Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors

Abstract

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.

Keywords: Activation loop; Inhibitor; Kinase; RIPK2; Receptor-interacting protein kinase 2.

Graphical abstract

Fig. 1

Superimposed crystal structure of docked regorafenib (pink) in RIPK2 lacking a resolved activation loop (gray; PDB ID: 4C8B) and biaryl urea (yellow) in the RIPK2 (blue; PDB ID: 5AR7) structure with a resolved activation loop (highlighted in deep pink).

Fig. 2

(A) Sequence alignment of the kinase domains of human RIPK1 and RIPK2. The kinase domains were aligned based on sequence similarity (http://www.uniprot.org); (B) Overlay structure showing positions of Ser161 (green) and Arg171 (deep pink) residues in the activation loops of RIPK1 (yellow) and RIPK2 (blue), respectively.

Fig. 3

Docking of regorafenib (pink) in RIPK2 (purple; PDB ID: 5AR7) structure with a resolved activation loop (highlighted in deep pink). Hydrophobic residues are highlighted in yellow. Distances from meta- and para-positions of urea phenyl to Arg171 shown.

Scheme 1

Synthesis of intermediates 10a–d. Reagents and conditions: (a) CH₃SCH₂CH₂OH, DIAD, PPh₃, THF, 0 °C to rt, 24 h (76%); (b) CH₃I, NaH, THF, 0 °C to rt, 16 h (30%); (c) H₂SO₄, reflux, 16 h (92%); (d) SOCl₂, MeOH, DME, 0–40 °C, 18 h (78%); (e) i) SOCl₂, reflux, 16 h, ii) NH₄OH, 0 °C, 1 h (87%); (f) (F₃CCO₂) ₂PhI, H₂O/MeCN, rt, 18 h (99%); (g) Boc₂O, NaHCO₃, THF, 0 °C to rt, 16 h (86%); (h) NH₄Cl, Fe, EtOH/H₂O, reflux, 1 h (76–99%).

Scheme 2

Synthesis of 1,2,5-thiadiazolidin-3-one 1,1-dioxide intermediate 10e. Reagents and conditions: (a) methyl 2-bromoacetate, Bu₄NBr, NaHCO₃, DMF, 90 °C, 18 h (62%); (b) 1) BocNHSO₂Cl, Et₃N, CH₂Cl₂, 0 °C, 4 h, 2) TFA, CH₂Cl₂, rt, 2 h (27% over two steps); (c) NaH, THF, rt, 1 h (96%); (d) NH₄Cl, Fe, EtOH/H₂O, reflux, 1 h (81%).

Scheme 3

Synthesis of intermediates 10f–h. Reagents and conditions: (a) methyl chloroacetate, K₂CO₃, MeCN, rt, 3.5 h (83–99%); (b) SOCl₂, MeOH, 0 °C to rt, 16 h (93%).

Scheme 4

Synthesis of CSR analogs with hydrophilic moieties on phenyl ring A. Reagents and conditions: (a) ^tBuOK, DMF, rt to 100 °C, 16 h (87%); (b) phenyl chloroformate, Py, CH₂Cl₂, 0 °C to rt, 1.5 h (28–99%); (c) 19, Py, 90 °C, 16 h (28–61%); (d) mCPBA, CH₂Cl₂, rt, 1 h (31%); (e) TFA, CH₂Cl₂, rt, 16 h (84%); (f) H₂, 10% Pd/C, MeOH, rt, 2 d (99%); (g) LiOH, THF/H₂O, 60 °C, 18 h (61–98%).

Fig. 4

(A and B) Co-crystal structure of CSR35 (pink) with RIPK2 (gray). Inhibitor CSR35 forms hydrogen bonds to the backbone of Met98, Glu66 in the αC-helix, and Asp164 in the DFG motif. Lys169 forms an ionic interaction with CSR35’s carboxylate side-chain. In addition, a salt bridge between Glu66 on the αC-helix and β3-Lys47 is evident in the DFG-out/αC-helix-in conformation. Red dashed lines indicate hydrogen bonds and red solid lines displays the ionic–ionic interactions. (PDB ID: 6ES0); C) Superimposed structure of RIPK2·CSR35 (gray and pink) and VEGFR2 (orange; PDB ID: 3WZE).

Fig. 5

Chemical structure of CSR35 fragments.