Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket

Abstract

Janus kinases (JAKs) are a family of cytoplasmatic tyrosine kinases that are attractive targets for the development of anti-inflammatory drugs given their roles in cytokine signaling. One question regarding JAKs and their inhibitors that remains under intensive debate is whether JAK inhibitors should be isoform selective. Since JAK3 functions are restricted to immune cells, an isoform-selective inhibitor for JAK3 could be especially valuable to achieve clinically more useful and precise effects. However, the high degree of structural conservation makes isoform-selective targeting a challenging task. Here, we present picomolar inhibitors with unprecedented kinome-wide selectivity for JAK3. Selectivity was achieved by concurrent covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. We confirmed that in vitro activity and selectivity translate well into the cellular environment and suggest that our inhibitors are powerful tools to elucidate JAK3-specific functions.

Keywords: JAK3; Janus kinases; chemical probe; covalent reversible inhibitor; kinome selectivity.

Graphical abstract

Figure 1

Structures of Tofacitinib 1, Lead Compound 2, and Novel JAK3 Inhibitors 3–8 See also Tables S1, S2, S3, and S6.

Figure 2

Co-crystal Structures of JAK3 and Compounds 4 and 5 (A) 4 non-covalently bound to JAK3 (PDB: 5LWM). (B) 5 non-covalently bound to JAK3 (PDB: 5LWN). (C) 5 covalently bound to JAK3 (PDB: 5LWN). (D) 2F_o − F_c omitted electron density map of 4-JAK3. (E) 2F_o − F_c omitted electron density map of 5-JAK3. (F) Dose-dependent BRET experiment showing displacement of the fluorescent tracer in NanoLuc tagged JAK3 in HeLa cells. (G) Residence time experiment using BRET. HeLa cells expressing NanoLuc JAK3 were equilibrated with 1 μM of 4, washed out, and treated with high concentrations of tracer. Displacement of 4 was monitored by BRET. BRET levels of full occupancy control were reached after approx. 1 hr. Data shown are means ± SD of quadruplicates. See also Figures S1, S2, and Table S4.

Figure 3

Induced Binding Pocket Around R911 (A) Protein surface of 1 bound to JAK3 (PDB: 3LXK). (B) Protein surface of 5 bound to JAK3 (PDB: 5LWN). N-terminal lobes are omitted for clarity and heteroatoms of the residues of R911, D912, and R953 are colored. Comparison of (A) and (B) shows the rearrangement of these amino acid side chains upon formation of the arginine pocket. (C) Alignment of 16 JAK3 crystal structures with 4-JAK3. Residues of R911, D912, and R953 are shown as sticks (4-JAK3, PDB: 5LWM) or as lines (other structures, PDB: 3LXK, 4QT1, 4QPS, 4RIO, 4ZEP, 4I6Q, 3ZC6, 4HVD, 4HVG, 4HVH, 4HVI, 3PJC, 3LXL, 1YVJ, 4V0G, 4Z16). The deviating conformation of these side chains is unique to our structure, while it is relatively conserved among the other JAK3 structures. (D and E) BRET experiments measuring residence time of 4 in HeLa cells expressing the NanoLuc mutant JAK3 R953A (D) or the NanoLuc double mutant JAK3 R911A/R953A (E). BRET traces of vehicle-treated cells are shown as filled black spheres (D) and diamonds (E), traces of inhibitor-treated cells are shown in red, and no tracer control is shown as empty squares. Washout experiments show that both mutants retain slow binding kinetics of 4. Data shown are mean ± SD of quadruplicates. See also Figure S1, Tables S4, and S5.

Figure 4

Selective Inhibition of JAK3-Mediated Cytokine Signaling by Compounds 4 and 5 Human CD4⁺ T cells were pre-incubated for 1 hr with the indicated concentrations of the JAK inhibitors (JAKi) 1, 4 or 5 and stimulated for 30 min with IL-2 (activates JAK3/JAK1) (A), IL-4 (activates JAK3/JAK1) (B), IL-6 (activates JAK2/JAK1/TYK2) (C) or IFN-α (activates JAK1/TYK2) (D). Phosphorylation of STAT5 (A), STAT6 (B), STAT3 (C), or STAT1 (D) was determined by phospho-specific Abs and immunoblotting (ns, no cytokine stimulation). Levels of actin were determined to show equal loading. See also Table S1.