Identification of imidazo[1,2- b]pyridazine TYK2 pseudokinase ligands as potent and selective allosteric inhibitors of TYK2 signalling

Abstract

As a member of the Janus (JAK) family of non-receptor tyrosine kinases, TYK2 mediates the signaling of pro-inflammatory cytokines including IL-12, IL-23 and type 1 interferon (IFN), and therefore represents an attractive potential target for treating the various immuno-inflammatory diseases in which these cytokines have been shown to play a role. Following up on our previous report that ligands to the pseudokinase domain (JH2) of TYK2 suppress cytokine-mediated receptor activation of the catalytic (JH1) domain, the imidazo[1,2-b]pyridazine (IZP) 7 was identified as a promising hit compound. Through iterative modification of each of the substituents of the IZP scaffold, the cellular potency was improved while maintaining selectivity over the JH1 domain. These studies led to the discovery of the JH2-selective TYK2 inhibitor 29, which provided encouraging systemic exposures after oral dosing in mice. Phosphodiesterase 4 (PDE4) was identified as an off-target and potential liability of the IZP ligands, and selectivity for TYK2 JH2 over this enzyme was obtained by elaborating along selectivity vectors determined from analyses of X-ray co-crystal structures of representative ligands of the IZP class bound to both proteins.

Fig. 1. Known TYK2 JH1 ligands.

Fig. 2. Janus family kinase architecture and structure of TYK2 kinase and pseudokinase domains. (a) Schematic illustrating the entire structure of the Janus kinase family (JAKs). (b) Superposition of TYK2 JH2 domain structure (green) PDB code 4WOV with the TYK2 JH1 domain structure complexed with ADP (magenta ribbons and ADP carbons in cyan), PDB code ; 4GVJ. (c) TYK2 pseudokinase domain residues corresponding to those of protein kinases normally involved in catalytic machinery are shown in stick. Key residues of the ATP-pocket are differentiated from the JH2 to the JH1 domains, see ref. 14 for additional details.

Fig. 3. High throughput screen hits for IL-23 inhibition.

Fig. 4. Previously reported TYK2 JH2 ligands.

Scheme 1

Scheme 2

Fig. 5. X-ray crystal structure representation of 18 co-complexed with TYK2 JH2 (PDBID 5TKD). (a) Close-up of the binding site. Hydrogen bond interactions are shown with dotted lines with key residues and observed water labelled. TYK2 JH2 ribbon and carbons in green. Carbons of 18 in magenta. (b) Surface representation of the protein binding site highlighting space off of the C3 amide and part of the C6 aniline (yellow arrows). Hydrogens added for clarity.

Fig. 6. Selectivity profile of 29. Selectivity was determined from binding data generated using a fluorescent probe displacement assay. Illustration reproduced courtesy of Cell Signaling Technology, Inc.

Fig. 7. X-ray crystal structure representation of 30 co-complexed with PDE4 (PDBID 5TKB). Surface representation of the protein binding site is shown highlighting that space off of both meta positions of the C6 aniline are hindered (yellow arrows). Carbons of PDE4 are coloured green and carbons of 30 in magenta. Hydrogens added for clarity.