KH-CB19

Cellular activity of the compound was confirmed at 1-10 microM by reduced level of phosphorylated SRp55, SRp75 or other SR proteins. KH-CB19 should be used carefully for dissecting splicing machinery in the cell because (1) the effective concentration level in cell culture is double-digits higher than in cell-free kinase assays, and (2) there are multiple substrates downstream of CLK family member with different phosphorylation intensities. I recommend using other inhibitors, such as TG003, or knockdown techniques.

I would use KH-CB19 with caution; the profile of this compound needs to be developed further. The compound inhibits multiple CLKs (CLK1&3 - although it is somewhat selective for CLK1 over CLK3 in vitro). The compound also inhibits the related kinase DYRK1. KH-CB19 was active in cells but at signifiantly higher concentrations than in the enzymatic assay. I would like to see the compound screened against the full kinome to assess kinase selectivity more thoroughly. I would also like to see it screened against the recommended off-target panel of GPCRs, ion-channels, transporters, etc. 

Users should also be mindful of the chemical structure of KH-CB19. For example, if the double bond in KH-CB19 isomerizes, it may form 4-cyanoBauerine C (compound 3 in the authors' manuscript). No data has been presented on the stability of KH-CB19 under biological conditions, so this transformation cannot be ruled out in a cellular or in vivo setting. KH-CB19 also contains an ester that can possibly hydrolyze to an acid. Such hydrolysis could happen in vivo or in cells in the presence of intracellular esterases. It may be that the two ortho-substituents retard any ester hydrolysis; however, in the absense of data related to this transformation, it remains a possibility. It would be interesting to see if the acid counterpart to KH-CB19 has any relevant activity; if not, it could serve as a negative control.

Target-related cellular activity was demonstrated at concentrations of 1-10 uM, which seems high relative to the reported enzymatic activity (IC50 <100 nM). Given that there are no data on the compound's permeability, solubility, stability in cell media or binding to the cell media, it is difficult to establish if the enzyme to cell shift can be explained without invoking off-target effects. Stability data would be useful in light of the enamine ester, which may cyclize to provide a compound, which based on the Thermal Shift data available in the supplemental material, is expected to be a potent CLK1, CLK4 and possibly DYRK1A, PIM and TGFbetaR2 inhibitor. In addition to the reported enzymatic inhibition values for CLK1, CLK3 and DYRK1A, KH-CB19 is expected to be a potent inhibitor of CLK4 based on the TSA data. Additional weak interactions based on TSA were seen with PIM1/3 and SGK085, which could potentially translate into relevant enzymatic inhibition.