In vitro activity: WHI-P97 is a novel, rationally designed, and potent inhibitor of Janus kinase (JAK)-3 with an estimated Ki value of 0.09 μM in modeling studies and a measured IC50 value of 2.5 μM in EGFR kinase inhibition assays. Treatment of mast cells with WHI-P97 inhibited the translocation of 5-lipoxygenase (5-LO) from the nucleoplasm to the nuclear membrane and consequently 5-LO-dependent leukotriene (LT) synthesis after IgE receptor/FcepsilonRI crosslinking by>90% at low micromolar concentrations. WHI-P97 did not directly inhibit the enzymatic activity of 5-LO, but prevented its translocation to the nuclear membrane without affecting the requisite calcium signal. WHI-P97 was very well tolerated in mice, with no signs of toxicity at dose levels ranging from 5 microg/kg to 50 mg/kg, and LD(10) was not reached at a 50 mg/kg dose level when administered as a single i. p. or i.v. bolus dose. Therapeutic WHI-P97 concentrations, which inhibit mast cell leukotriene synthesis in vitro, could easily be achieved in vivo after the i.v. or i.p. administration of a single nontoxic 40 mg/kg bolus dose of WHI-P97. Notably, WHI-P97 showed promising biological activity in a mouse model of allergic asthma at nontoxic dose levels. Treatment of ovalbumin-sensitized mice with WHI-P97 prevented the development of airway hyper-responsiveness to methacholine in a dose-dependent fashion. Furthermore, WHI-P97 inhibited the eosinophil recruitment to the airway lumen after the ovalbumin challenge in a dose-dependent fashion. Further development of WHI-P97 may therefore provide the basis for new and effective treatment as well as prevention programs for allergic asthma in clinical settings

Kinase Assay: WHI-P97 is a novel, rationally designed, and potent inhibitor of Janus kinase (JAK)-3 with an estimated Ki value of 0.09 μM in modeling studies and a measured IC50 value of 2.5 μM in EGFR kinase inhibition assays.

Cell Assay: RBL-2H3 cells were cultured overnight on 22- × 22-mm coverslips at a cell density of 0.01 × 106/ml with 0.24 mg/ml DNP-IgE. Sensitized RBL-2H3 cells were then treated with 30 μM WHI-P97, WHI-P131, or vehicle before challenge with 20 ng/ml DNP-BSA at 37°C. After stimulation with DNP-BSA, the cells were fixed in cold methanol for 30 min, permeabilized in cold acetone, and blocked with 1% BSA containing nonimmune goat serum. Staining of mast cells with primary and secondary antibodies followed by confocal laser scanning microscopy.