In an effort to devise strategies for overcoming bacterial ␤-lactamases, we studied LN-1-255, a 6-alkylidene-2-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among ␤-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing bla SHV extended-spectrum and inhibitor-resistant ␤-lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 ␤-lactamases with nM affinity (K I ‫؍‬ 110 ؎ 10 and 100 ؎ 10 nM, respectively). The rapidly increasing number of antibiotic-resistant Gramnegative microorganisms, including the Enterobacteriaceae family and the Pseudomonas, Acinetobacter, and Klebsiella genera, represents a grave threat to human health. The first-line treatments for such infections are ␤-lactam antibiotics, and the most common mechanism of resistance to such agents is bacterial production of ␤-lactamases. Regrettably, Enterobacteriaceae resistant to penicillins and extended-spectrum cephalosporins (e.g. ceftazidime, ceftriaxone and cefepime) are continuing to threaten the efficacy of our available ␤-lactam antibiotics (1-4). Currently, up to 30% of the Enterobacter spp., Escherichia coli, and Klebsiella spp. recovered from patients with infections in hospitals, long-term care facilities, and intensive care units in the United States are resistant to ceftazidime (1, 2, 5-9). Of greatest concern is the emerging number of community-acquired E. coli and Klebsiella spp. that are resistant to these cephalosporins (3, 4, 8 -11). This latter group presents a very significant future danger to the current use of ␤-lactam antibiotics to treat common infections in the ambulatory setting (11).Resistance to extended-spectrum cephalosporins in E. coli and Klebsiella pneumoniae is typically caused by a plasmid or chromosomal class A bla gene that encodes for extended-spectrum ␤-lactamases (ESBLs) 4 (1, 5, 12, 13). Among the ␤-lactam class of antibiotics, only carbapenems are recommended for the therapy of infections caused by ESBL-producing bacteria (1, 2, 7). Many ESBL-producing bacteria possess ␤-lactamases that are susceptible to ␤-lactamase inhibitors, but the clinical efficacy of ␤-lactam/␤-lactamase inhibitor combinations still remains to be established conclusively (5, 12). ␤-Lactam/␤-lactamase inhibitor combinations offer an extremely attractive approach to combating infections caused by ESBL-producing bacteria. Unfortunately, current commercial ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) narrowly target class A enzymes. Thus, there is an urgent need for the * This work was supported, in whole or in part, by National Institutes of Health Grants R01 AI062968 (to F. V. D. A.), RO1 AI063517-01 (to R. A. B.), and the R...

A set of designed internally quenched £uorescence peptide substrates has been used to probe the e¡ects of insertion of L L-peptide bonds into peptide sequences. The test sequence chosen corresponds to a proteolytically susceptible site in hemoglobin K K-chain, residues 32^37. Fluorescence and mass spectral measurements demonstrate that the insertion of an L L-residues at the potential cleavage sites completely abolishes the action of proteases; in addition, the rate of cleavage of the peptide bond preceding the site of modi¢cation is also considerably reduced.

No abstract

Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blueshifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-Å crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid.