The present study focuses on the direct interaction of chemically modified tetracyclines (CMTs) with the active site of the matrix metalloproteinase 2 (MMP-2). Molecular docking, molecular dynamics (MD) simulations, and free energy calculations were accomplished for seven CMT derivatives. New sets of parameters are proposed for structural and catalytic zinc atoms in order to study MMPs and their complexes by means of the AMBER force field. Our computational results show that six CMTs studied bind to the catalytic zinc of the MMP-2 enzyme at the O11-O12 site as proposed experimentally. The exception was the CMT-3 analogue that is found embedding within the active site, enhancing the van der Waals and hydrophobic contacts with the hydrophobic S1' pocket in the MMP-2 enzyme. The binding energy calculated in solution predicts the CMT-3 complexes as the most favorable, followed by the CMT-7 and CMT-8 analogues, respectively, which is in line with experimental findings. This work is the first step toward understanding the mechanism of CMTs as MMP inhibitors at a molecular level.
The increasing number of new psychoactive substances (NPS) and their quick worldwide spreading, often only slightly modified in the form of new derivatives and analogues, have brought the need for fast, wide-ranging, and unequivocal identification methods in clinical and forensic investigations. Because it usually provides secure results, gas chromatography coupled to mass spectrometry (GC-MS) has been routinely employed as the standard technique for the detection of NPS in blotter papers. For 25I-NBOH (N-(2-hydroxybenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-aminium), however, GC-MS analysis of an blotter paper extract leads to incorrect results. In this work, we investigated whether easy ambient sonic-spray mass spectrometry imaging (EASI-IMS), and ambient ionization MS method can be applied directly to the surface of the sample requiring therefore no extraction or sample preparations, would serve as an efficient, sensitive, and secure alternative for 25I-NBOH screening.
Protein precipitation is the most commom sample preparation method applied in studies involving biological matrices, such as plasma and serum, in metabolomics. However, a poor protein precipitation step may reduce the lifetime of chromatographic columns, may cause interferences in Mass Spectrometry, and may result in limited access to sample metabolites. In this work different protein precipitation approaches were evaluated in order to assess the best protocol for untargeted metabolomics studies. For this purpose, solvents selection; sample volumes; sample/solvent ratio and temperature were evaluated by a factorial planning. Serum samples were analyzed by Bradford assay.
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