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Cell-Based Assays to Detect Inhibitors of Fungal mRNA Capping Enzymes and Characterization of Sinefungin as a Cap Methyltransferase Inhibitor

Departments of Immunology, Merck Research Laboratories, Rahway, NJ

Douglas Wisniewski

Departments of Human and Animal Infectious Disease Research, Merck Research Laboratories, Rahway, NJ

Ann L. Perkins

Departments of Immunology, Merck Research Laboratories, Rahway, NJ

Qiaolin Deng

Departments of Molecular Systems, Merck Research Laboratories, Rahway, NJ

Myra B. Kurtz

Departments of Microbial Vaccine Research, Merck Research Laboratories, Rahway, NJ

Alice Marcy

Departments of Human and Animal Infectious Disease Research, Merck Research Laboratories, Rahway, NJ

Stephen A. Parent

Departments of Immunology, Merck Research Laboratories, Rahway, NJ

The m7GpppN cap at the 5' end of eukaryotic mRNAs is important for transcript stability and translation. Three enzymatic activities that generate the mRNA cap include an RNA 5'-triphosphatase, an RNA guanylyltransferase, and an RNA (guanine-7-) -methyltransferase. The physical organization of the genes encoding these enzymes differs between mammalian cells and yeast, fungi, or viruses. The catalytic mechanism used by the RNA triphosphatases of mammalian cells also differs from that used by the yeast, fungal, or viral enzymes. These structural and functional differences suggest that inhibitors of mRNA capping might be useful antifungal or antiviral agents. The authors describe several whole-cell yeast-based assays developed to identify and characterize inhibitors of fungal mRNA capping. They also report the identification and characterization of the natural product sinefungin in the assays. Their characterization of this S-adenosylmethionine analog suggests that it inhibits mRNA cap methyltransferases and exhibits approximately 5- to 10-fold specificity for the yeast ABD1 and fungal CCM1 enzymes over the human Hcm1 enzyme expressed in yeast cells.

Key Words: mRNA cap • (guanine-7-)-methyltransferase • sinefungin • Candida • yeast

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