CEP-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activa tion of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultur es and differentiated PC12 cells after trophic withdrawal and in mice treat ed with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify mole cular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream reg ulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1 347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induce d by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, du al leucine zipper kinase, and leucine zipper kinase), The response was sele ctive because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC,, values ran ging from 23 to 51 nM, comparable to inhibitory potencies observed in intac t cells. In addition, overexpression of MLK3 led to death in Chinese hamste r ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as target s of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 ca n block MLK-induced cell death.