Nakanishi S, Yamada K, Iwahashi K, Kuroda K, Kase H
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
Mol Pharmacol. 1990 Apr;37(4):482-8.
KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
KT5926，即(8R*,9S*,11S*)-(-)-9-羟基-9-甲氧基羰基-8-甲基-14-正丙氧基-2,3,9,10-四氢-8,11-环氧-1H,8H,11H-2,7b,11a-三氮杂二苯并[a,g]环辛[cde]三茚-1-酮，被发现是肌球蛋白轻链激酶的一种强效且选择性的抑制剂。该化合物对Ca2+/钙调蛋白依赖性和非依赖性平滑肌肌球蛋白轻链激酶的抑制程度相似。这种抑制不受钙调蛋白浓度的影响。动力学分析表明，其抑制模式对于ATP而言是竞争性的（Ki为18 nM），对于肌球蛋白轻链而言是非竞争性的（Ki为12 nM）。这些结果表明KT5926在催化位点直接与该酶相互作用。KT5926也抑制其他蛋白激酶，但Ki值相对较高；蛋白激酶C、cAMP依赖性蛋白激酶和cGMP依赖性蛋白激酶的Ki值分别为723、1200和158 nM。在浓度低于10 microM时，KT5926不会抑制Ca2(+)-ATP酶、Na+/K(+)-ATP酶、己糖激酶和5'-核苷酸酶。在兔血小板中研究了KT5926对血小板活化因子或佛波酯诱导的5-羟色胺分泌和蛋白磷酸化的影响。KT5926抑制了一种20-kDa蛋白的磷酸化，但对一种40-kDa蛋白的磷酸化没有影响，从而表明该化合物在完整细胞中对肌球蛋白轻链激酶发挥其选择性抑制作用。该化合物抑制了血小板活化因子诱导的5-羟色胺分泌，但其效力明显低于K-252a，即(8R*,9S*,11S*)-(-)-9-羟基-9-甲氧基羰基-8-甲基-2,3,9,10-四氢-8,11-环氧-1H,8H,11H-2,7b,11a-三氮杂二苯并[a,g]环辛[cde]三茚-1-酮，后者抑制了20-kDa蛋白和40-kDa蛋白的磷酸化。KT5926不会抑制佛波酯诱导的分泌。这些结果提供了证据，表明肌球蛋白轻链激酶对20-kDa蛋白的磷酸化和蛋白激酶C对40-kDa蛋白的磷酸化在血小板的分泌反应中都起着重要作用。
