Substituting c-Jun N-terminal kinase-3 (JNK3) ATP-binding site amino acid residues with their p38 counterparts affects binding of JNK- and p38-selective inhibitors.
c-Jun N-terminal kinase (JNK) activation is linked to the aberrant cell death in several neurodegenerative disorders, including Parkinson's and Alzheimer's disease. The sequence similarity among the JNK isoforms and fellow MAP kinase family member p38 has rendered the challenge of producing JNK3-specific inhibitors difficult. Using the crystal structure of JNK3 complexed with JNK inhibitors, potential compound-interacting amino acid residues were mutated to the corresponding residues in p38. The effects of these mutations on the kinetic parameters with three compounds were examined: a JNK3- (vs. p38-) selective inhibitor (SP 600125); a p38-selective inhibitor (Merck Z); and a potent combined JNK3 and p38 inhibitor (Merck Y). The data confirm the role of the JNK3 residues Ile-70 and Val-196 in both inhibitor and ATP-binding. Remarkably, the Ile-70-Val and Val-196-Ala mutations caused an increase and decrease, respectively, in the binding affinity of the p38-specific compound, Merck Z, of 10-fold. The Ile-70-Val effect may be due to the increased capacity of the active site to accommodate Merck Z, whereas the Val-196-Ala mutant may induce an unfavourable conformational change. Conservative mutations of the Asn-152 and Gln-155 residues inactivated the JNK3 enzyme, possibly interfering with protein folding in a critical hinge region of the protein.
Fricker
Michael
M
Department of Molecular and Cellular Neuroscience, Neuroscience Research Centre, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, England, UK.
Lograsso
Philip
P
Ellis
Semantha
S
Wilkie
Neil
N
Hunt
Peter
P
Pollack
Scott J
SJ
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D001665:Binding Sites; D019921:Biotinylation; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D020935:MAP Kinase Signaling System; D048057:Mitogen-Activated Protein Kinase 10 / Q000737:chemistry*; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017510:Protein Folding; D011743:Pyrimidines / Q000494:pharmacology; D014633:Valine / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D000596:Amino Acids; D000873:Anthracenes; D004791:Enzyme Inhibitors; D007093:Imidazoles; C502553:Merck Y compound; C502554:Merck Z compound; D011743:Pyrimidines; D007532:Isoleucine; C432165:pyrazolanthrone; D000255:Adenosine Triphosphate; D048057:Mitogen-Activated Protein Kinase 10; D048051:p38 Mitogen-Activated Protein Kinases; D005978:Glutathione; D014633:Valine; D000409:Alanine