Cross-interactions of Two p38 Mitogen-activated Protein (MAP) Kinase Inhibitors and Two Cholecystokinin (CCK) Receptor Antagonists with the CCK1 Receptor and P38 MAP Kinase

Caroline Morel,Géraldine Ibarz,Catherine Oiry,Eric Carnazzi,Gilbert Bergé,Didier Gagne,Jean-Claude Galleyrand,Jean Martinez

doi:10.1074/jbc.m408851200

Abstract

Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

Highlights

Cholecystokinin (CCK)1 is a peptide involved in a wide range of biological functions
We showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity
Pharmacological Studies of the Interaction of SB202190 and SB203580 with the Rat CCK1 Receptor—We investigated the effect of the two p38 MAP kinase inhibitors (SB202190 and SB203580) on the binding of 125I-BH-CCK8s to rat CCK1 transiently transfected in COS7 cells

Summary

Introduction

Cholecystokinin (CCK) is a peptide involved in a wide range of biological functions. The development of p38 MAP kinase inhibitors is regarded as the main therapeutic strategy for stopping the production of pro-inflammatory cytokines involved in inflammatory and immunoresponsive diseases [14, 15] Among such compounds, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4pyridyl)-imidazole) and SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole) were described as potent and selective inhibitors of p38 MAP kinase, having no effect on ERKs and JNKs [16, 17]. SB compounds were described as preventing nucleoside transport by interacting with the NBMPR-sensitive equilibrative nucleoside transporter ENT1 [22, 23] This emphasizes the need to ensure the specificity of SB203580 when this compound is used in studies involving the p38 MAP kinase signaling pathway. We showed that the two nonpeptide CCK antagonists, L364,718 and L365,260, respectively selective for the CCK1 and the CCK2 receptors (Fig. 1) [3, 4], were able to directly interact with p38 MAP kinase to inhibit its activity

Results

Discussion

Conclusion