JNK2_3 COV56d

In cell based assays, JNK2_3 Cov56d should provide selective inhibition of JNK2 verse JNK1 when used at concentrations of 1-5 uM. However, the potency of the probe against JNK3 in cells in not known and was reported as "not determinable", despite being calculated for other compounds reported in the article. No data regarding cellular cytotoxicity is provided. No inactive control compounds are available. In vitro, the probe is selective for JNK2 and JNK3 verse 95 other kinases. However, cell based selectivity against the human kinome has not been determined, and can differ from selectivity profiles determined in vitro (e.g., PMID: 25038787). Additional profiling using kinase-directed (e.g., PMID: 28051857) and cysteine-directed activity-based protein profiling platforms (e.g., PMID: 27309814) would clarify the proteome wide selectivity of the probe and appropriate use concentration. 56d is a covalent inhibitor, evidences by time dependent inhibition, sustained inhibition upon washout, and formation of a stable adduct with JNK2 (LC-MS). Cys116 is the expected nucleophile in JNK2, but this has not been definitely shown. The authors reported a hook-effect, likely due to poor solubility, when using JNK2_3 Cov56d in biochemical assays in vitro (at 125 nM, ~5-fold IC50 concentration). Compound solubility should be carefully monitored when performing experiments.

56d is likely a useful compound to probe MAPK9 and 10 (JNK2 and 3) function. It has an ATP-competitive, covalent mechanism of action with impressive kinact/KI. This compound shows very limited in vitro inhibition towards MAPK8 (JNK1) although the cell data shows this differentiation may be less pronounced in a biologically relevant setting (~11-fold selective). Caveats of use include the lack of data on the majority of the human kinome (97 kinases were tested and the covalently modified cysteine is conserved in other kinases) and potential solubility issues as indicated by the hook effect observed in in vitro assays. The ability of this compound to distinguish between effects of MAPK8 vs MAPK9/10 are likely to be useful particularly when combined with the use of tanzisertib as a MAPK8/9/10 pan inhibitor.