title: A novel retro-inverso peptide is a preferential JNK substrate-competitive inhibitor.
abstract: A novel 18 amino acid peptide PYC98 was demonstrated to inhibit JNK1 activity toward c-Jun. We observed a 5-fold increase in the potency of the retro-inverso form, D-PYC98 (a D-amino acid peptide in the reversed sequence) when compared with the inhibition achieved by L-PYC98, prompting our further evaluation of the D-PYC98 inhibitory mechanism. In vitro assays revealed that, in addition to the inhibition of c-Jun phosphorylation, D-PYC98 inhibited the JNK1-mediated phosphorylation of an EGFR-derived peptide, the ATF2 transcription factor, and the microtubule-regulatory protein DCX. JNK2 and JNK3 activities toward c-Jun were also inhibited, and surface plasmon resonance analysis confirmed the direct interaction of D-PYC98 and JNK1. Further kinetics analyses revealed the non-ATP competitive mechanism of action of D-PYC98 as a JNK1 inhibitor. The targeting of the JNK1 common docking site by D-PYC98 was confirmed by the competition of binding by TIJIP. However, as mutations of JNK1 R127 and E329 within the common docking domain did not impact on the affinity of the interaction with D-PYC98 measured by surface plasmon resonance analysis, other residues in the common docking site appear to contribute to the JNK1 interaction with D-PYC98. Furthermore, we found that D-PYC98 inhibited the related kinase p38 MAPK, suggesting a broader interest in developing D-PYC98 for possible therapeutic applications. Lastly, in evaluating the efficacy of this peptide to act as a substrate competitive inhibitor in cells, we confirmed that the cell-permeable D-PYC98-TAT inhibited c-Jun Ser63 phosphorylation during hyperosmotic stress. Thus, D-PYC98-TAT is a novel cell-permeable JNK inhibitor.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Kevin R W
authors.initials: KR
authors.lastname: Ngoei
authors.forename: Bruno
authors.initials: B
authors.lastname: Catimel
authors.forename: Nadia
authors.initials: N
authors.lastname: Milech
authors.forename: Paul M
authors.initials: PM
authors.lastname: Watt
authors.forename: Marie A
authors.initials: MA
authors.lastname: Bogoyevitch
mesh_terms: D051698:Activating Transcription Factor 2 / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D000089662:Doublecortin Protein; D057075:Enzyme Assays; D066246:ErbB Receptors / Q000378:metabolism; D055767:Immobilized Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D050807:ets-Domain Protein Elk-1 / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D051698:Activating Transcription Factor 2; C000714847:Dcx protein, mouse; D000089662:Doublecortin Protein; D055767:Immobilized Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D050807:ets-Domain Protein Elk-1; D066246:ErbB Receptors; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: Activity assays; Binding assays; Peptide inhibitor; Substrate selectivity; c-Jun N-terminal kinase