Biochemical assays

The kinase domains of human JAK1 (837-1142), JAK2 (828-1132), JAK3 (781-1124), and Tyk2 (873-1187) are cloned by PCR with N-terminal epitope tags. Recombinant proteins are expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays used a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction is carried out with INCB018424 or control, JAK enzyme, 500 nM peptide, adenosine triphosphate (ATP; 1 mM), and 2.0% dimethyl sulfoxide (DMSO) for 1 hour. The 50% inhibitory concentration (IC50) is calculated as the compound concentration required for inhibition of 50% of the fluorescent signal. Biochemical assays for CHK2 and c-MET enzymes are performed using standard conditions (Michaelis constant [K_m] ATP) with recombinantly expressed catalytic domains from each protein and synthetic peptide substrates. An additional panel of kinase assays (Abl, Akt1, AurA, AurB, CDC2, CDK2, CDK4, CHK2, c-kit, c-Met, GFR, EphB4, ERK1, ERK2, FLT-1, HER2, IGF1R, IKKα, IKKβ, JAK2, JAK3, JNK1, Lck, MEK1, p38α, p70S6K, PKA, PKCα, Src, and ZAP70) is performed using standard conditions using 200 nM INCB018424. Significant inhibition is defined as more than or equal to 30% (average of duplicate assays) compared with control values.

Cell Lines

Ba/F3-EpoR-JAK2V617F or HEL cells

Concentrations

0-4 nM

Incubation Time

48 hours

Methods

Ba/F3-EpoR-JAK2V617F or HEL cells are seeded at 2000/well of white bottom 96-well plates, treated with INCB018424 from DMSO stocks (0.2% final DMSO concentration), and incubated for 48 hours at 37 °C with 5% CO₂. Viability is measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent or viable cell counting. Values are transformed to percent inhibition relative to vehicle control, and IC50 curves are fitted according to nonlinear regression analysis of the data.

Animal Models

6- to 8-week-old female BALB/c mice with Ba/F3-JAK2V617F tumors

Formulation

5% dimethyl acetamide, 0.5% methocellulose

Doses

180 mg/kg

Administration

Oral gavage