Inhibition of Inositol Phosphorylceramide Synthase by the Cyclic Peptide Aureobasidin A

ABSTRACTBy using a detergent-washed membrane preparation, the interaction of the fungal natural product inhibitor aureobasidin A (AbA) with inositol phosphorylceramide synthase (IPC synthase) was studied by kinetic analysis of wild-type and mutant enzyme-catalyzed reactions. AbA inhibited the wild-type enzyme from both Candida albicansand Saccharomyces cerevisiaein an irreversible, time-dependent manner, with apparent Kivalues of 183 and 234 pM, respectively. Three synthetic chemistry-derived AbA derivatives, PHA-533179, PHA-556655, and PHA-556656, had affinities 4 to 5 orders of magnitude lower and were reversible inhibitors that competed with the donor substrate phosphatidylinositol (PI). AbA was a reversible, apparently noncompetitive inhibitor, with a Kiof 1.4 μM, of the IPC synthase from an AbA-resistant S. cerevisiaemutant. The Kmvalues for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the Vmaxfor the mutant enzyme was less than 10% of that for the wild-type enzyme. A comparison of the results obtained with AbA with those obtained with two other natural products inhibitors, rustmicin and khafrefungin, revealed that while rustmicin appeared to be a reversible, noncompetitive inhibitor of the wild-type enzyme, with a Kiof 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent Kiof 0.43 nM. An evaluation of the efficiencies of these compounds as inhibitors of the mutant enzyme revealed for both a drop in the apparent affinity for the enzyme of more than 2 orders of magnitude.