Inhibition of inositol phosphorylceramide synthase by the cyclic peptide aureobasidin A.
By 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 albicans and Saccharomyces cerevisiae in an irreversible, time-dependent manner, with apparent K(i) values 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 K(i) of 1.4 microM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The K(m) values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the V(max) for 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 K(i) of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent K(i) of 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.
Aeed
Paul A
PA
Pharmacia Corp., Kalamazoo, Michigan 49001, USA.
Young
Casey L
CL
Nagiec
Marek M
MM
Elhammer
Ake P
AP
D000465:Algorithms; D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects; D047630:Depsipeptides / Q000494:pharmacology*; D025141:Drug Resistance, Fungal; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006017:Glycolipids / Q000494:pharmacology; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D013997:Time Factors
D000935:Antifungal Agents; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D006017:Glycolipids; C109747:khafrefungin; C071398:aureobasidin A; D006602:Hexosyltransferases; C088714:phosphatidylinositol-ceramide phosphoinositol transferase