Paula M. D. Fitzgerald

Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamase

BACKGROUND High level resistance to carbapenem antibiotics in gram negative bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-beta-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-beta-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. RESULTS Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-beta-lactamase identified through screening and predicted using molecular modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water molecule. Inhibition of metallo-beta-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis. CONCLUSIONS BPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-beta-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.

[30] Macromolecular crystallographic information file

Publisher Summary A variety of approaches for improved scientific data representation is being explored. The approach described in this chapter, which has been developed under the auspices of the International Union of Crystallography (IUCr), is to extend the Crystallographic Information File (CIF) data representation used for describing small-molecule structures and associated diffraction experiments. This extension is referred to as the “macromolecular Crystallographic Information File (mmCIF)” and is discussed in the chapter. The chapter covers the history of mmCIF, similarities to and differences from the Protein Data Bank (PDB) format, contents of the mmCIF dictionary, and the way to represent structures using mmCIF. The mmCIF home page contains a historic description of the development of the dictionary, current versions of the dictionary in text and HyperText markup language (HTML) formats, software tools, archives of the mmCIF discussion list, and a detailed online tutorial. It is anticipated that full use of the expressive power of mmCIF will be made only when existing structure solution, refinement programs are modified to maintain mmCIF data items and software tools are developed to help prepare and use an mmCIF effectively.

Discovery of MK-7655, a β-lactamase inhibitor for combination with Primaxin®

β-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C β-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C β-lactamases in vitro. It effectively restored imipenems activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.

Side Chain Sar of Bicyclic Beta-Lactamase Inhibitors (Blis). 1. Discovery of a Class C Bli for Combination with Imipinem.

Bridged monobactam beta-lactamase inhibitors were prepared and evaluated as potential partners for combination with imipenem to overcome class C beta-lactamase mediated resistance. The (S)-azepine analog 2 was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.

HIV protease-ligand complexes: Current Opinion in Structural Biology 1993, 3: 868–874

Abstract HIV-1 protease remains a promising target for the treatment of AIDS, and research continues in many laboratories to exploit a knowledge of the structure of the protease and its interactions with inhibitors in the development of an AIDS therapy. Structures of HIV-1 protease in complex with peptide-based inhibitors continue to be reported, and recently structures have appeared for a number of compounds that are not fundamentally peptidyl in nature, compounds that were identified as inhibitors either by screening methods or by structure-based database searches. Three structures of the closely related HIV-2 protease in complex with peptide-based inhibitors have now also been determined.

Side chain SAR of bicyclic β-lactamase inhibitors (BLIs). 2. N-Alkylated and open chain analogs of MK-8712

The bridged monobactam β-lactamase inhibitor MK-8712 (1) effectively inhibits class C β-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C β-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.

Macromolecular dictionary (mmCIF)

This is version 2.0.09 of the macromolecular CIF dictionary (mmCIF). The history and philosophy of this dictionary are described in Chapter 1.1 and a commentary on its use may be found in Chapter 3.6. The data names defined here include a modified representation of those in the core CIF dictionary (Chapter 4.1).

Binding of a Reduced-Peptide Inhibitor and a Statine-Containing Inhibitor to the Protease from the Human Immunodeficiency Virus

In the replication cycle of human immunodeficiency virus, viral mRNA is translated as a polyprotein precursor. Cleavage by a viral-encoded protease is required for release of mature viral structural proteins and enzymes from the precursor; the key role of the protease in the maturation of this virus, the causative agent in acquired immune deficiency syndrome (AIDS), makes the it an attractive target for therapeutic intervention in the treatment of AIDS. To aid in the design of safe and effective inhibitors, we have studied the structure of the protease from human immunodeficiency virus type 1 (HIV-1), both in its native state1 and in complex with a statine-containing inhibitor, acetyl-pepstatin2. We report here the determination of the structure of the protease in complex with a reduced-peptide inhibitor, L365,862 (Ser-Gln-Asn-PheΨ(CH2-N)Pro-Ile-Val-Gln). The amino acid sequence of this peptide corresponds to the cleavage site between the membrane-associated and capsid proteins in the polyprotein (with the substitution of Phe for Tyr); the replacement of the scissile bond in this octapeptide with a reduced-peptide linkage generates an inhibitor that has been used as an affinity ligand in the purification of the protease3. Although the structure of this complex is still being refined, a preliminary comparison to the structure of the complex between HIV-1 protease and acetyl-pepstatin reveals considerable difference in the length of hydrogen bonds between the protein and the inhibitor. The conformation of the backbone of most of the inhibitor is extended, but the backbone has a turn conformation at the position of the amino-terminal serine residue; turn-forming at this position may facilitate binding between the protease and its natural substrates.

Expression, purification, crystallization and preliminary X-ray analysis of Aeromonas hydrophilia metallo-β-lactamase

The CphA metallo-beta-lactamase from Aeromonas hydrophilia has been expressed, purified and crystallized by the hanging-drop vapor-diffusion method using ammonium sulfate as the precipitant. The crystals exhibit orthorhombic symmetry (P2(1)2(1)2), with unit-cell parameters a = 40.75, b = 42.05, c = 128.88 A. There is one monomer in the asymmetric unit and the solvent content is estimated to be 44% by volume. A data set extending to 1.8 A has been measured.

Classification and use of core data

Each data item in a CIF has a unique identity and properties, and these are described in the DDL dictionary definitions. The name or tag of a data item is its primary identifier and it serves as the unique access key to a data value. Although each data item is unique, it is often closely related to other data items. These relationships, which are specified in the dictionaries using particular DDL attributes, can require certain items to be grouped into common lists, and others, because of specific relational dependencies, to be present in a CIF when another data item is used. Such relationships lead to data being classified into groups, or categories of data. The data name is chosen to be as self-descriptive of the data item as possible and contains the category name at the front. For example, data describing atom sites in a structural model have names that start with the string _atom_site and are placed into the category group of the same name. This chapter describes the rationale for the classification of crystallographic data into the existing categories used in the core dictionary.