A sensitive coupled HPLC/electrospray mass spectrometry assay for SPM-1 metallo-beta-lactamase inhibitors.

Antibiotic-resistant bacteria continue to threaten human health through multiple mechanisms, including hydrolytic inactivation of beta-lactam antibiotics by metallo-beta-lactamases (MBLs). The SPM-1 enzyme, originally identified from a Pseudomonas aeruginosa clinical isolate, is a Class B beta-lactamase responsible for resistance in bacteria against antibiotics such as penicillins, cephalosporins, and carbapenems. Unlike Class A, C, and D beta-lactamases, which employ a serine residue in their active site, Class B enzymes possess one or two Zn atoms in the active site that play both a structural and catalytic role. A beta-lactamase inhibitor with co-administration of a beta-lactam antibiotic has proven to be an effective treatment against antibiotic-resistant bacteria whose resistance is due to serine-based beta-lactamases (e.g., amoxicillin/clavulanic acid). A similar clinical approach has not yet been developed for resistant bacteria possessing MBLs. The identification and development of specific and effective MBL inhibitors to combat this resistance could extend the utility of currently prescribed antibiotics such as cephalosporins and carbapenems. To discover MBL inhibitors, compound libraries are screened typically by enzymatic hydrolysis of a chromogenic substrate such as nitrocefin monitored by absorbance. Spectrophotometric assays, while valuable, lack the sensitivity and selectivity to screen natural product extract libraries because of the strongly absorbing nature of some extracts and the dilute concentrations of active components. An assay is described herein that monitors the SPM-1-catalyzed hydrolysis of penicillin G by high-performance (high-pressure) liquid chromatography-electrospray mass spectroscopy, which permits investigations with greater sensitivity and selectivity allowing the screening of natural product extracts for inhibitors of MBLs.

Oleic acid and peanut oil high in oleic acid reverse the inhibitory effect of insulin production of the inflammatory cytokine TNF-alpha both in vitro and in vivo systems.

BACKGROUND: Chronic inflammation is a key player in pathogenesis. The inflammatory cytokine, tumor necrosis factor-alpha 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-alpha 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, KKAy. METHODS: The rat pancreatic beta cell line INS-1 was used as a cell biological model since it exhibits glucose dependent insulin secretion. Insulin production assessment was carried out using enzyme linked immunosorbent assay and cAMP quantification with competitive ELISA. Viability of TNF-alpha and oleic acid treated cells was evaluated using flow cytometry. PPAR-gamma translocation was assessed using a PPRE based ELISA system. In vivo studies were carried out on adult male KKAy mice and glucose levels were measured with a glucometer. RESULTS: Oleic acid and peanut oil high in oleic acid were able to enhance insulin production in INS-1. TNF-alpha inhibited insulin production but pre-treatment with oleic acid reversed this inhibitory effect. The viability status of INS-1 cells treated with TNF-alpha and oleic acid was not affected. Translocation of the peroxisome proliferator- activated receptor transcription factor to the nucleus was elevated in oleic acid treated cells. Finally, type II diabetic mice that were administered a high oleic acid diet derived from peanut oil, had decreased glucose levels compared to animals administered a high fat diet with no oleic acid. CONCLUSION: Oleic acid was found to be effective in reversing the inhibitory effect in insulin production of the inflammatory cytokine TNF-alpha. This finding is consistent with the reported therapeutic characteristics of other monounsaturated and polyunsaturated fatty acids. Furthermore, a diet high in oleic acid, which can be easily achieved through consumption of peanuts and olive oil, can have a beneficial effect in type II diabetes and ultimately reverse the negative effects of inflammatory cytokines observed in obesity and non insulin dependent diabetes mellitus.

Age dependence of glucose tolerance in adult KK-Ay mice, a model of non-insulin dependent diabetes mellitus.

Yellow KK mice carrying the 'yellow obese' gene Ay are a well established polygenic model for human non-insulin dependent diabetes mellitus. These animals develop marked adiposity and decreased glucose tolerance relative to their control littermates, KK mice. The authors monitored glucose tolerance in KK-Ay mice over time and observed a significant (P

Novel IMP-1 metallo-beta-lactamase inhibitors can reverse meropenem resistance in Escherichia coli expressing IMP-1.

IMP-1 metallo-beta-lactamase is a zinc metalloenzyme that confers antibiotic resistance to bacteria through the hydrolysis of beta-lactam antibiotics. Pathogens that express the enzyme show reduced susceptibility to carbapenems, such as meropenem and imipenem. In order to identify novel IMP-1 inhibitors, the National Cancer Institute (NCI) chemical diversity set was screened using 96-well high throughput screening format. The collection yielded several novel succinic acid derivatives that exhibited mixed inhibition of IMP-1 with compound 20707 having the highest affinity with a Ki value of 3.3 microM+/-1.7. The compounds are moderately potent inhibitors of IMP-1 with IC50 values ranging from 5.0 to 17 microM. An original chemical class of IMP-1 inhibitor, 2-((E)-(1,3-dihydroxy-2-methylpropan-2-ylimino)methyl)-4,6-diiodophenol, was discovered and was the most potent with an IC50 of 1.2 microM. NCI compounds, 20707, 140905 and 9746 sensitized a carbapenem-resistant laboratory strain of Escherichia coli to clinically achievable levels of meropenem.

Expression, purification, crystallization and preliminary X-ray analysis of Aeromonas hydrophilia metallo-beta-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.

Sabadinine: a potential non-peptide anti-severe acute-respiratory-syndrome agent identified using structure-aided design.

A novel human coronavirus has been reported to be the causative agent of severe acute respiratory syndrome (SARS). Since replication of HcoVs depends on extensive proteolytic processing, the main proteinase, 3CLpro, is an attractive drug target for anti-SARS agents. We have employed molecular docking of a chemical database into the active site of 3CLpro to search for non-peptidyl inhibitors. One compound was identified to be the natural product sabadinine, isolated from a historical herbal remedy.

Iseganan (IntraBiotics pharmaceuticals).

Iseganan (IB-367) is a protegrin under development by IntraBiotics, as part of a larger protegrin program, for the potential treatment of oral mucositis, a frequent side effect of anticancer therapies. The company is developing three formulations of the drug: A rinse for the potential treatment of mucositis, an aerosolized liquid for the potential treatment of respiratory infection and a gel formulation for the potential treatment of pneumonia [376325]. Iseganan kills a broad-spectrum of bacteria and fungi, including those resistant to conventional antimicrobial drugs, by attaching to and destroying the integrity of the lipid cell membrane [241594]. Until August 1999, Pharmacia & Upjohn was a codeveloper of iseganan. IntraBiotics re-acquired the global rights to iseganan in December 1999, and both companies agreed to terminate the collaboration [335766]. In May 2000, analysts at SG Cowen predicted the drug's potential market at US $100 to US $200 million [376325].

Metallo-beta-lactamase inhibitors: promise for the future?

Carbapenem resistance continues to erode the effectiveness of antibiotics such as imipenem and meropenem in the clinic. Resistance mechanisms can include interplay between porin loss (membrane permeability), mutation of penicillin binding proteins necessary for cell division, and expression of class A, B and D beta-lactamases. Bacterial resistance to beta-lactams such as penicillin or amoxicillin has been overcome in the clinic using several strategies, including development of antibiotics not susceptible to hydrolysis by beta-lactamases, or co-administration of the antibiotic with beta-lactamase inhibitors. This overview will focus on progress since 2000 in identifying inhibitors of class B, or metallo-beta-lactamases with the aim of reversing carbapenem resistance.

A "mix and read" assay for insulin using fluorometric microvolume assay technology.

We describe a novel, "mix and read" immunoassay for insulin in biological samples using FMAT. Current commercial assays for insulin require multiple washing steps and can be expensive. The insulin assay described is a simple two-step, time-saving assay and amenable to robotics. The linear response for the fluorometric signal is comparable to that observed using classical ELISA and RIA. A series of mouse plasma samples were tested for insulin levels and yielded results comparable to that measured using a commercial ELISA for insulin.