The availability and posttranslational modification status of these regulatory proteins define the outcome of the splicing reaction and link it to extracellular signaling (Blaustein et?al., 2007). of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF). Highlights ? We report a highly selective nanomolar inhibitor KH-CB19 for the kinases CLK1/4 and DYRK1 ? KH-CB19 cocrystal structures revealed an ATP competitive but not ATP mimetic binding mode ? KH-CB19 formed halogen bonds with the kinase hinge region ? KH-CB19 led to dephosphorylation of SR proteins and effected splicing of TF isoforms in cells Introduction There are about 23,000 protein-coding genes in the human genome. However, the human proteome consists of a far larger number of unique protein sequences. In fact, some 90% of all transcribed genes may undergo alternative splicing and more than 80% may have at least 15% abundance of minor splicing forms (Shi et?al., 2008). In many cases, alternative splicing leads to the expression of several protein isoforms with different and sometimes antagonistic functions (Pajares et?al., 2007). Notable examples include pro- and antiapoptotic isoforms of Bcl-2 family members (Akgul et?al., 2004) and pro- and antiangiogenic forms of VEGFA (Harper and Bates, 2008). This plasticity plays a fundamental role in tissue development and the cellular response to external stimuli, for example in the control of blood clotting (Eisenreich et?al., 2009) and insulin action (Jiang et?al., 2009). Not surprisingly, the deregulation of alternative splicing has also been linked to numerous human pathologies (Ward and Cooper, 2010). The regulation of alternative splicing is highly complex. In addition to the essential enzymatic step of RNA breakage and ligation, the spliceosome must recognize the exon and intron boundaries precisely and in a controlled fashion. Not surprisingly, the splicing machinery involves hundreds of auxiliary factors that control splice site selection, spliceosome assembly and the splice reaction (Wahl et?al., Mebhydrolin napadisylate 2009; Bourgeois et?al., 2004). Indeed, the spliceosome alone rivals the ribosome and chromatin remodeling complexes in its complexity (Ritchie et?al., 2009). But what distinguishes the spliceosome is its very dynamic nature. During the different stages of the splicing process, dozens of proteins get recruited or dissociated from the spliceosomal complex (Wahl et?al., 2009). The availability and posttranslational modification status of these regulatory proteins define the outcome of the splicing reaction and link it to extracellular signaling (Blaustein et?al., 2007). One group of proteins regulating the selection of alternatively spliced Mebhydrolin napadisylate exonic or intronic premessenger (mRNA) sequences in response to environmental changes are serine/arginine-rich (SR) proteins (Bourgeois et?al., 2004). The group name relates to the serine/arginine-rich sequences present in these proteins (Long and Caceres, 2009). The serine residues in these sequence patches are phosphorylated by several protein kinase families, most notably the serine/arginine-rich protein kinases (SRPKs) and the CDC2-like kinase family (CLKs) (Colwill et?al., 1996; Gui et?al., 1994). The phosphorylation status of SR proteins regulates in turn their cellular localization and activity (Stamm, 2008). The phosphorylation-dependent signal transduction Mebhydrolin napadisylate is?a recurrent theme in cell signaling and the control of alternative splicing appears to be no exception. Given the recent success in designing selective kinase inhibitors, several efforts have been made to target CLKs. Muraki et?al. (2004) reported a cell permeable benzothiazole compound (TG003) with 20 nM and 15 nM potency for CLK1 and CLK4, respectively. However, more comprehensive profiling of this compound revealed strong inhibition of TG003 for all CLK family members except for CLK3 but also cross reactivity with casein kinase (CK1 and CK1?), dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1B), Yeast Sps1/Ste20-related kinase (YSK4) and proviral insertion site in Moloney Murine Leukemia Virus (PIM) kinase isoforms (Mott et?al., 2009). The latter paper reported also a series of substituted 6-arylquinazolines with low nM potencies inhibiting all CLKs as well as DYRK1A and DYRK1B and Mebhydrolin napadisylate the tyrosine kinase EGFR. In addition, a number of nonselective inhibitors have been reported together with the crystal structures of CLK1 and CLK3 (Bullock et?al., 2009). However, to date there are still no potent and highly selective CLK inhibitors with the submicromolar cellular activity available that would be required for use in in?vivo experiments. Chemical probes with such characteristics may help to decipher the role of Col4a2 CLKs not only in splicing regulation, but also in the control of viral infections (Karlas et?al., 2010) as well as cellular metabolism (Rodgers et?al., 2010). Here, we describe a novel class of CLK inhibitors (dichloroindolyl enaminonitriles), with high specificity for CLK1/CLK4 isoforms and a unique binding mode to the kinase hinge region. The lead compound shows single-digit nanomolar activity in.