Gustafsson A B, Brunton L L
Department of Pharmacology, Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California, USA.
Mol Pharmacol. 1999 Aug;56(2):377-82. doi: 10.1124/mol.56.2.377.
The fungal metabolite balanol is a potent inhibitor of protein kinase A (PKA) and protein kinase C (PKC) in vitro that acts by competing with ATP for binding (K(i) approximately 4 nM); congeners of balanol show specificity for PKA over PKC. We have characterized the effects of balanol and 10"-deoxybalanol in intact cells to determine whether these compounds cross the cell membrane and whether the potency and specificity noted in vitro are preserved in vivo. In neonatal rat myocytes and cultured A431 cells transiently transfected with a cyclic AMP response element-luciferase reporter construct, balanol inhibits the induction of luciferase activity by isoproterenol, indicating inhibition of PKA. Western analysis shows that both balanol and 10"-deoxybalanol reduce phosphorylation of cAMP response element-binding protein in isoproterenol-stimulated A431 cells; inhibition is concentration dependent with an IC(50) value of approximately 3 microM. Balanol, but not 10"-deoxybalanol, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, a PKC substrate, in phorbol ester-stimulated A431 cells (IC(50) approximately 7 microM). Our data demonstrate that balanol is a potent inhibitor of PKA and PKC in several whole-cell systems and causes no obvious toxicity. In addition, balanol congeners inhibit PKA and PKC with the specificity and potency predicted by in vitro experiments.
真菌代谢产物巴拉诺醇在体外是蛋白激酶A（PKA）和蛋白激酶C（PKC）的有效抑制剂，其作用方式是与ATP竞争结合（抑制常数约为4 nM）；巴拉诺醇的同系物对PKA的特异性高于PKC。我们已对巴拉诺醇和10″-脱氧巴拉诺醇在完整细胞中的作用进行了表征，以确定这些化合物是否能穿过细胞膜，以及体外观察到的效力和特异性在体内是否得以保留。在新生大鼠心肌细胞和用环磷酸腺苷反应元件-荧光素酶报告基因构建体瞬时转染的A431培养细胞中，巴拉诺醇抑制异丙肾上腺素诱导的荧光素酶活性，表明其对PKA有抑制作用。蛋白质印迹分析表明，巴拉诺醇和10″-脱氧巴拉诺醇均可降低异丙肾上腺素刺激的A431细胞中环磷酸腺苷反应元件结合蛋白的磷酸化；抑制作用呈浓度依赖性，半数抑制浓度（IC50）值约为3 μM。在佛波酯刺激的A431细胞中，巴拉诺醇（而非10″-脱氧巴拉诺醇）抑制豆蔻酰化富含丙氨酸的C激酶底物蛋白（一种PKC底物）的磷酸化（IC50约为7 μM）。我们的数据表明，巴拉诺醇在多个全细胞系统中是PKA和PKC的有效抑制剂，且不会引起明显毒性。此外，巴拉诺醇同系物以体外实验预测的特异性和效力抑制PKA和PKC。
