Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes

<div><p>Monocyte activation and polarization play essential roles in many chronic inflammatory diseases. An imbalance of M1 and M2 macrophage activation (pro-inflammatory and alternatively activated, respectively) is believed to be a key aspect in the etiology of these diseases, thus a therapeutic approach that regulates macrophage activation could be of broad clinical relevance. Methylglyoxal-bis-guanylhydrazone (MGBG), a regulator of polyamine metabolism, has recently been shown to be concentrated in monocytes and macrophages, and interfere with HIV integration into the DNA of these cells <i>in vitro</i>. RNA expression analysis of monocytes from HIV+ and control donors with or without MGBG treatment revealed the only gene to be consistently down regulated by MGBG to be osteopontin (OPN). The elevated expression of this pro-inflammatory cytokine and monocyte chemoattractant is associated with various chronic inflammatory diseases. We demonstrate that MGBG is a potent inhibitor of secreted OPN (sOPN) in cultured monocytes with 50% inhibition achieved at 0.1 μM of the drug. Furthermore, inhibition of OPN RNA transcription in monocyte cultures occurs at similar concentrations of the drug. During differentiation of monocytes into macrophages <i>in vitro</i>, monocytes express cell surface CD16 and the cells undergo limited DNA synthesis as measured by uptake of BrdU. MGBG inhibited both activities at similar doses to those regulating OPN expression. In addition, monocyte treatment with MGBG inhibited differentiation into both M1 and M2 classes of macrophages at non-toxic doses. The inhibition of differentiation and anti-OPN effects of MGBG were specific for monocytes in that differentiated macrophages were nearly resistant to MGBG activities. Thus MGBG may have potential therapeutic utility in reducing or normalizing OPN levels and regulating monocyte activation in diseases that involve chronic inflammation.</p></div

Kinetic Characterization of Hydrolysis of Nitrocefin, Cefoxitin, and Meropenem by β‑Lactamase from <i>Mycobacterium tuberculosis</i>

The constitutively expressed, chromosomally encoded β-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to β-lactam antibiotics in <i>Mycobacterium tuberculosis</i>. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity [Tremblay, L. W., et al. (2008) <i>Biochemistry 47</i>, 5312–5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal velocity depends on the ionization state of two groups: a general base exhibiting a p<i>K</i> value of 4.5 and a general acid exhibiting a p<i>K</i> value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose <i>k</i>_cat values differ by 5.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both <i>V</i> and <i>V</i>/<i>K</i>. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on <i>V</i> of 3.9 but a small SKIE of 1.8 on <i>V</i>/<i>K</i> in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of β-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation steps of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate-limiting steps for β-lactam hydrolysis of these substrates are analyzed, and the chemical steps responsible for the observed SKIE are discussed

MGBG inhibited monocyte CD16 expression and BrdU incorporation.

<p>Isolated human monocytes were cultured in suspension with or without MGBG treatment. CD16 expression was measured by flow cytometry. (A). Monocytes differentiate spontaneously in culture. CD16 expression level increases in monocyte culture. n = 4, means and SEM. (B). MGBG inhibited CD16 expression in 1 day cultured monocytes in a dose-dependent manner. The average CD16 geometric mean fluorescence of 1 day untreated monocytes was measured at 108 units. n = 4, means and SEM. (C). MGBG inhibited BrdU incorporation in cultured monocytes. Isolated human monocytes were cultured for 3–6 days for BrdU incorporation using a FITC BrdU flow kit. The average percentage of BrdU incorporated untreated monocytes was measured at 4.7%. MGBG inhibited monocyte BrdU uptake in a dose-dependent manner. n = 5, means and SEM.</p

SPM reversed the MGBG inhibitory effects on sOPN and CD16 expression in monocytes.

<p>PBMCs were cultured for 3 days with or without treatment. Cells were cultured with 10 μM MGBG and various concentrations of SPM. (A). MGBG significantly decreased sOPN; SPM reversed the MGBG inhibitory effects on sOPN. The average sOPN level of 3 day untreated and 10 μM MGBG treated cells was 30 and 2 ng/ml, respectively. n = 6, means and SEM. (B). MGBG inhibited monocyte CD16 expression; SPM reversed the MGBG inhibitory effects on CD16 expression. Cultured PBMCs were double stained with CD14 and CD16 antibodies for flow cytometry analysis. The average CD16 geometric mean in untreated and MGBG treated CD14+ monocytes were measured at 510 and 249, respectively. n = 3, means and SEM. Data was presented as a percentage of inhibition of MGMG treatment only.</p

MGBG potently inhibited sOPN production in monocytes, but showed limited inhibitory effect in differentiated macrophages.

<p>(A). MGBG significantly inhibited sOPN production in monocytes. The degree of MGBG inhibition on sOPN was related to time of exposure. Isolated human monocytes were cultured at 0.1 million cells per ml with various concentrations of MGBG for 16 hr, 1, 2 and 3 days. MGBG showed potent inhibition on monocyte sOPN production with IC50 < 0.1 μM on day 3. Data were presented as percentage of untreated controls. The average sOPN levels of untreated monocytes were approximately 110, 130, 670 and 5300 pg/ml at 16 hr, 1, 2, and 3 days, respectively. The inhibition is independent of cytotoxicity (e.g. little cell death at 16 hours and day 1). The data is normalized to number of live cells in the culture. n = 2. (B). MGBG showed limited inhibitory effect on sOPN production in mature macrophages. Isolated monocytes were cultured to allowed to differentiate into macrophages for 6 days prior to drug treatment. Fresh media was replaced and cells were treated with 10 μM MGBG for 3 days. n = 4, means and SEM.</p

Hierarchical illustration of the eight semantic classes (with some of the constituent SN types and CUIs omitted for conciseness)

<p><b>Copyright information:</b></p><p>Taken from "Using contextual and lexical features to restructure and validate the classification of biomedical concepts"</p><p>http://www.biomedcentral.com/1471-2105/8/264</p><p>BMC Bioinformatics 2007;8():264-264.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC2014782.</p><p></p

MGBG inhibited sOPN levels and gene expression in cultured human monocytes.

<p>1 day cultured monocytes were collected for total RNA extraction and iOPN expression, and culture supernatants for OPN ELISA; quantitative real-time PCR was performed for OPN gene expression. iOPN expression was measured by flow cytometry. MGBG decreased OPN gene expression and sOPN level in a dose-dependent manner. MGBG slightly decreased iOPN expression. The data were normalized against that of untreated controls. The average sOPN level for 1 day untreated monocytes was approximately 3 ng/ml. The iOPN geometric mean florescence of 1 day untreated cells varies among individuals ranging from 209–689. The OPN RNA level was calculated from β-actin normalized Ct difference of 1D cultured cells versus cells at isolation. The OPN Ct difference of 1 day untreated monocytes also varies among individuals ranging from 1.7 to 9.9. ED50 of MGBG on OPN cytokine level and gene expression on day 1: ~ 0.1 μM, n = 4, means and SEM.</p

MGBG showed greater inhibition on CD16 expression in monocytes than that in differentiated macrophages.

<p>Monocytes were isolated from PBMCs using CD14 microbeads. Cells were cultured for 1, 3, and 6 days with various concentrations of MGBG, and collected for CD16 expression measurement using flow cytometry. MGBG showed greater inhibitory effect on CD 16 expression in 1 and 3 day monocytes than that in differentiated macrophages. The average CD16 geometric mean fluorescence was measured at 108, 448, and 249 for 1, 3, and 6 day untreated cells, respectively. n = 4, means and SEM.</p

NXL104 Irreversibly Inhibits the β-Lactamase from <i>Mycobacterium tuberculosis</i>

NXL104 is a novel β-lactamase inhibitor with a non-lactam structural scaffold. Our kinetic and mass spectrometric analysis demonstrates that NXL104 quantitatively inhibits BlaC, the only chromosomally encoded β-lactamase from <i>Mycobacterium tuberculosis</i>, by forming a carbamyl adduct with the enzyme. The inhibition efficiency (<i>k</i>₂/<i>K</i>) of NXL104 was shown to be more than 100-fold lower than that of clavulanate, a classical β-lactamase inhibitor, which is probably caused by the bulky rings of NXL104. However, the decarbamylation rate constant (<i>k</i>₃) was determined to be close to zero. The BlaC–NXL104 adduct remained stable for at least 48 h, while the hydrolysis of the BlaC–clavulanate adduct was observed after 2 days. The three-dimensional crystal structure of the BlaC–-NXL104 carbamyl adduct was determined at a resolution of 2.3 Å. Interestingly, the sulfate group of NXL104 occupies the position of a phosphate ion in the structure of the BlaC–clavulanate adduct and is hydrogen bonded to residues Ser128, Thr237, and Thr239. Favorable interactions are also seen in the electrostatic potential map. We propose that these additional interactions, as well as the intrinsic stability of the carbamyl linkage, contribute to the extraordinary stability of the BlaC–NXL104 adduct

Tebipenem, a New Carbapenem Antibiotic, Is a Slow Substrate That Inhibits the β‑Lactamase from <i>Mycobacterium tuberculosis</i>

The genome of Mycobacterium tuberculosis contains a gene, <i>blaC</i>, which encodes a highly active β-lactamase (BlaC). We have previously shown that BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. We have shown that carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme–drug covalent complexes that are hydrolyzed extremely slowly. In the current study, we have determined apparent <i>K</i>_m and <i>k</i>_cat values of 0.8 μM and 0.03 min^–1, respectively, for tebipenem, a novel carbapenem whose prodrug form, the pivalyl ester, is orally available. Tebipenem exhibits slow tight-binding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin. FT-ICR mass spectrometry demonstrated that the tebipenem acyl–enzyme complex remains stable for greater than 90 min and exists as mixture of the covalently bound drug and the bound retro-aldol cleavage product. We have also determined the high-resolution crystal structures of the BlaC–tebipenem covalent acylated adduct (1.9 Å) with wild-type BlaC and the BlaC–tebipenem Michaelis–Menten complex (1.75 Å) with the K73A BlaC variant. These structures are compared to each other and to other carbapenem–BlaC structures