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

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.

Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors

“…Analyses of kinase inhibitor selectivity often focus on selectivity versus other protein kinases (32) and methods are available to test this (3). Our findings and others (6,7) show that additional, non-ATP-dependent proteins may also mediate off-target effects of kinase inhibitors.…”

“…Studies of inhibitor specificity typically examine cross-reactivity against other protein kinases (3) or low-potency effects against target panels involved in liabilities (4). However, recent publications illustrate that kinase inhibitors are also capable of potent interactions that affect the function of additional protein classes (5)(6)(7)(8). For example, an inhibitory activity of imatinib and nilotinib on the oxidoreductase enzyme Nqo2 has been shown; this activity is not shared with dasatinib (5, 7), a third kinase inhibitor approved in chronic myelogenous leukemia.…”

Nuclear factor kappa B–dependent gene transcription in cholecystokinin- and tumor necrosis factor-α–stimulated isolated acinar cells is regulated by p38 mitogen-activated protein kinase

“…Previous studies using pancreatic fragments stimulated by the CCK analog, cerulein, have shown that pro-inflammatory mediator production increased in parallel with activation of p38 and that these changes were attenuated by chemical inhibitors of p38 (22). However, chemical inhibitors of p38 MAP kinase may lack specificity as suggested by recent reports that they also inhibit the CCK-A cell surface receptor (23). Therefore, utilizing genetic manipulation via over expression of an adenoviral vector expressing DN p38 is a superior method of modulating p38 MAP kinase with respect to the non-specificity of chemical inhibitors.…”

Purinergic signaling induces thrombospondin-1 expression in astrocytes

“…Because SB202190 affects both Akt and p38͞MAPK phosphorylation, the complete reduction in TSP-1 induced by ATP could be a result of the combinatorial effect of blocking both Akt and p38͞MAPK signaling. We have considered the possibility that MAPK pathway inhibitors may affect functions of P2Y receptors such as receptor activation or G protein coupling because it has been reported that SB202190 can directly interact with the G protein-coupled receptor subtype CCK1, although not with the CCK2 subtype (45). However, the other kinase inhibitors we have used, U0126, wortmannin, and SP600125, are structurally unrelated to SB202190.…”