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Antimicrobial Agents and Chemotherapy, February 2009, p. 496-504, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00633-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Inhibition of Inositol Phosphorylceramide Synthase by the Cyclic Peptide Aureobasidin A{triangledown}

Paul A. Aeed,{dagger} Casey L. Young,{ddagger} Marek M. Nagiec,§ and Åke P. Elhammer*

Pharmacia Corp., 7000 Portage Rd., Kalamazoo, Michigan 49001

Received 14 May 2008/ Returned for modification 21 September 2008/ Accepted 23 November 2008

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 Ki 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 Ki of 1.4 µM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The Km values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the Vmax 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 Ki of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent Ki 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.

* Corresponding author. Present address: AureoGen Biosciences, Inc., 6475 Technology Avenue, Suite C, Kalamazoo, MI 49009. Phone: (269) 353-3805. Fax: (269) 585-6083. E-mail: ake.p.elhammer{at}aureogen.com

{triangledown} Published ahead of print on 1 December 2008.

{dagger} Present address: Pfizer, Inc., 333 Portage Street, Kalamazoo, MI 49007.

{ddagger} Present address: Replidyne Corporation, 1450 Infinite Drive, Louisville, CO 80026.

§ Present address: Global Research and Development, Pfizer, Inc., 700 Chesterfield Parkway West AA4E, Chesterfield, MO 63017-1732.

Antimicrobial Agents and Chemotherapy, February 2009, p. 496-504, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00633-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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