Functional and structural characterization of a novel class of MAP-kinase inhibitors

p38 MAP kinases are a class of Ser/Thr kinases that link a number of extracellular stimuli to intracellular signaling pathways with effects on cellular processes such as cell cycle regulation, cell death, differentiation, senescence, cytokine production, etc. p38 kinase activation is nvolved in the pathogenesis of several inflammatory and oxidative stress diseases so that developing novel and more efficient pharmacological modulators of p38 activity is of great importance. In this context, we tested in HT29 human colorectal adenocarcinoma cells two novel pyrazolobenzothiazine inhibitors (COXP4M12 and COXH11) of p38 MAPK activity previously characterized in vitro in cell-free experiments. The inhibitory activity of the two test compounds was confirmed during the stimulation of cellular p38 activity with either LPS or H 2 O 2 . Interestingly enough, in LPS stimulated cells, these compounds were also found to be efficient inhibitors of JNK stress kinase, but not of ERK1/2 kinase. Of the two test compounds COXH11 showed the better inhibitory efficacy combined with low cytotoxicity and thus it was selected for further biochemical investigation that included successful crystallographic experiments for structural and molecular characterization of the protein-inhibitor complex. These preliminary data encourages us to further investigate COXH11 MAPK inhibitor in vivo.