title: Development of JNK2-selective peptide inhibitors that inhibit breast cancer cell migration.
abstract: Despite their lack of selectivity toward 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 (JIP(10)-Δ-TAT(i)) or to a poly arginine sequence (JIP(10)-Δ-R(9)) enabled the potent inhibition of JNK2 (IC(50) ≈ 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 overexpression of GFP-JNK2α. Both JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) 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 JIP(10)-Δ-TAT(i) 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 JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) 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.
authors: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, TX 78712, United States.
authors: Tamer S
authors: TS
authors: Kaoud
authors: Shreya
authors: S
authors: Mitra
authors: Sunbae
authors: S
authors: Lee
authors: Juliana
authors: J
authors: Taliaferro
authors: Michael
authors: M
authors: Cantrell
authors: Klaus D
authors: KD
authors: Linse
authors: Carla L
authors: CL
authors: Van Den Berg
authors: Kevin N
authors: KN
authors: Dalby
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology*; D002465:Cell Movement / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D048056:Mitogen-Activated Protein Kinase 9 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009362:Neoplasm Metastasis / Q000188:drug therapy / Q000473:pathology; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D010455:Peptides; D048056:Mitogen-Activated Protein Kinase 9