Poster Presentation 10th Australian Peptide Conference 2013

D-PYC98, a novel substrate-selective peptide inhibitor of c-Jun N-terminal Kinase (JNK) (#106)

Kevin R Ngoei 1 , Bruno Catimel 2 , Nadia Milech 3 , Paul M Watt 3 , Marie A Bogoyevitch 1
  1. Bio21 Institute, University of Melbourne, Parkville, VIC, Australia
  2. Ludwig Institute of Cancer Research, Heidelberg, VIC, Australia
  3. Phylogica Ltd., Subiaco, WA, Australia

Protein kinase have remained attractive drug targets in the treatment of various pathological conditions with recent attention moving from ATP-competitive inhibitors towards the discovery and development of peptide inhibitors targeting the protein kinase substrate-binding site. Initially discovered in yeast two-hybrid screen, PYC98 is a JNK inhibitory peptide with a 5-fold increased potency to inhibit c-Jun phosphorylation in its D- retroinverso form. In vitro JNK activity assays revealed that D-PYC98 inhibited phosphorylation of an EGFR-derived peptide substrate, transcription factors c-Jun and ATF2, and the microtubule-regulatory protein DCX (doublecortin), but not phosphorylation of the transcription factor Elk1 or the microtubule-destabilising proteins SCG10 and Op18 (Stathmin). Surface Plasmon Resonance analysis confirmed D-PYC98 binding to both non-phosphorylated (inactive) and active JNK1, but not the substrates c-Jun and Elk1. Further biochemical analyses to determine the kinetics of inhibition revealed the non-ATP competitive mechanism of action of D-PYC98. The targeting the common docking site of JNK1 by D-PYC98 was further confirmed by BIAcore competition assay in presence of TIJIP and the failure of JNK1 common docking site mutants (JNK1-R127A-E329A) to bind D-PYC98. Lastly, in evaluating the efficacy of this peptide to act as a substrate competitive inhibitor in cell culture system, we observed the action of a cell-permeable version of D-PYC98 (D-PYC98-TAT) to inhibit c-Jun phosphorylation at Ser63 under hyperosmotic stress conditions. Overall, D-PYC98 is a novel substrate-selective, non-ATP competitive inhibitor of JNK, and in its modified form as D-PYC98-TAT represents an effective JNK-inhibitor suitable for use as a probe to discern particular biological functions of this important stress-responsive kinase in cellular systems.