Regulation and inhibition of MAP kinases
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Due to their role in cellular signaling, mitogen activated protein (MAP) kinases represent targets of pharmaceutical interest. Most MAP kinase inhibitors target the highly conserved ATP binding site. This conservation promotes cross-reactivity and toxicities that may limit their potential as drugs. These drawbacks motivate the search for non-ATP competitive inhibitors with acceptable specificity and potency and also drive efforts to understand MAPK regulation. We applied a virtual screening (VS) workflow to discover novel scaffolds for ATP-independent JNK (C-Jun N-terminal Kinase) inhibitors targeting the JNK-JIP (JNK Interacting Protein) interaction. (-)-Zuonin A was identified as an inhibitor of JNK, exhibiting 100-fold selectivity for the JNKs over other MAP kinases. (-)-Zuonin A was characterized extensively both in vitro and in cell-based assays. The JNK2 isoform has been reported to regulate breast cancer cell migration. Accordingly, we engineered a JNK2-selective peptide inhibitor. Peptides derived from the JIP scaffolds linked to the cell-penetrating peptide TAT are used widely to investigate JNK-mediated signaling events without exibiiting isoform selectivity. Herein, 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 [mathematical equation], or to a poly-arginine sequence [mathematical equation] enabled the potent inhibition of JNK2 (IC₅₀~90 nM) with 10-fold selectivity over JNK1 and JNK3. Both peptides revealed a potent ability to inhibit the induction of JNK activation and c-Jun phosphorylation in HEK293 cells treated with anisomycin, and inhibited the migration of Polyoma Middle-T Antigen Mammary Tumor (P[subscript y]VMT) cells through the selective inhibition of JNK2. ERK2 dimerization has been reported to regulate its nuclear translocation and signaling. Our analysis using light scattering, analytical ultracentrifugation and NMR provide strong evidence that ERK2 is monomeric under physiological conditions.