacological DYRK/CLK inhibitors emerged as a promising anticancer strategy. Investigation of the pyrido[3,4-
g
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Serine-threonine protein kinases of the DYRK and CLK families regulate a variety of vital cellular functions. In particular, these enzymes phosphorylate proteins involved in pre-mRNA splicing. Targeting splicing with pharmacological DYRK/CLK inhibitors emerged as a promising anticancer strategy. Investigation of the pyrido[3,4-
g]quinazoline scaffold led to the discovery of DYRK/CLK binders with differential potency against individual enzyme isoforms. Exploring the structure–activity relationship within this chemotype, we demonstrated that two structurally close compounds, pyrido[3,4-
g]quinazoline-2,10-diamine
1 and 10-nitro pyrido[3,4-
g]quinazoline-2-amine
2, differentially inhibited DYRK1-4 and CLK1-3 protein kinases in vitro. Unlike compound
1, compound
2 efficiently inhibited DYRK3 and CLK4 isoenzymes at nanomolar concentrations. Quantum chemical calculations, docking and molecular dynamic simulations of complexes of
1 and
2 with DYRK3 and CLK4 identified a dramatic difference in electron donor-acceptor properties critical for preferential interaction of
2 with these targets. Subsequent transcriptome and proteome analyses of patient-derived glioblastoma (GBM) neurospheres treated with
2 revealed that this compound impaired CLK4 interactions with spliceosomal proteins, thereby altering RNA splicing. Importantly,
2 affected the genes that perform critical functions for cancer cells including DNA damage response, p53 signaling and transcription. Altogether, these results provide a mechanistic basis for the therapeutic efficacy of
2 previously demonstrated in in vivo GBM models.
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