Targeting metallo-carbapenemases via modulation of electronic properties of cephalosporins.

The global proliferation of metallo-carbapenemase-producing Enterobacteriaceae has created an unmet need for inhibitors of these enzymes. The rational design of metallo-carbapenemase inhibitors requires detailed knowledge of their catalytic mechanisms. Nine cephalosporins, structurally identical except for the systematic substitution of electron-donating and withdrawing groups in the para position of the styrylbenzene ring, were synthesized and utilized to probe the catalytic mechanism of New Delhi metallo-ß-lactamase (NDM-1). Under steady-state conditions, K(m) values were all in the micromolar range (1.5-8.1 µM), whereas k(cat) values varied widely (17-220 s(-1)). There were large solvent deuterium isotope effects for all substrates under saturating conditions, suggesting a proton transfer is involved in the rate-limiting step. Pre-steady-state UV-visible scans demonstrated the formation of short-lived intermediates for all compounds. Hammett plots yielded reaction constants (ρ) of -0.34 ± 0.02 and -1.15 ± 0.08 for intermediate formation and breakdown, respectively. Temperature-dependence experiments yielded ΔG(‡) values that were consistent with the Hammett results. These results establish the commonality of the formation of an azanide intermediate in the NDM-1-catalysed hydrolysis of a range cephalosporins with differing electronic properties. This intermediate is a promising target for judiciously designed ß-lactam antibiotics that are poor NDM-1 substrates and inhibitors with enhanced active-site residence times.

Engineering ß-lactamase zymogens for use in protease activity assays.

In this study, the concept of autoinhibition, a mechanism of protein activity regulation, was applied to the design and engineering of a ß-lactamase zymogen. Using this zymogen, a sensitive protease assay method was developed in which activation of the zymogen by proteases produces an amplified absorbance signal. The approach reported here can be adapted for engineering of zymogens as biological sensors and components of synthetic signaling pathways.

Real-time PCR assay and a synthetic positive control for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-ß-lactamase-1 (bla(NDM-1)).

Carbapenems are important last-line antibiotics for the treatment of hospital infections. Enterobacteriaceae (such as Klebsiella pneumoniae or Escherichia coli) expressing the "New Delhi Metallo-ß-lactamase" gene bla(NDM-1) are resistant to carbapenems and were predicted to become a major global health problem. To cope with this emerging threat, there is a need for rapid and sensitive molecular assays to detect bla(NDM-1) in carbapenem-resistant Enterobacteriaceae from clinical isolates. In diagnostic laboratories, real-time PCR is the current gold standard for the sensitive and rapid detection of pathogens. We describe a real-time PCR assay as well as two conventional PCR assays to detect bla(NDM-1). Only minute amounts of total DNA extracted from one bacterial colony are sufficient to allow detection of bla(NDM-1) by real-time PCR within less than 1 h. We also introduce a chemically synthesized bla(NDM-1) gene as a convenient positive control for those laboratories wishing to setup in-house assays for bla(NDM-1) detection. Importantly, our study represents a proof of principle for the usefulness of rapidly synthesized genes serving as positive controls for novel diagnostic PCR assays of emerging pathogens during the initial phase after their discovery when biological isolates are still rare and not commonly available.

Hydrolysis of organophosphate esters: phosphotriesterase activity of metallo-beta-lactamase and its functional mimics.

The phosphotriesterase (PTE) activity of a series of binuclear and mononuclear zinc(II) complexes and metallo-beta-lactamase (mbetal) from Bacillus cereus was studied. The binuclear complex 1, which exhibits good mbetal activity, shows poor PTE activity. In contrast, complex 2, a poor mimic of mbetal, exhibits much higher activity than 1. The replacement of Cl(-) ligands by OH(-) is important for the high PTE activity of complex 2 because this complex does not show any catalytic activity in methanol. The natural enzyme mbetal from B. cereus is also found to be an inefficient catalyst in the hydrolysis of phosphotriesters. These observations indicate that the binding of beta-lactam substrates at the binuclear zinc(II) center is different from that of phosphotriesters. Furthermore, phosphodiesters, the products from the hydrolysis of triesters, significantly inhibit the PTE activity of mbetal and its functional mimics. Although the mononuclear complexes 3 and 4 exhibited significant mbetal activity, these complexes are found to be almost inactive in the hydrolysis of phosphotriesters. These observations indicate that the elimination of phosphodiesters from the reaction site is important for the PTE activity of zinc(II) complexes.

Correlation between catalytic efficiency and the transcription read-out in chemical complementation: a general assay for enzyme catalysis.

High-throughput assays for enzyme catalysis that can be applied to a broad range of chemical reactions are key to advances in directed evolution and proteomics. Recently, we reported such a general assay, chemical complementation, which links enzyme catalysis to reporter gene transcription in vivo using the yeast three-hybrid assay. In this proof-of-principle experiment, it was shown that a wild-type beta-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen. Ideally, however, such an assay should be able to distinguish enzymes based on their catalytic activity. Thus, here, we set out to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level of transcription activation in the chemical complementation assay. First, the reaction mechanism for the cleavage of the beta-lactam substrate used in the chemical complementation proof-of-principle experiment was determined. Then a series of beta-lactamase variants was designed to span several orders of magnitude in k(cat)/K(m). The activity of each variant was determined both in vitro using purified enzyme and in vivo in the chemical complementation transcription assay. Beta-lactamase variants spanning three-orders of magnitude in k(cat)/K(m) could be distinguished in the assay, and the catalytic efficiency of each variant correlated with its level of transcription activation in vivo. These results establish the suitability of chemical complementation for the directed evolution of enzymes with improvements in catalytic activity and for profiling the relative substrate specificities of a group of enzymes in proteomics applications.

Convergent synthesis of the E'FGH' ring fragment of ciguatoxin 1B via an acetylene cobalt complex strategy.

[reaction: see text] A convergent synthesis of the E'FGH' ring fragment of ciguatoxin has been accomplished through (i) coupling between the E' ring-acetylide and the H' ring-aldehyde, (ii) stereoselective F ring cyclization via an acetylene cobalt complex, (iii) conversion to a carbonyl function, and (iv) reductive hydroxy-ketone cyclization to construct the G ring.

Protein minimization by random fragmentation and selection.

Protein-protein interactions are involved in most biological processes and are important targets for drug design. Over the past decade, there has been increased interest in the design of small molecules that mimic functional epitopes of protein inhibitors. BLIP is a 165 amino acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.1 nM). To aid in the development of new inhibitors of beta-lactamase, the gene encoding BLIP was randomly fragmented and DNA segments encoding peptides that retain the ability to bind TEM-1 beta-lactamase were isolated using phage display. The selected peptides revealed a common, overlapping region that includes BLIP residues C30-D49. Synthesis and binding analysis of the C30-D49 peptide indicate that this peptide inhibits TEM-1 beta-lactamase. Therefore, a peptide derivative of BLIP that has been reduced in size by 88% compared with wild-type BLIP retains the ability to bind and inhibit beta-lactamase.

A model for ZnII-containing-beta-lactamase: synthesis, X-ray crystal structure of a zinc(II) complex bearing thiol group and hydrolysis of phosphate diester.

A novel N3S1 typed tripodal ligand bearing an SH group, N-(mercaptoethyl)-di(2-pyridylmethyl)amine, DPASH, was prepared and its zinc(II) complex, [ZnII(DPAS)Cl], was structurally characterized by X-ray crystallography. [ZnII(DPAS)Cl] promoted hydrolysis of sodium bis(p-nitrophenyl)hydrogenphosphate), BNP-.

Synthesis and structure-activity relations in the class of 2-(pyridyl)penems.

The isosteric CH----N substitution in the class of 2-arylpenems results in improved antibacterial activity, with retention of the favorable characteristic of stability towards renal dehydropeptidase. High therapeutic efficacy was demonstrated in experimental mice septicemias with the 2-(3-pyridyl) derivative 2b and its orally absorbed acetoxymethyl ester prodrug 4n.