Development of JNK2-Selective Peptide Inhibitors That Inhibit Breast Cancer Cell Migration

Abstract

Despite their lack of selectivity towards c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP10-Δ-TATi), or to a poly-arginine sequence (JIP10-Δ-R9) enabled the potent inhibition of JNK2 (IC50~90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the over-expression of GFP-JNK2α. Both JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2α. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP10-Δ-TATi containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined and found to inhibit p38MAPKα in an in vitro cell-free assay with little propensity to inhibit JNK isoforms.

Novel tumor-suppressor function of KLF4 in pediatric T-cell acute lymphoblastic leukemia

“…Therefore, it is essential to develop selective inhibitors against specific JNK proteins to efficiently eliminate cancer cells with limited side effects. For example, two JNK2-specific peptide inhibitors showed the in vivo inhibition of migration in breast cancer cells through selective inhibition of JNK2 (96). AV7, an inhibitor selective for JNK1, has been tested in murine embryonic fibroblasts in vitro (97).…”

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

“…Precise structural models enhance the reliability of molecular docking to predict the binding modes of new lead compounds [85,[102][103][104][105][106]. Understanding water structure and energetics within the relatively shallow binding sites on the surface of MAP kinases may be critical to the further optimization of the non-ATP competitive inhibitors reviewed here [91].…”

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

“…Precise structural models enhance the reliability of molecular docking to predict the binding modes of new lead compounds [85, 102–106]. Understanding water structure and energetics within the relatively shallow binding sites on the surface of MAP kinases may be critical to the further optimization of the non-ATP competitive inhibitors reviewed here [91]. …”

“…In one of our recent reports we engineered new JIP-based peptide inhibitors that demonstrate specificity for the JNK2-isoform with an IC 50 ~90 nM. Using a flexible six carbon linker 6-aminohexanoyl (AHX) these peptides fused the JIP sequence to either the N -terminus of an inverted TAT sequence (JIP 10 -Δ-TAT i : Ac-PKRPTTLNLF-AHX-RRRQRRKKRG-amide) or to a poly-arginine sequence (JIP 10 -Δ-R 9 : Ac-PKRPTTLNLF-AHX-RRRRRRRRR-amide) [91]. Both peptides were shown to significantly inhibit JNK activation and c-Jun phosphorylation in HEK293T cells.…”