KN-62, 1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine, a specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine (KN-62), a selective inhibitor of rat brain Ca²⁺/calmodulin-dependent protein kinase II (Ca²⁺/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca²⁺/CaM kinase II with K(i) value of 0.9 μM, but no significant effect up to 100 μM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca²⁺/calmodulin-dependent autophosphorylation of both α (50 kDa) and β (60 kDa) subunits of Ca²⁺/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca²⁺/CaM kinase II. Moreover, Ca²⁺/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca²⁺/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 μM)-induced autophosphorylation of the 53-kDa subunit of Ca²⁺/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca²⁺/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca²⁺/CaM kinase II activity in vivo.