JH295 hydrate is a potent, irreversible, and selective NIMA-related kinase 2 (Nek2) inhibitor that inhibits cellular Nek2 by Cys22 alkylation (IC50 = 770 nM). JH295 hydrate is not active against the mitotic kinases Cdk1, Aurora B, or Plk1, and does not interfere with bipolar spindle assembly or the spindle assembly checkpoint [1].

Treating RPMI7951 cells with JH295 (Compound 16; concentration range 0.08-20 μM; for 45 minutes) significantly inhibits the activity of wild-type Nek2, achieving an IC50 value of approximately 1.3 μM. Conversely, this treatment exhibits minimal impact on the C22V mutant version of Nek2. Western Blot analysis, employing the same cell line and varying concentrations of JH295 (0.08 μM to 20 μM over a 45-minute incubation period), corroborates these findings, underscoring the compound's potent inhibition of WT Nek2 while demonstrating negligible effects on the C22V mutant.