SB-203207 and SB-203208, two novel isoleucyl tRNA synthetase inhibitors from a Streptomyces sp. II. Structure determination.

Two novel isoleucyl tRNA synthetase inhibitors, SB-203207 and SB-203208 have been isolated from a Streptomyces sp. and found to be structurally related to altemicidin. Structures of SB-203207 and SB-203208 have been deduced by a combination of spectroscopic techniques, derivatisation, hydrolysis studies and found to be 4-(aminocarbonyl)-7-[[(2-amino-3-methylpentanoyl)aminosul phonyl]acetamido]-2,4a,5,6,7,7a-hexahydro-6-hydroxy-2-methyl-1H-2- pyrindine-7-carboxylic acid (1) and 4-(aminocarbonyl)-7-[[(2-amino-3-methyl pentanoyl)-aminosulphonyl]acetamido]-2,4a,5,6,7,7a-hexahydro-6-(2- amino-3-phenylbutanoyl oxy)-2-methyl-1H-2-pyrindine-7-carboxylic acid (2), respectively.

Inhibition of metallo-beta-lactamases by a series of mercaptoacetic acid thiol ester derivatives.

A series of mercaptoacetic acid thiol esters have been identified as metallo-beta-lactamase inhibitors. Electrospray mass spectrometry (ESMS) has shown that irreversible inhibition of the Bacillus cereus II metallo-beta-lactamase by SB214751, SB214752, and SB213079 was concomitant with a 90-Da increase in mass of the enzyme. Tryptic digestion of the B. cereus II inhibited with SB214751 illustrated that the peptide fragment, containing the only cysteine of the enzyme, had undergone a mass increment of 90 Da. It was further demonstrated that B. cereus II hydrolyzed this type of compound across the thiol ester bond to yield mercaptoacetic acid. Mercaptoacetic acid is the only molecular fragment common to SB214751, SB214752, and SB213079, and free mercaptoacetic acid does not bind covalently to B. cereus II. Therefore, it is concluded that these compounds inhibit B. cereus II by the mechanism-based delivery of mercaptoacetic acid, forming a disulfide linkage with the active sites cysteine (predicted mass shift = +90 Da) under the aerobic conditions of the assay. The different thiol esters examined had a broad range of potencies against the metallo-beta-lactamases tested. For example SB214751, SB214752, and SB213079 all had 50% inhibitory concentrations of < 10 and > 1,000 microM for the Stenotrophomonas maltophilia L-1 and Bacteroides fragilis CfiA enzymes, respectively. SB216968 was particularly active against the Aeromonas hydrophila CphA metallo-beta-lactamase and was found to be an uncompetitive inhibitor of this enzyme (Ki = 3.9 microM), whereas it exhibited irreversible inhibition of the L-1 enzyme. These observations with this series of compounds have revealed subtle differences between the active sites of different metallo-beta-lactamases. Finally, a novel application for isothermal titration calorimetry for assessing the zinc chelating activity of candidate inhibitors is also presented.

beta-Lactamase ragged ends detected by electrospray mass spectrometry correlates poorly with multiple banding on isoelectric focusing.

Purified preparations of TEM-2, P99, Bacillus cereus I and B. cereus II beta-lactamases were examined by electrospray (ES) mass spectrometry. The ES mass spectra of the B. cereus enzymes revealed the presence of four to five components of different mass, corresponding to the loss of different numbers of N-terminal amino acids (ragged ends). The ES mass spectra of both TEM-2 and P99 consisted of a single component with no evidence of ragged ends. All four beta-lactamase preparations were visualized on isoelectric focusing (IEF) gels stained with nitrocefin to investigate a possible correlation between IEF patterns and ragged ends. Multiple banding patterns were seen with each beta-lactamase preparation. Although these may correlate with the presence of ragged ends in the two B. cereus preparations, the satellite bands seen with P99 and TEM-2 were not associated with differences detected by ES mass spectrometry. In this study we have shown for the first time that beta-lactamase satellite bands seen on IEF are not always associated with ragged ends. Furthermore, we have illustrated the use of ES mass spectrometry to characterize the extent of ragged end formation in protein samples. This is of particular significance if the sample is required for detailed biochemical or crystallography experiments.