Metallo beta-lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of beta-lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo beta-lactamases. Reported here are the crystal structures of the native IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4 -(phenylb utyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 A (native) and 2.0 A (complex) resolution. Binding of the inhibitor in the active site induces a conformational change that results in closing of the flap and transforms the active site groove into a tunnel-shaped cavity enclosing 83% of the solvent accessible surface area of the inhibitor. The inhibitor binds in the active site through interactions with residues that are conserved among metallo beta-lactamases; the inhibitor's carboxylate group interacts with Lys161, and the main chain amide nitrogen of Asn167. In the "oxyanion hole", the amide carbonyl oxygen of the inhibitor interacts through a water molecule with the side chain of Asn167, the inhibitor's thiolate bridges the two Zn(II) ions in the active site displacing the bridging water, and the phenylbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 A compared to the unbound structure, allowing Trp28 to interact edge-to-face with the inhibitor's thiophene ring. The similarities between this inhibitor and the beta-lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo beta-lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the beta-lactam substrates. These interactions are complemented by additional nonspecific binding between the more variable groups in the substrates and the flexible flap. This unique mode of binding of the mercaptocarboxylate inhibitor in the enzyme active site provides a binding model for metallo beta-lactamase inhibition with utility for future drug design.

The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-Å resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S 2 subsite, and do not bind in the caspase primary aspartic acid binding pocket (S 1 ). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

No abstract

When collecting data using oscillation photography,