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-]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-]quinazoline-2,10-diamine and 10-nitro pyrido[3,4-]quinazoline-2-amine , differentially inhibited DYRK1-4 and CLK1-3 protein kinases in vitro. Unlike compound , compound efficiently inhibited DYRK3 and CLK4 isoenzymes at nanomolar concentrations. Quantum chemical calculations, docking and molecular dynamic simulations of complexes of and with DYRK3 and CLK4 identified a dramatic difference in electron donor-acceptor properties critical for preferential interaction of with these targets. Subsequent transcriptome and proteome analyses of patient-derived glioblastoma (GBM) neurospheres treated with revealed that this compound impaired CLK4 interactions with spliceosomal proteins, thereby altering RNA splicing. Importantly, 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 previously demonstrated in in vivo GBM models.