Structure-Activity Studies of Cationic Bilayer Stabilizing Inhibitors of Protein Kinase C

Abstract

Several compounds possessing diverse chemical structures have been shown to inhibit the action of protein kinase C (PKC). A general property of some of these compounds is that they inhibit bilayer to hexagonal phase interconversion in phosphatidylethanolamine bilayers. In addition, a large number of PKC inhibitors are positively charged. To clarify the relationship between charge and enzyme inhibition, the effect of the cationic amphiphile sphingosine was studied at different pH's. Inhibition by sphingosine was found to be pH dependent. Above pH 7.75, sphingosine has little or no inhibitory effect. In fact, at pH 8.5 sphingosine slightly enhances enzyme activity above that which occurs when the enzyme is stimulated by diacylglycerol and phosphatidylserine. After correcting for electrostatic repulsion, the intrinsic pK for sphingosine in Triton micelles is 8.5. Inhibition of PKC by sphingosine at physiological pH's therefore correlates with the presence of positive charge. In an attempt to optimize the structural features necessary for inhibition of PKC, a number of compounds which incorporate both positive charge as well as bilayer stabilizing ability were designed. These compounds possess a hydrophobic backbone which does not perturb hydrocarbon packing and have a tertiary or quaternary nitrogen functionality in the head group. All designed amphiphiles inhibit PKC activity; the potency of the amphiphile correlates with the presence of positive charge. Quaternary ammonium bilayer stabilizers are 10 fold more potent than their tertiary amine precursors, generally inhibiting in the 10 - 60 (mu)M range using the Triton mixed micelle assay. Aside from charge, factors such as the structure of the amine containing head group and its length from the hydrocarbon moiety did not markedly influence inhibitor potency. In contrast, the hydrocarbon backbone did influence potency. Cationic amphiphiles containing asteroid backbone were more potent than their straight chain analogues. These amphiphiles do not appear to alter the partitioning of PKC from the aqueous phase to the membrane surface. A number of bilayer stabilizing compounds possessing carboxylate and sulfate anions in addition to the quaternary nitrogen functionality were also designed. Although inhibitor potency correlated with the amount of charge present on the amphiphile, charge could not account for all the observed effects. Changes in the position of the charged functionalities and hydrocarbon length resulted in marked differences in amphiphile potency. Some inhibited in the submicromolar range. The results of these studies suggest that a combination of positive charge and a bilayer stabilizing structural characterisitic provides a basis for the rational design of PKC inhibitors.