Inhibitors of nicotinamide:N -methyltransferase designed to mimic the methylation reaction transition state

Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyses the methylation of nicotinamide to form N'-methylnicotinamide. Both NNMT and its methylated product have recently been linked to a variety of diseases, suggesting a role for the enzyme as a therapeutic target beyond its previously ascribed metabolic function in detoxification. We here describe the systematic development of NNMT inhibitors derived from the structures of the substrates involved in the methylation reaction. By covalently linking fragments of the NNMT substrates a diverse library of bisubstrate-like compounds was prepared. The ability of these compounds to inhibit NNMT was evaluated providing valuable insights into the structural tolerances of the enzyme active site. These studies led to the identification of new NNMT inhibitors that mimic the transition state of the methylation reaction and inhibit the enzyme with activity on par with established methyltransferase inhibitors

Clarithromycin is an effective immunomodulator when administered late in experimental pyelonephritis by multidrug-resistant Pseudomonas aeruginosa

BACKGROUND: To apply clarithromycin as an immunomodulatory treatment in experimental urosepsis by multidrug-resistant Pseudomonas aeruginosa. METHODS: Acute pyelonephritis was induced in 40 rabbits after inoculation of the test isolate in the renal pelvis. Therapy was administered upon signs of sepsis in four groups: A, controls; B, intravenous clarithromycin; C, amikacin; and D, both agents. Survival and vital signs were recorded; blood was sampled for culture and estimation of pro-inflammatory mediators; monocytes were isolated for determination of apoptotic rate and ex vivo TNFα secretion. Quantitative cultures and biopsies of organs were performed after death. RESULTS: Increased rectal temperature and oxygen saturation were found in groups B and D compared to A and C. Mean survival of groups A, B, C and D was 2.65, 7.15, 4.25 and 8.70 days respectively. No differences were noted between groups concerning bacterial load in blood and tissues and serum endotoxins. Serum MDA and total caspase-3 activity of monocytes of group D decreased following treatment compared to other groups. Negative correlation was detected between cytoplasmic caspase-3 and ex vivo secretion of TNFα of blood monocytes of group A; similar correlation was not found for any other group. Pathology scores of liver and lung of group B were lower than group A. CONCLUSION: Clarithromycin administered late in experimental urosepsis by multidrug-resistant P. aeruginosa prolonged survival and ameliorated clinical findings. Its effect is probably attributed to immunomodulatory intervention on blood monocytes

Simultaneous determination of the tobacco smoke uptake parameters nicotine, cotinine and thiocyanate in urine, saliva and hair, using gas chromatography-mass spectrometry for characterisation of smoking status of recently exposed subjects

A method using gas chromatography (GC)-mass spectrometry ( MS) for the simultaneous determination of the smoke uptake parameters thiocyanate, nicotine and cotinine in human tissues is reported. Nicotine, cotinine and thiocyanate, in combination with a phase-transfer catalyst, were extracted from urine, saliva and hair into dichloromethane (DCM). Thiocyanate was alkylated in the DCM-layer to form a pentafluorobenzyl derivative. The biochemical markers in DCM were directly injected into the GC system and separated on a DB-1MS column using a 9.4 min temperature program. The method was validated in urine and saliva between the limits of quantitation (1.0-15 mug ml(-1) thiocyanate, 0.010 - 3.0 mug ml(-1) nicotine and cotinine in urine, 0.010 - 1.0 mug ml(-1) nicotine and cotinine in saliva). The calibration curves were found to be linear (r > 0.996), the within- and between-day accuracy's were 83 - 120%, the repeatability coefficients of variation were 3 - 20% and the limits of detection were 0.060 ng ml(-1) thiocyanate and 0.60 ng ml(-1) nicotine and cotinine. The results of the analysis of the biomarkers in the urine of 44 volunteers were used to develop a predictive model for smoking status, using discriminant analysis. The classification model correctly classified 93.2% of cross-validated grouped cases. Saliva samples were used to confirm the results of the classification metho

A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase

Nicotinamide <i>N</i>-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor <i>S</i>-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine nitrogen of nicotinamide to generate <i>N</i>-methylnicotinamide. Interestingly, NNMT is also able to N-methylate a variety of other pyridine-containing small molecules, suggesting a secondary role for the enzyme in the detoxification of xenobiotics. A number of recent studies have also revealed links between NNMT overexpression and a variety of diseases, including multiple cancers, Parkinson’s disease, diabetes, and obesity. To facilitate further study of both the substrate scope and potential for inhibitor development, we here describe the development of a new NNMT activity assay. The assay makes use of ultra-high-performance hydrophilic interaction chromatography, allowing for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection, providing for enhanced sensitivity and enabling high-throughput sample analysis. We successfully demonstrated the general applicability of the method by performing kinetic analyses of NNMT-mediated methylation for a range of pyridine-based substrates. These findings also provide new insight into the diversity of substrate recognition by NNMT in a quantitative manner. In addition, we further established the suitability of the assay for the identification and characterization of small molecule inhibitors of NNMT. To do so, we investigated the inhibition of NNMT by the nonspecific methyltransferase inhibitors sinefungin and <i>S</i>-adenosyl-l-homocysteine, revealing IC₅₀ values in the low micromolar range. The results of these inhibition studies are particularly noteworthy as they will permit future efforts toward the development of new NNMT-specific inhibitors

Optimization of in-line fritless solid-phase extraction for capillary electrophoresis-mass spectrometry

In this study, in-line frit-free solid-phase extraction (SPE) has been studied for the preconcentration of analytes prior to analysis by capillary electrophoresis-mass spectrometry (CE-MS). The mixed-mode sorbent Oasis HLB was selected for the trapping of compounds of different polarity. Using 2-ethylidene-1,5-dimethyl-3,3-diphenylpirrolidine (EDDP), dihydrocodeine and codeine as test compounds, SPE parameters such as the pH of the sample and composition of the washing and elution solvent were optimized. Trapping of the analytes was optimal at pH 8.0 or higher. For efficient elution of the SPE micro column, 85% of methanol in water with 2% (v/v) acetic acid was used, which also prevented current break down in subsequent CE analysis. CE resolution of the test compounds was highest for background electrolytes (BGEs) with a pH above 8. For optimal analysis, samples were 1:1 diluted with carbonate buffer (1. M, pH 8.0) prior to analysis, BGE was 60. mM ammonium acetate buffer (pH 10.0), and the injected sample volume was 60 μl (i.e., 30 capillary volumes). Good recoveries were found: 101% for EDDP, 88% for codeine and 90% for dihydrocodeine. Intraday RSDs for migration time and peak areas were below 0.56% and 15%, respectively. Peak widths at half height obtained with SPE-CE-MS were 12. s for EDDP, 3.7. s for dihydrocodeine and 7.4. s for codeine, and were comparable to those for CE-MS. LODs were 0.22. pg/ml for EDDP, 2.1. pg/ml for dihydrocodeine and 24. pg/ml for codeine. It is concluded that the applied fritless in-line preconcentration construct proved to be highly useful for improving the sensitivity of CE while maintaining separation

Bioanalysis of erlotinib, its O-demethylated metabolites OSI-413 and OSI-420, and other metabolites by liquid chromatography-tandem mass spectrometry with additional ion mobility identification

Erlotinib is a first-generation epithelial growth factor receptor inhibitor used in the treatment of non-small cellular lung cancers. Our previously published method on a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer for the quantitation of erlotinib, OSI-420, and OSI-413 and some other kinase inhibitors was transferred to a more sensitive Sciex QTRAP5500 system. Both methods showed comparable performance in the previous range (5–5000 and 1–1000 ng/mL for erlotinib and OSI-420) with comparable accuracies and precisions (98.9–106.2 vs 98.7.0–104.0, and 3.7–13.4 vs 4.6–13.2), and a high level of agreement between the methods (R2 = 0.9984 and 0.9951) for the quality control samples. The new system however was also capable of quantifying lower concentrations of both erlotinib and OSI-420 (0.5 and 0.1 ng/mL) with sufficient accuracy and precision. Along with the increased sensitivity we included the semi-quantitative determination of additional erlotinib metabolites M2, M3, M5, M6, M7, M8, M9, M10, M11, M12, M16 (hydroxy-erlotinib), M17, M18, M19, M20, M21 in a 0.1–1000 ng/mL range to the method. With a simple crash, dilute, and shoot sample preparation with acetonitrile and a 4.5 min analytical run time the method outperformed most other published methods in speed and simplicity and was suitable for TDM. Further, enhancement of the understanding of the pharmacokinetics of erlotinib and its metabolites was demonstrated

Orthoester Functionalized N-Guanidino Derivatives of 1,5-dideoxy-1,5-imino-D-xylitol as pH-Responsive Inhibitors of β-Glucocerebrosidase

FWN – Publicaties zonder aanstelling Universiteit Leide

Mass spectrometry for glycan biomarker discovery

The association between aberrant glycosylation of proteins and many cancers, and autoimmune and inflammatory diseases has been known for many years. Altered glycosylation can occur at the onset and during disease progression and identifying these changes at an early stage may greatly increase survival and improve quality of life. However, the identification of these biomarkers has not been easy, mainly due to the structural diversity and numerous possible glycan isomers. Fortunately, glycomics is becoming more feasible due to major improvements in mass spectrometry and separation science. The present review discusses recent methods for mass-spectrometry (MS) based glycomics for the identification of glycan biomarkers. Recent MS techniques with and without coupling to liquid chromatography, capillary electrophoresis or ion mobility spectrometry are described, and the most recent glycan biomarker studies are presented and future prospects discussed