Co-crystal structure determination and cellular evaluation of 1,4-dihydropyrazolo[4,3-c] [1,2] benzothiazine 5,5-dioxide p38α MAPK inhibitors

p38α mitogen-activated protein kinase (MAPK) is an attracting pharmacological target in inflammatory diseases and cancer. Searching for new and more efficient p38-MAPK inhibitors, two recently developed pyrazolobenzothiazine-based (COXP4M12 and COXH11) compounds were investigated in this study using a cellular model of p38 activation. This consisted of HT29 human colorectal adenocarcinoma cells exposed to H 2 O 2 or lipopolysaccharide (LPS). Immunoblot data confirmed the inhibitory effect of COXP4M12 and COXH11 on p38 substrate phosphorylation (MAPK-APK2 and ATF2 transcription factor). Compound cytotoxicity was very low and apparent efficacy of these inhibitors was comparable with that of SB203580, a commercially available type I inhibitor of p38. All these compounds also inhibit upstream kinases that promote p38-MAPK phosphorylation and co-activate the stress-activated protein kinase JNK, while ERK1/2 MAPK phosphorylation was unaffected. Compound-target kinase interaction was investigated by means of co-crystallization experiments that provided further structural and molecular insight on the inhibitory mechanism and optimization strategy of this new class of p38-MAPK inhibitors.