Tricyclic covalent inhibitors selectively target Jak3 through an active site thiol

Abstract

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.

Keywords: Covalent Inhibitor; Cytokine; Drug Discovery; Enzyme Inhibitor; Enzyme Kinetics; Jak3; Janus Kinase (JAK); Tyrosine Kinase.

FIGURE 1.

Sequence conservation of human Janus kinase active sites and other kinases carrying a reactive thiol analogous to Cys-909 of Jak3. Positions listed above are shown on the crystal structure of Jak3 in yellow. Cys-909 is highlighted in red.

FIGURE 2.

Janus kinase inhibitors used in these studies. Cmpd, compound.

FIGURE 3.

Compounds (Cmpd) 1–4 show preincubation time-dependent changes in Jak3 inhibition. Kinase reactions (60 min) were initiated by the addition of ATP (1 μm final) after the specified preincubation time with compounds and Jak3 in the presence of peptide substrate. a, inhibition curves using Compound 1. b, IC₅₀ as a function of preincubation time for tricyclic compounds. c, inhibition curves using Compound 3. d, IC₅₀ as a function of preincubation time for comparator compounds. Note that 0.003 μm is the lower limit of quantitation in the assay, as shown by the dotted line in b and d. Error bars, S.E.

FIGURE 4.

Association kinetics of Jak3 inhibitors. The association of inhibitor binding to Jak3 was monitored by the displacement of a trFRET probe. a, Compound (Cmpd) 3. b, Compound 5. Decay curves were fit to an exponential function from which values of k_obs and the amplitude changes at each inhibitor concentration were determined. cts, fluorescence counts. c, k_obs versus inhibitor concentration yields the association rate constant from the slope of the linear fit: Compound 3, m = 1.0 × 10⁷ m⁻¹ min⁻¹; Compound 5, m = 2.7 × 10⁷ m⁻¹ min⁻¹. Only k_obs values below 0.2 min⁻¹ were used to ensure that rates had no contribution from dissociation of the trFRET probe.

FIGURE 5.

Covalent inhibitors irreversibly inhibit Jak3. a, Compounds (Cmpd) 1 and 4 were preincubated for 30 min at 2 μm concentrations with 0.32 μm Jak3 in the absence of ATP and diluted 400-fold, where kinase reactions in 1 μm ATP were performed. b, Compounds 3 and 6 were preincubated for 30 min at 2.8 and 15.2 μm concentrations, respectively, with 0.32 μm Jak3 plus 10 μm ATP and diluted 400-fold, where kinase reactions in 10 μm ATP were performed. Error bars, S.E.

FIGURE 6.

a, mass spectra of the Jak3 kinase domain shows that Compounds (Cmpd) 3 and 4 are covalent inhibitors. Jak3 was treated with inhibitor for 2 h (5-fold excess: 3 μm Jak3 and 15 μm inhibitor) in MOPSO kinase assay buffer in the absence of ATP at room temperature prior to mass spectrometry analysis. The known reversible inhibitor (Compound 6) does not modify the molecular weight. The predicted molecular mass for the Jak3 construct used carrying two phosphates is 33,550 Da. The predicted molecular mass of Compounds 3 and 4 is 241.3 and 356.4 Da, respectively. b, mass spectrometry mapping shows that Cys-909 is modified by Compound 3. Shown is the MS/MS spectrum (orbitrap/ion trap mode using an LTQ-Orbitrap) of peptide LVMEYLPSGCLR (m/z 811.40 {+2}) depicting modification of Cys-909 by Compound 3.

FIGURE 7.

Cellular activity of Jak3 inhibitors. Upper panel, phosphorylation of STAT5 (pSTAT5) in IL-2-stimulated T-blasts (dependent on signaling from both Jak1 and Jak3). Lower panel, phosphorylation of STAT5 in EPO-stimulated UT-7 cells (dependent on signaling from Jak2). Covalent tricyclic compounds (Compounds (Cmpd) 1 and 3) are inactive (>50 μm) in this assay. Error bars, S.E.

FIGURE 8.

a, crystal structure of Compound 4 covalently bound to Jak3 (monomer C). Electron density from F_O − F_C maps is shown contoured at 2σ. b, x-ray structure of Compound 4 (dark orange) and Jak3 overlaid with tricyclic core (light orange) modeled into the Jak3 active site. The structure of basic tricyclic core is shown on the right. c, Compound 3 modeled into Jak3 using a covalent docking method from the program Glide using 4QPS monomer C as a reference model (41).