Structure of PolC reveals unique DNA binding and fidelity determinants.

PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-A resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique beta-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.

Discovery and analysis of 4H-pyridopyrimidines, a class of selective bacterial protein synthesis inhibitors.

Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyl-tRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC(50)s) ranging from 3 to 14 µM. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five- or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 µg/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition.

Spectrum of activity and mode of action of REP3123, a new antibiotic to treat Clostridium difficile infections.

OBJECTIVES: The aim of this study was to characterize the antimicrobial profile of REP3123, a novel inhibitor of methionyl-tRNA synthetase (MetRS) in development for the treatment of Clostridium difficile infection. METHODS: The spectrum of activity of REP3123 was determined by susceptibility testing of C. difficile and non-target organisms. The mode of action was studied by enzyme inhibition assays, macromolecular synthesis assays, target overexpression and selection of spontaneous resistant mutants. RESULTS: REP3123 was active against a collection of 108 clinical isolates of C. difficile and against epidemic, moxifloxacin-resistant BI/NAP1/027 strains (MIC range=0.5-1 mg/L and MIC(90) = 1 mg/L). The spectrum of activity included clinically important aerobic Gram-positive cocci such as Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Enterococcus faecium (MIC(90)s < 1 mg/L), but REP3123 was not active against most Gram-negative bacteria. REP3123 targeted C. difficile MetRS with a calculated inhibition constant (K(i)) of 0.020 nM, and selectivity was >1000-fold over human mitochondrial and cytoplasmic MetRS. The specific mode of action within bacterial cells was demonstrated by macromolecular synthesis assays that showed inhibition of protein synthesis by REP3123, and by metS overexpression, which resulted in a 16-fold increase in MIC for REP3123. Spontaneous REP3123-resistant mutants of C. difficile (MICs, 4-128 mg/L) arose with frequencies of 10(-8)-10(-9) and harboured distinct point mutations within the metS gene, resulting in 13 different amino acid substitutions. Most of the MetRS substitutions caused reduced catalytic efficiency and a growth fitness burden. CONCLUSIONS: REP3123 demonstrated a favourable microbiological profile and was found to target C. difficile with high specificity and selectivity.

A Visual Tagging Process for Mix and Sort Combinatorial Chemistry.

Colored glass beads and caps provide a simple color-coding strategy adapted to porous plastic containers and allow visual identification of the individual constituents of combinatorial libraries.

Development of 4H-pyridopyrimidines: a class of selective bacterial protein synthesis inhibitors.

BACKGROUND: We have identified a series of compounds that inhibit protein synthesis in bacteria. Initial IC50's in aminoacylation/translation (A/T) assays ranged from 3 to14 µM. This series of compounds are variations on a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ol scaffold (e.g., 4H-pyridopyrimidine). METHODS: Greater than 80 analogs were prepared to investigate the structure-activity relationship (SAR). Structural modifications included changes in the central ring and substituent modifications in its periphery focusing on the 2- and 6-positions. An A/T system was used to determine IC50 values for activity of the analogs in biochemical assays. Minimum inhibitory concentrations (MIC) were determined for each analog against cultures of Enterococcus faecalis, Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli tolC mutants and E. coli modified with PMBN. RESULTS: Modifications to the 2-(pyridin-2-yl) ring resulted in complete inactivation of the compounds. However, certain modifications at the 6-position resulted in increased antimicrobial potency. The optimized compounds inhibited the growth of E. faecalis, M. catarrhalis, H. influenzae, S. pneumoniae, S. aureus, E. coli tolC, mutants and E. coli modified with PMBN with MIC values of 4, ≤ 0.12, 1, 2, 4, 1, 1 µg/ml, respectively. IC50 values in biochemical assay were reduced to mid-nanomolar range. CONCLUSION: 4H-pyridopyrimidine analogs demonstrate broad-spectrum inhibition of bacterial growth and modification of the compounds establishes SAR.