In vitro activity: ML167 (also known as CID 44968231) is a highly selective Cdc2-like kinase 4 (Clk4) inhibitor with IC50 of 136 nM,>10-fold selectivity for closely related kinases Clk1-3 and Dyrk1A/1B. ML167 is found as the first fully selective inhibitor of Clk4 but not other related kinases including Clk1, Clk2 and Clk3 and Dyrk1A/1B. It is 10-fold more potent against Clk4 over other kinases. The treatment of ML167 on Caco-2 cells proves that ML167 is highly cell-permeable. Besides that, ML167 is also found to be an ATP-competitive inhibitor.

Kinase Assay: All compounds were screened using a qHTS approach, where compounds are assayed using at least seven concentrations to generate concentration-response curves for every compounds. The methodology for creating a concentration-titration series between successive copies of library plates for the purpose of large-scale titration-based screening has been described. Briefly, qHTS uses an inter-plate dilution method where the first plate contains the highest concentration of a set of compounds in DMSO, while subsequent plates contain the same compounds in the same well locations, but at successive lower concentrations. Using the protocol outlined above, we calculated a plate throughput of 18 plates/hr, or approximately 7 samples/sec, on the Kalypsys robotic system, which means that a 7 point CRC was obtained every second on the robotic system.

Cell Assay: ML167, as a probe of Clk4, shows high cell permeability in a Caco-2 assay.