A set of four non-heme iron(II) and 2-oxoglutarate-dependent enzymes catalyze the post-translational modification of a transcription factor, hypoxia inducible factor (HIF), that mediates the hypoxic response in animals. Hydroxylation of HIF both causes its degradation and limits its activity. We describe how the use of structural data coupled to solid-phase synthesis led to the discovery of a selective inhibitor of one of the HIF hydroxylases. The inhibitor N-oxalyl-d-phenylalanine was shown to inhibit the HIF asparaginyl hydroxylase (FIH) but not a HIF prolyl hydroxylase. A crystal structure of the inhibitor complexed to FIH reveals that it binds in the 2OG and, likely, in the dioxygen binding site. The results will help to enable the modulation of the hypoxic response for the up-regulation of specific genes of biomedical importance, such as erythropoietin and vascular endothelial growth factor.

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The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.

Resistance to ␤-lactam antibiotics mediated by metallo-␤-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-␤-lactamase inhibitors. The K i values (Bacillus cereus enzyme) are 0.09 M for R-thiomandelic acid and 1.28 M for the Sisomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-␤-lactamases, with a submicromolar K i value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg 91 . Amide chemical shift perturbations for residues 30 -40 (the ␤ 3 -␤ 4 loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.The ␤-lactam antibiotics are among the most useful antibacterial chemotherapeutic agents, but their efficiency is being continuously challenged by the emergence of resistant strains of pathogenic bacteria. ␤-Lactamases, which inactivate these antibiotics by hydrolyzing their endocyclic amide bond, play a major role in this resistance (1). ␤-Lactamases have been divided into four classes on the basis of their amino acid sequences and catalytic mechanisms (2). The mechanisms of class A, C, and D enzymes, which contain a nucleophilic serine side chain as a key component of their active site, have been extensively studied, due to their established clinical importance. Class B enzymes are metalloproteins that require one or two zinc ion(s) for their activity (3). The first metallo-␤-lactamase (MBL) 1 to be discovered was produced by an innocuous strain of Bacillus cereus (4), but in the last 20 years, MBLmediated resistance has appeared in several pathogenic strains including Bacteroides fragilis, Aeromonas hydrophila, Stenotrophomonas maltophilia, and Serratia marcescens (5). Even more threatening is the rapid dissemination of some metallo-␤-lactamase genes by horizontal transfer, involving both plasmid-and integron-borne genetic elements. For instance, the IMP enzymes that were first isolated in clinical isolates of S. marcescens and Pseudomonas aeruginosa have also been found in Klebsiella, Alcalinogenes, Acinetobacter, and Shigella strains (6), and up to four variants of IMP-1 have been described so far (7-10).The MBLs with known sequences share a small number of conserved motifs, but otherwise they show significant sequence diversity and have th...

The crystal structure of the class-B ␤-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, D-captopril, has been solved at 1.5-Å resolution. The enzyme has the typical ␣␤/␤␣ metallo-␤-lactamase fold and the characteristic two metal binding sites of members of the subclass B1, in which two Zn 2؉ ions were identified. D-Captopril, a diastereoisomer of the commercial drug, captopril, acts as an inhibitor by displacing the catalytic hydroxyl ion required for antibiotic hydrolysis and intercalating its sulfhydryl group between the two Zn 2؉ ions. Interestingly, D-captopril is located on one side of the active site cleft. The x-ray structure of the complex of the closely related enzyme, IMP-1, with a mercaptocarboxylate inhibitor, which also contains a sulfhydryl group bound to the two Zn 2؉ ions, shows the ligand to be located on the opposite side of the active site cleft. A molecule generated by fusion of these two inhibitors would cover the entire cleft, suggesting an interesting approach to the design of highly specific inhibitors.