Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

Abstract

JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics.

Fig. 1.

In vitro characterization of pepJIP1 and BI-78D3. (A) Surface representation of JNK1 in complex with pepJIP1 (RPKRPTTLNLF) and the ATP mimic SP600125 (PDB-ID 1UKI). Surface generated with MOLCAD (28) and color coded according to cavity depth (blue, shallow; yellow, deep). (B) Chemical structure of BI-78D3. (C) Dose dependent displacement of biotinylated pepJIP1 from GST-JNK1. (D) Kinase inhibition assay for BI-78D3.

Fig. 2.

Docking studies and NMR analysis. (A) Chemical structure and predicted binding mode of SP600125 derived indazole-TEMPO compound. (B) 1D ¹H-NMR T_1ρ spectra of BI-78D3 (500 μM) in the presence of 5 μM JNK-2 (blue) or 5 μM JNK-2 and 200 μM ATP-TEMPO (red) at 100 ms. NMR resonance assignments for the hydrogen nuclei of the small molecule are reported. Signal reductions are as follows: H1 46%, H2 56%, H3/H5 65%, and H4 72%. (C) Docked structure of BI-78D3 into the x-ray structure of JNK1 (PDB ID 1UKI). Predicted hydrogen bonding interactions between the compound and the residue Arg-127 (displayed) are highlighted with yellow dashed lines. Computational docking studies were performed with GOLD 2.1 (The Cambridge Crystallographic Data Centre) (28, 29) and analyzed with Sybyl. Molecular models were generated with CONCORD (29) and energy minimized with Sybyl.

Fig. 3.

Binding affinity data. The dissociation constants for BI-78D3 as determined by isothermal titration calorimetry are reported for JNK2 (A), JNK2 (B) R127A, and JNK2 C162S (C).

Fig. 4.

Bioevaluation studies with BI-78D3. (A) TR-FRET analysis of c-Jun phosphorylation upon TNF-alpha stimulation of HeLa cells in the presence of increasing BI-78D3. (B) BI-78D3 effect on serum alanine-aminotransferase levels after 7.5 h of exposure to ConA compared with DMSO control. Results shown are averages ± SD (n = 7). (C) Effect of BI-78D3 (25 mg/kg) on insulin resistance in 11-week-old BKS.Cg-+Lepr^db/+Lepr^db/OlaHsd db/db mice (Harlan Sprague–Dawley). Closed diamonds, vehicle control; closed squares, 25 mg/kg BI-78D3. Data shown as means ± SD (n = 5). *, P < 0.0003; **, P < 0.0001.