Stability of Novel Siderophore Cephalosporin S-649266 against Clinically Relevant Carbapenemases.

To better understand the antibacterial activity of S-649266 against carbapenemase producers, its stability against clinically relevant carbapenemases was investigated. The catalytic efficiencies (kcat/Km) of IMP-1, VIM-2, and L1 for S-649266 were 0.0048, 0.0050, and 0.024 µM(-1) s(-1), respectively, which were more than 260-fold lower than that for meropenem. Only slight hydrolysis of S-649266 against KPC-3 was observed. NDM-1 hydrolyzed meropenem 3-fold faster than S-649266 at 200 µM.

Synthesis and in vitro/in vivo antibacterial activity of oxazolidinones having thiocarbamate at C-5 on the A-ring and an amide- or urea-substituted [1,2,5]triazepane or [1,2,5]oxadiazepane as the C-ring.

Oxazolidinones bearing a seven-membered [1,2,5]triazepane or [1,2,5]oxadiazepane heterocycle substituted with an amide or urea functionality as the C-ring and having a [1,2,3]triazole, a thiocarbamate, an isoxazole-3-ylamino, or a thioacetamide C-5 side chain unit on the A-ring instead of the typical acetamide were synthesized and their in vitro antibacterial activities towards various pathogens were evaluated. Several derivatives exhibited potent in vitro antibacterial activity toward not only Gram-positive, but also Gram-negative and linezolid-resistant pathogens. The in vivo therapeutic effects of amide 11a and ureas 16e, 17a were 2- to 3-fold greater than that of linezolid in a systemic mouse infection model treated by intravenous administration. Furthermore, compounds 11a and 17a showed lower monoamine oxidase (MAO)-inhibitory activity than our previously reported potent oxazolidinone antibacterials 3a and 3b.

Antibacterial oxazolidinone analogues having a N-hydroxyacetyl-substituted seven-membered [1,2,5]triazepane or [1,2,5]oxadiazepane C-ring unit.

We synthesized a series of oxazolidinone analogues bearing a N-hydroxyacetyl-substituted [1,2,5]triazepane or [1,2,5]oxadiazepane C-ring unit as homologues of an earlier drug candidate, eperezolid. Several of these compounds exhibited potent in vitro antibacterial activities towards not only Gram-positive, but also Gram-negative and linezolid-resistant pathogens. Compounds 21a and 21b, bearing a thiocarbamate side chain, showed high in vivo activity against methicillin-resistant Staphylococcus aureus SR3637, together with a promising safety profile in terms of weak inhibition of monoamine oxidase and cytochrome P450 isozymes.

Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue.

The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.

Control of bacteria adhesion by cell-wall engineering.

UDP-MurNAc-pentapeptide derivative bacterial cell-wall precursors were synthesized as effective tools for surface display on living bacteria. Lactobacilli were incubated in the ketone-modified precursor-containing medium, and the ketone moiety was displayed on the bacterial surface through cell-wall biosynthesis. Oligomannose was coupled with the ketone moiety on the bacterial surface via a aminooxyl linker, thereby displaying this oligosaccharide on the surface of the bacteria. The increase in the adhesion of the sugar-displaying bacteria onto a concanavalin A-attached film compared to that of native bacteria was confirmed by microscopic observation and surface plasmon resonance measurement. The incorporation of the artificial cell-wall precursors was enhanced when incubated with fosfomycin, an inhibitor of cell-wall precursor biosynthesis.

Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus.

The murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the murB gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity.