No abstract

Background: Chronic inflammation is a key player in pathogenesis. The inflammatory cytokine, tumor necrosis factoralpha is a well known inflammatory protein, and has been a therapeutic target for the treatment of diseases such as Rheumatoid Arthritis and Crohn's Disease. Obesity is a well known risk factor for developing non-insulin dependent diabetes melitus. Adipose tissue has been shown to produce tumor necrosis factor-alpha, which has the ability to reduce insulin secretion and induce insulin resistance. Based on these observations, we sought to investigate the impact of unsaturated fatty acids such as oleic acid in the presence of TNF-α in terms of insulin production, the molecular mechanisms involved and the in vivo effect of a diet high in oleic acid on a mouse model of type II diabetes, KKA y .

IMP-1 metallo-␤-lactamase (class B) is a plasmid-borne zinc metalloenzyme that efficiently hydrolyzes ␤-lactam antibiotics, including carbapenems, rendering them ineffective. Because IMP-1 has been found in several clinically important carbapenem-resistant pathogens, there is a need for inhibitors of this enzyme that could protect broad spectrum antibiotics such as imipenem from hydrolysis and thus extend their utility. We have identified a series of 2,3-(S,S)-disubstituted succinic acids that are potent inhibitors of IMP-1. Determination of high resolution crystal structures and molecular modeling of succinic acid inhibitor complexes with IMP-1 has allowed an understanding of the potency, stereochemistry, and structure-activity relationships of these inhibitors.Carbapenems such as imipenem (Scheme 1) have proven useful for the treatment of a variety of Gram-negative and Gram-positive infections (1, 2). Carbapenems and other ␤-lactam antibiotics covalently modify penicillin-binding proteins (PBPs) 1 involved in the peptidoglycan biosynthetic pathway of cell wall assembly in bacteria (3, 4). Resistance to carbapenems can arise because of acquisition of low affinity PBPs (3) (e.g. PBP2a of Staphylococcus aureus), altered membrane permeability (5), and expression of class A, B, and D ␤-lactamases (6 -10). Class B ␤-lactamases (metallo-␤-lactamases or MBLs) can hydrolyze a wide variety of substrates of the ␤-lactam class, including carbapenems, penicillins, and cephalosporins, rendering them ineffective as antibiotics.The IMP-1 gene encoding an MBL has been identified on a plasmid and in Japan has transferred among clinical isolates such as Pseudomonas aeruginosa (11,12), Klebsiella pneumoniae, Serratia marcescens, and other members of the Enterobacteriaceae (13,14). In addition, carbapenem-resistant clinical isolates expressing MBLs related to IMP-1 have been identified recently in Singapore (15), Italy (16), and Hong Kong (10). Such reports of plasmid-borne imipenem resistance highlight the need for inhibitors of IMP-1 that can restore the activity of carbapenems in resistant bacteria.Several classes of MBL inhibitors have been reported (for reviews, see Refs. 17 and 18)) including phenazines (19), trifluoromethyl alcohol and ketone derivatives of L-and D-alanine (20), thioesters (18, 21-23), thiols (24 -28), biphenyl tetrazoles (29, 30), and amino acid-derived hydroxamates (31). Biphenyl tetrazoles have been shown to reverse imipenem resistance in a clinical isolate of Bacteroides fragilis (29), and thioesters have been shown to reverse resistance to the carbapenem L-742,728 in a laboratory strain of Escherichia coli expressing IMP-1 (32). A 1␤-methylcarbapenem substituted at C-2 with a benzothienylthio moiety has been reported to be a potent IMP-1 inhibitor that can reverse resistance to imipenem in an IMP-1-producing strain of Serratia marcescens (33). Although the inhibitors described above have been reported to have good activity against a specific MBL, only certain thiols (e.g. SB 264218) exhibit broad spe...

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.

As part of a structure-aided effort to design clinically useful inhibitors of metallo-beta-lactamases, the X-ray crystal structure of a complex between the metallo-beta-lactamase from Bacteroides fragilis and 4-morpholinoethanesulfonic acid (MES) has been determined and a model for the structure has been refined to a crystallographic R-factor of 0.151 for data between 10.0- and 1.85-A resolution. Although the binding of MES was an adventitious result of the use of MES as a buffer in the crystallization mixture, MES was subsequently shown to be a competitive inhibitor of the enzyme, with a Ki of 23 +/- 5 mM. MES binds in the same fashion to both of the molecules in the crystallographic asymmetric unit; both direct and solvent-mediated hydrogen bonds to the protein and to the binuclear zinc cluster are observed, involving the oxygens of the sulfonic acid group and the nitrogen of the morpholino ring. In addition, there are hydrophobic interactions between the morpholino ring and residues in the flexible beta-strand of the enzyme between residues 26 and 36. Comparison of this structure with the previously reported unliganded structures of the same enzyme [Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. -M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] reveals that although the overall conservation of structure in the three different crystal lattices is very high, binding of MES is correlated with a significant change in the conformation of this beta-strand. The flexibility of this beta-strand will be an important consideration in the design of inhibitors of the metallo-beta-lactamases.