Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2) and a molecule currently in clinical trials (Phase II) for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks) in vitro and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR) proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC50 = 3–90 nM) was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.

DYRK1A is important in neuronal development and function, and its excessive activity is considered a significant pathogenic factor in Down syndrome and Alzheimer's disease. Thus, inhibition of DYRK1A has been suggested to be a new strategy to modify the disease. Very few compounds, however, have been reported to act as inhibitors, and their potential clinical uses require further evaluation. Here, we newly identify CX-4945, the safety of which has been already proven in the clinical setting, as a potent inhibitor of DYRK1A that acts in an ATP-competitive manner. The inhibitory potency of CX-4945 on DYRK1A (IC50=6.8 nM) in vitro was higher than that of harmine, INDY or proINDY, which are well-known potent inhibitors of DYRK1A. CX-4945 effectively reverses the aberrant phosphorylation of Tau, amyloid precursor protein (APP) and presenilin 1 (PS1) in mammalian cells. To our surprise, feeding with CX-4945 significantly restored the neurological and phenotypic defects induced by the overexpression of minibrain, an ortholog of human DYRK1A, in the Drosophila model. Moreover, oral administration of CX-4945 acutely suppressed Tau hyperphosphorylation in the hippocampus of DYRK1A-overexpressing mice. Our research results demonstrate that CX-4945 is a potent DYRK1A inhibitor and also suggest that it has therapeutic potential for DYRK1A-associated diseases.

Enteroviruses are major causative agents of various human diseases, and some of them are currently considered to be an enormous threat to public health. However, no effective therapy is currently available for the treatment of these infections. We identified gemcitabine, a nucleoside-analog drug used for cancer treatment, from a screen of bioactive chemicals as a novel inhibitor of coxsackievirus B3 (CVB3) and enterovirus 71 (EV71). Gemcitabine potently inhibited the proliferation of CVB3 and EV71, as well as the replication of CVB3 and EV71 replicons, in cells with a low micromolar IC50 (1-5 μM). Its strong inhibitory effect was also observed in cells infected with human rhinoviruses, demonstrating broad-spectrum antiviral effects on enteroviruses. Mechanistically, an extensive analysis excluded the involvement of 2C, 3A, IRES-dependent translation, and also that of polyprotein processing in the antiviral effects of gemcitabine. Importantly, gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, exhibited a synergistic antiviral effect on the replication of CVB3 and EV71 replicons. Consequently, our results clearly demonstrate a new indication for gemcitabine as an effective broad-spectrum inhibitor of enteroviruses and strongly suggest a new therapeutic strategy using gemcitabine alone or in combination with ribavirin for the treatment of various diseases associated with enterovirus infection.

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