Discovery of potent and selective covalent inhibitors of JNK

Abstract

The mitogen-activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here, we report the discovery of irreversible inhibitors of JNK1/2/3. We describe two JNK3 cocrystal structures at 2.60 and 2.97 Å resolution that show the compounds form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK, in cells exposed to submicromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochemical, cellular, and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggests that the compound will be broadly useful as a pharmacological probe of JNK-dependent signal transduction. Potential lead compounds have also been identified for kinases, including IRAK1, PIK3C3, PIP4K2C, and PIP5K3.

Figure 1. Chemical structures for JNK inhibitors

(A) Representatives JNK inhibitors (B) Structural modifications relative to JNK-IN-1 for JNK-IN-1 to 6 or relative to JNK-IN-7 for JNK-IN-7 to 12 are highlighted in red.

Figure 2. Mass spectra for THZ-IN-2 labeled JNK1 protein

(A) Untreated JNK1 protein (B) JNK-IN-2 treated recombinantly produced JNK1 kinase domain. Inset mass spectra were obtained after deconvolution and show addition of 493 daltons after incubation with JNK-IN-2. (C) HCD MS/MS spectrum of the peptide LMDANLC*QVIQME (JNK residues 110-122; C* indicates labeled cysteine). Identification of ions of type b and y are indicated with lines above and below the sequence, respectively. See also Figure S2.

Figure 3. Crystal structure of complex JNK-IN-2 and JNK-IN-7 with JNK3

Crystal structures of JNK3 residues 39-402 modified at Cys-154 by (A) JNK-IN-2 and (B) JNK-IN-7. The covalent inhibitors (green stick figure for JNK-IN-2 and orange stick figure for JNK-IN-7) and the key residues of JNK3 that are involved in hydrophobic and hydrogen bonding interactions with the covalent inhibitors are labeled and are shown in stick models (grey). The hydrogen bonds between the kinase ‘hinge’ residue Met-149 and the aminopyrimidine-motif of the covalent inhibitors are represented as orange dotted lines (Figure S3). See also Figures S1 and S4.

Figure 4. Cellular pathway profiling with Western-blot

Western blot analysis of inhibition of JNK, cJun, MSK1 and p38 for JNK-IN-7, 8 and 11 following anisomycin stimulation of HEK293-IL1R cells. See also Figures S5 and S6.

Figure 4. Cellular pathway profiling with Western-blot

Western blot analysis of inhibition of JNK, cJun, MSK1 and p38 for JNK-IN-7, 8 and 11 following anisomycin stimulation of HEK293-IL1R cells. See also Figures S5 and S6.

Figure 4. Cellular pathway profiling with Western-blot

Western blot analysis of inhibition of JNK, cJun, MSK1 and p38 for JNK-IN-7, 8 and 11 following anisomycin stimulation of HEK293-IL1R cells. See also Figures S5 and S6.

Figure 5. Evaluation of the functional selectivity with mutagenesis

Mutation of the conserved Cys116 to Ser increases the IC50 for inhibition of JNK2 by over 100-fold for JNK-IN-7 and JNK-IN-8 but only by approximately 10-fold for JNK-IN-11.

Figure 5. Evaluation of the functional selectivity with mutagenesis

Mutation of the conserved Cys116 to Ser increases the IC50 for inhibition of JNK2 by over 100-fold for JNK-IN-7 and JNK-IN-8 but only by approximately 10-fold for JNK-IN-11.