Application of Affinity Selection/Mass Spectrometry to Determine the Structural Isomer of Parnafungins Responsible for Binding Polyadenosine Polymerase Gregory C. Adam,* Craig A. Parish, Douglas Wisniewski, Juncai Meng, Min Liu, Kathleen Calati, Benjamin D. Stein, † John Athanasopoulos, Paul Liberator, Terry Roemer, Guy Harris, and Kevin T. Chapman Target Validation Chemistry, Natural Products Chemistry, and Infectious Diseases, Merck Research Laboratories, Merck and Company, P.O. Box 2000, Rahway, New Jersey 07065 Received July 16, 2008; E-mail: gregory_adam@merck.com Abstract: To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the “straight” parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the “bent” structural isomer (parnafungin B). Introduction With the higher prevalence of invasive medical procedures as well as an increased number of immuno-compromised patients due to factors such as cancer and organ transplant chemotherapy and diseases such as AIDS, life-threatening fungal infections have become a more prominent clinical issue. 1 The current arsenal of antifungal agents could be enhanced by discovering new structural classes that possess broad spectrum activity, have a strong safety profile, and are orally bioavailable. New screening methods have been developed to identify novel chemical scaffolds that specifically target unexploited biochemi- cal pathways in fungi. One such method is the Candida albicans Fitness Test (CaFT), a whole cell screening platform that can be used to define the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus C. albicans. 2,3 In the CaFT, a pool of 2868 molecularly bar coded C. albicans strains have been genetically engineered to be heterozygous for a unique gene. These genes have been selected based on their documented importance for normal growth and/or viability in Saccharomyces cereVisiae 4 and/or C. albicans. 3,5 This pool is screened for hypersensitivity against sublethal concentrations of chemical agents. Compound-specific growth effects on individual mutant strains provide crucial information to the identity of the inhibitor’s cellular target and/ or the affected biochemical pathway. Traditionally, the CaFT assay had been applied to screening purified libraries and collections of synthetic compounds with antifungal activity. 5,6 Recently, however, the CaFT technology has been extended to the analysis of crude fermentation extracts to discover natural products with novel modes of action. 7 Historically, natural products screening has provided a wealth of structurally diverse antifungal agents from which two major classes have become commercial drugs. These include the enchinocandin lipopeptides (caspofungin, 8 micafungin, 9 anidu- lafungin 10 ), which target fungal cell wall biosynthesis, 11 and the macrocyclic polyenes (amphotericin), which target mem- brane ergosterol. 12 From the screening of a large set of fermentation broth extracts, the CaFT identified a sample derived † Current address: The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037. (1) Aperis, G.; Myriounis, N.; Spanakis, E. K.; Mylonakis, E. Expert Opin. InVest. 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Published on Web 11/17/2008 10.1021/ja805531w CCC: $40.75  2008 American Chemical Society 16704 9 J. AM. CHEM. SOC. 2008, 130, 16704–16710