Pyrazolo[1,5-a]pyrimidine-based inhibitors of HCV polymerase.

The present paper describes a novel series of HCV RNA polymerase inhibitors based on a pyrazolo[1,5-a]pyrimidine scaffold bearing hydrophobic groups and an acidic functionality. Several compounds were optimized to low nanomolar potencies in a biochemical RdRp assay. SAR trends clearly reveal a stringent preference for a cyclohexyl group as one of the hydrophobes, and improved activities for carboxylic acid derivatives.

Discovery of 4H-pyrazolo[1,5-a]pyrimidin-7-ones as potent inhibitors of hepatitis C virus polymerase.

A series of 4H-pyrazolo[1,5-a]pyrimidin-7-one derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase. A number of these compounds exhibited potent activity in enzymatic assay. The synthesis and structure-activity relationship are also described.

High-throughput assays for yeast RNA 5' triphosphatase (Cet1p).

The 5' cap on eukaryotic messenger RNA (mRNA) is critical for the stabilization, processing, nuclear transport, and translation of the transcript. Before capping can occur, the gamma-phosphate from the 5' end of newly synthesized RNA must be removed. In Saccharomyces cerevisiae, this reaction is catalyzed by Cet1p, an RNA triphosphatase. Because Cet1p is both essential for fungal growth and sufficiently different from its human counterpart in terms of three-dimensional structure and catalytic mechanism, it represents an unexplored target for antifungal drug discovery. To this end, we characterized the steady-state kinetics of Cet1p using both synthetic RNA oligos and nucleoside triphosphates. Nucleotide triphosphatase activity was measured in a scintillation proximity assay (SPA)-based high-throughput screen using [gamma-(33)P]biotin-11 GTP as substrate (GTP-SPA); the format is sensitive, accurate, robust, and compatible with automation. A charcoal absorption method was used to measure the release of free inorganic phosphate from an RNA substrate; the method was adapted to fit a 96-well plate format. The performance of the GTP-SPA and RNA assays was tested against a panel of commercially available compounds and found to be comparable. The charcoal absorption method run in the 96-well plate format has general utility for any phosphatase using nucleotides, nucleic acids, or proteins as substrate.

Sphingolipid biosynthesis in pathogenic fungi: identification and characterization of the 3-ketosphinganine reductase activity of Candida albicans and Aspergillus fumigatus.

An early step in sphingolipid biosynthesis, the reduction of 3-ketosphinganine, is catalyzed in the yeast Saccharomyces cerevisiae by Tsc10p (TSC10 (YBR265W)). We have identified orthologs of TSC10 in two clinically important fungal pathogens, Candida albicans and Aspergillus fumigatus. The translated sequences of the putative C. albicans ortholog, KSR1 (orf6.5112), and the putative A. fumigatus ortholog, ksrA, show significant homology to the yeast protein. All three proteins contain the signature motifs of NAD(P)H-dependent oxidoreductases in the short-chain dehydrogenase/reductase family and a conserved putative substrate-binding domain. Despite being essential in S. cerevisiae, we demonstrate that the C. albicans ortholog, KSR1, is not required for cell viability. However, ksr1 null mutants produce lower levels of inositolphosphorylceramides, are significantly more sensitive than the wildtype to an inhibitor of a subsequent step in sphingolipid biosynthesis, and are defective for the transition from yeast to filamentous growth, a key virulence determinant. Recombinant, purified Ksr1p and KsrA can carry out the reduction of 3-ketosphinganine in an NADPH-dependent manner. Molecular modeling of Ksr1p with bound substrates suggests that a significant portion of the aliphatic chain of 3-ketosphinganine protrudes from the enzyme. Guided by this molecular model, we developed shorter, water-soluble derivatives of 3-ketosphinganine that are substrates for 3-ketosphinganine reductase.

Aminothiazole inhibitors of HCV RNA polymerase.

Aminothiazole-based inhibitors designed for HCV polymerase display low micromolar potencies in biochemical assays. These compounds show a stringent preference for a cyclohexyl hydrophobe at the 2-amino position. The composition of these compounds suggests that they may be interacting at a recently discovered allosteric site on the polymerase.