Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria. Because of its unique physiological and biochemical properties, it serves as a potential target for development of novel antibacterial agents. In this study, we report the production, isolation, and structure determination of a family of structurally related novel lipoglycopeptides from a Streptomyces sp. as inhibitors of SPase I. Detailed spectroscopic analyses, including MS and NMR, revealed that these lipoglycopeptides share a common 14-membered cyclic peptide core, an acyclic tripeptide chain, and a deoxy-␣-mannose sugar, but differ in the degree of oxidation of the N-methylphenylglycine residue and the length and branching of the fatty acyl chain. Biochemical analysis demonstrated that these peptides are potent and competitive inhibitors of SPase I with K i 50 to 158 nM. In addition, they showed modest antibacterial activity against a panel of pathogenic Gram-positive and Gram-negative bacteria with minimal inhibitory concentration of 8 -64 M against Streptococcus pneumonniae and 4 -8 M against Escherichia coli. Notably, they mechanistically blocked the protein secretion in whole cells as demonstrated by inhibiting ␤-lactamase release from Staphylococcus aureus. Taken together, the present discovery of a family of novel lipoglycopeptides as potent inhibitors of bacterial SPase I may lead to the development of a novel class of broad-spectrum antibiotics.Proteins destined for secretion in both prokaryotic and eukaryotic organisms are initially synthesized as precursors with an amino-terminal extension known as signal (or leader) peptide. The signal sequence is removed by a signal peptidase (SPase) 1 that is localized in the cytoplasmic membrane in bacteria. Cleavage of precursors by SPase leads to the release of secreted proteins from the outer surface of cytoplasmic membrane. In bacteria, two major SPases, SPase I and SPase II with different cleavage specificities, have been identified. SPase I is responsible for processing the majority of secreted proteins (1-3), and SPase II is exclusively involved in processing glyceride-modified lipoproteins (4).SPase I is an attractive target for development of antibacterial agents because of its unique biochemical and physiological properties. It is essential for bacterial viability and growth as demonstrated by gene knockout and other genetic experiments (5-8). It is widely distributed in both Gram-positive and Gramnegative bacteria, as well as in Chlamydia. Genes encoding SPase I have been cloned and sequenced from different bacterial species, including many of clinically relevant bacteria (8, 9). The active domain of bacterial SPase I is exposed to the surface of cytoplasmic membrane as revealed by sequence and topological analysis (10 -12), and thus is accessible to potential inhibitors. In addition, SPases from bacteria and eukaryotic cells are different in composition, location, and possibly catalytic mechanism (13-17). These differences make it ...

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broadspectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a ␤-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and ␤-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

A novel, potent series of indole analogs were recently developed as MR antagonists, culminating in 14. This compound represents the first MR antagonist in this class of molecules, exhibiting picomolar binding affinity and in vivo blood pressure lowering at pharmaceutically relevant doses.

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Cdc25A assay-guided fractionation of a fermentation broth derived from a Streptomyces sp. resulted in the isolation of four novel naphthoquinones 1~4. Structures of these compounds were deduced by NMRand mass spectrometry. Two of them, 3 and 4, incorporate a modified cysteine residue which is observed for the first time in this class of natural products. Naphthoquinones 1~4 showed weak activity against cdc25A phosphatase.Cyclin/cdk (cyclin-dependent kinase) complexes serve as regulatory checkpoints for cell cycle progression.1}Cell cycle progression is delayed when the activities of cdks are suppressed through phosphorylation of their critical threonine and tyrosine residues. Cdc25, a family of dual specificity phosphatases, activate cdks by dephosphorylation of threonine and tyrosine residues and thus play a significant role in cell cycle progression.Cdc25Ais a memberof this family of phosphatases that appears to function early in the cell cycle.2) Furthermore, it has been shown that abnormal expression of cdc25A in the presence of RASmutants or RBdeletion mutants can lead to oncogenic transformation.3'4) Therefore, cdc25Aappears to be an attractive target for the discovery of novel cancer therapeutic agents. During the screening of microbial fermentations for cdc25A phosphatase inhibitors, an extract derived from a Streptomyces sp. was discovered to be active. Weherein report the production, purification, structure determination and cdc25A phosphatase activity of four novel napthoquinones. Experimental General MethodsHPLCseparations were performed on Rainin SD-1 and HPXLsystems with an UV-1absorbance detector (/l=240nm) using Dynamax 60A Microsorb phenyl (41.4 x 250mm, flow rate 45ml/minute) and Dynamax 60A C18 (21.4x250mm, flow rate 12ml/minute) column.UV absorption spectra were measured as the compounds eluted in 0.5% NH4OAc-CH3CN gradient using a Waters 717 system equipped with a Waters 996 photodiode array detector. IR spectra were determined on a Nicolet 510 P spectrophotometer. Optical rotations were measured on a JASCOmodel DIP-70 polarimeter.FAB-MS and HRFAB-MS were obtained on a VG Analytical ZAB-2-SE mass spectrometer. NMRspectra were determined on a Varian Unity (1H: 500MHz; 13C: 125.7MHz) spectrometer using a Z-SPEC microdual microsampleprobe. Carbon multiplicities were assigned by DEPTexperiments. Standard pulse sequences were employed for all 2D NMRexperiments. FermentationThe producing organism was isolated from a soil sample collected in Curacao. Stock cultures were kept as a suspension in 10%glycerol-5%lactose in the vapor phase of liquid nitrogen. Thawed aliquots (1 ml each)were inoculated into 3x50ml portions of medium containing 3% Trypticase Soy Broth (BBL), 0.5% glucose, 0.4% maltose, 0.3% yeast extract, and 0.2%MgSO4à"7H2O in de-ionized water. Each portion of inoculated mediumwas contained in a 250 ml Erlenmeyer flask and was incubated at 250rpm for 48 hours at 30°C