Summary activity statement /

Post-transcriptional and post-translational processes are required to expand the 20,000–25,000 genes into the estimated 50,000–500,000 human protein variants. Alternate gene splicing is one mechanism by which this rich diversity of protein isoforms is achieved, and novel tools to help researchers understand and control gene splicing outcomes continue to be of considerable interest. Since the report of the first Clk inhibitor probe from this project (CID 44968231/ML167), there have been a limited number of reports on additional inhibitors of the Clk kinases. Each has a distinctive signature and ML315 represents a new chemotype with Clk activity and a unique structural and phamacological signature with which to interogate this complex process. Like the other inhibitors of this target, ML315 will be used to interrogate the role that the Clk kinases play in altering SR protein phosphorylation and the resulting consequences on gene splicing. Apart from Clk4 (inhibited by ML167), no selective inhibitors have been reported for the other Clk isoforms (Clk1- Clk3); published Clk inhibitors either potently inhibit more than one isoform or have not been tested against all four isoforms. Using the available panel of inhibitors, including ML315, in parallel would allow biological investigations involving these kinases to proceed until isoform-selective inhibitors become available. One approach to using the three probes in parallel is described here: Using previous probe ML167 in Assay 1, one can inhibit Clk4 (136 nM) and observe the effect. Using previous probe ML106 in Assay 2, one can inhibit Clk1, Clk4, and Dyrk1A (all <100 nM) and observe the effect. Phenotypic or biochemical differences between Assay 1 and Assay 2 could be attributed to the lack of Clk1 or Dyrk1A activity. Using the new probe (ML315) in Assay 3, one can potently inhibit Clk1 and Clk4 (<100 nM), while only moderately inhibiting Clk2 and Dyrk1A (>200 nM). Phenotypic or biochemical differences between Assay 2 and Assay 3 could be attributed to the lack of Clk1 activity, allowing the roles Clk1 and Dyrk1A to be further distinguished. The differences observed between Assay 2 and Assay 3 might also be the result of moderate Clk2 inhibition. The parallel use of these probes is further discussed in Section 4.1. The previously reported probe (ML167) has been requested by numerous researchers and we plan to offer ML315 to researchers alongside this agent. We have additionally made other Clk inhibitors from the literature and offer each to researchers in the hopes that off-target activities associated with the divergent poly-pharmacology of each chemotype will be mitigated by parallel assessment of the activity of multiple Clk inhibitors (Coombs 2010, Coombs 2013).