Exploring the Mechanisms behind the Anti-Tumoral Effects of Model C-Scorpionate Complexes.

The growing worldwide cancer incidence, coupled to the increasing occurrence of multidrug cancer resistance, requires a continuous effort towards the identification of new leads for cancer management. In this work, two C-scorpionate complexes, [FeCl2(κ3-Tpm)] (1) and [Co(κ3-TpmOH)2](NO3)2 (2), (Tpm = hydrotris(pyrazol-1-yl)methane and TpmOH = 2,2,2-tris(pyrazol-1-yl)ethanol), were studied as potential scaffolds for future anticancer drug development. Their cytotoxicity and cell migration inhibitory activity were analyzed, and an untargeted metabolomics approach was employed to elucidate the biological processes significantly affected by these two complexes, using two tumoral cell lines (B16 and HCT116) and a non-tumoral cell line (HaCaT). While [FeCl2(κ3-Tpm)] did not display a significant cytotoxicity, [Co(κ3-TpmOH)2](NO3)2 was particularly cytotoxic against the HCT116 cell line. While [Co(κ3-TpmOH)2](NO3)2 significantly inhibited cell migration in all tested cell lines, [FeCl2(κ3-Tpm)] displayed a mixed activity. From a metabolomics perspective, exposure to [FeCl2(κ3-Tpm)] was associated with changes in various metabolic pathways involving tyrosine, where iron-dependent enzymes are particularly relevant. On the other hand, [Co(κ3-TpmOH)2](NO3)2 was associated with dysregulation of cell adhesion and membrane structural pathways, suggesting that its antiproliferative and anti-migration properties could be due to changes in the overall cellular adhesion mechanisms.

The Impact of the Serum Extraction Protocol on Metabolomic Profiling Using UPLC-MS/MS and FTIR Spectroscopy.

Biofluid metabolomics is a very appealing tool to increase the knowledge associated with pathophysiological mechanisms leading to better and new therapies and biomarkers for disease diagnosis and prognosis. However, due to the complex process of metabolome analysis, including the metabolome isolation method and the platform used to analyze it, there are diverse factors that affect metabolomics output. In the present work, the impact of two protocols to extract the serum metabolome, one using methanol and another using a mixture of methanol, acetonitrile, and water, was evaluated. The metabolome was analyzed by ultraperformance liquid chromatography associated with tandem mass spectrometry (UPLC-MS/MS), based on reverse-phase and hydrophobic chromatographic separations, and Fourier transform infrared (FTIR) spectroscopy. The two extraction protocols of the metabolome were compared over the analytical platforms (UPLC-MS/MS and FTIR spectroscopy) concerning the number of features, the type of features, common features, and the reproducibility of extraction replicas and analytical replicas. The ability of the extraction protocols to predict the survivability of critically ill patients hospitalized at an intensive care unit was also evaluated. The FTIR spectroscopy platform was compared to the UPLC-MS/MS platform and, despite not identifying metabolites and consequently not contributing as much as UPLC-MS/MS in terms of information concerning metabolic information, it enabled the comparison of the two extraction protocols as well as the development of very good predictive models of patient's survivability, such as the UPLC-MS/MS platform. Furthermore, FTIR spectroscopy is based on much simpler procedures and is rapid, economic, and applicable in the high-throughput mode, i.e., enabling the simultaneous analysis of hundreds of samples in the microliter range in a couple of hours. Therefore, FTIR spectroscopy represents a very interesting complementary technique not only to optimize processes as the metabolome isolation but also for obtaining biomarkers such as those for disease prognosis.

Protocol to study in vitro drug metabolism and identify montelukast metabolites from purified enzymes and primary cell cultures by mass spectrometry.

We present an optimized protocol set to study the production of drug metabolites in different in vitro systems. We detail the necessary steps to identify the metabolites of xenobiotics produced in different metabolic-competent systems, from purified enzymes to primary cell cultures. It is coupled to a high-resolution mass spectrometry analytical approach and can be adapted to study any xenobiotic. This protocol was optimized using montelukast, an antagonist of the cysteinyl leukotriene receptor 1, widely used for asthma management. For complete details on the use and execution of this protocol, please refer to Marques et al. (2022).1.

The mechanisms underlying montelukast's neuropsychiatric effects - new insights from a combined metabolic and multiomics approach.

AIMS: Montelukast (MTK) is an antagonist of the cysteinyl leukotrienes receptor 1 widely used to manage asthma symptoms among adults and children. However, it has been associated with an increasing number of neuropsychiatric adverse drug reactions (ADRs), particularly among children, including depression, sleep disturbance, and suicidal ideation. The aims of this work were to characterize MTK metabolism in vitro and in vivo and to identify its effects at the metabolome and proteome levels in order to explain its toxicity. MAIN METHODS: An extensive study of montelukast metabolism was carried out using in vitro systems, an embryonic neuron-enriched cell model, and a mouse model. Metabolites were identified by high-resolution mass spectrometry, and a combined mass spectrometry-based metabolomics and proteomics approach was employed to assess the effect of MTK on mice and isolated chicken neurons. KEY FINDINGS: Eighteen new MTK metabolites were identified. MTK's ability to react with glutathione was confirmed. The multi-omics approach employed confirmed that montelukast interferes with the glutathione detoxification system in the brain. Moreover, montelukast is also able to dysregulate various neurotransmitter and neurosteroid pathways, particularly those involved in regulation of the hypothalamic-pituitary-adrenal axis, also interfering with mitochondrial function in neuronal cells. SIGNIFICANCE: Results clearly indicate that montelukast therapeutic effects are accompanied by a strong modulation of specific processes in the central nervous system that may explain the observed neuropsychiatric reactions. Moreover, the results also suggest that adverse drug reactions are more likely to occur in children, due to the early maturation stage of their brains.

Optimized protocol for obtaining and characterizing primary neuron-enriched cultures from embryonic chicken brains.

We present here an optimized protocol to obtain primary neuron-enriched cultures from embryonic chicken brains with no need for an animal facility. The protocol details the steps to isolate a neuron-enriched cell fraction from chicken embryos, followed by characterization of the chicken neurons with mass spectrometry proteomics and cell staining. Because of the high homology between chicken and human amyloid precursor protein processing machinery, these chicken neurons can be used as an alternative to rodent models for studying Alzheimer disease.

Leukotrienes vs. Montelukast-Activity, Metabolism, and Toxicity Hints for Repurposing.

Increasing environmental distress is associated with a growing asthma incidence; no treatments are available but montelukast (MTK)-an antagonist of the cysteinyl leukotrienes receptor 1-is widely used in the management of symptoms among adults and children. Recently, new molecular targets have been identified and MTK has been proposed for repurposing in other therapeutic applications, with several ongoing clinical trials. The proposed applications include neuroinflammation control, which could be explored in some neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases (AD and PD). However, this drug has been associated with an increasing number of reported neuropsychiatric adverse drug reactions (ADRs). Besides, and despite being on the market since 1998, MTK metabolism is still poorly understood and the mechanisms underlying neuropsychiatric ADRs remain unknown. We review the role of MTK as a modulator of leukotriene pathways and systematize the current knowledge about MTK metabolism. Known toxic effects of MTK are discussed, and repurposing applications are presented comprehensively, with a focus on AD and PD.

Molecular dynamics simulations and analysis for bioinformatics undergraduate students.

A computational biochemistry laboratory, fitted for bioinformatics students, is presented. The molecular dynamics package GROMACS is used to prepare and simulate a solvated protein. Students analyze the trajectory with different available tools (GROMACS and VMD) to probe the structural stability of the protein during the simulation. Students are also required to make use of Python libraries and write their own code to probe non-covalent interactions between the amino acid side chains. Based on these results, students characterize the system in a qualitatively approach but also assess the importance of each specific interaction through time. This work mobilizes biochemical concepts and programming skills, fostering critical thinking and group work and developing presenting skills.

Flexible ZnO-mAb nanoplatforms for selective peripheral blood mononuclear cell immobilization.

Cancer is the second cause of death worldwide. This devastating disease requires specific, fast, and affordable solutions to mitigate and reverse this trend. A step towards cancer-fighting lies in the isolation of natural killer (NK) cells, a set of innate immune cells, that can either be used as biomarkers of tumorigenesis or, after autologous transplantation, to fight aggressive metastatic cells. In order to specifically isolate NK cells (which express the surface NKp30 receptor) from peripheral blood mononuclear cells, a ZnO immunoaffinity-based platform was developed by electrodeposition of the metal oxide on a flexible indium tin oxide (ITO)-coated polyethylene terephthalate (PET) substrate. The resulting crystalline and well-aligned ZnO nanorods (NRs) proved their efficiency in immobilizing monoclonal anti-human NKp30 antibodies (mAb), obviating the need for additional procedures for mAb immobilization. The presence of NK cells on the peripheral blood mononuclear cell (PBMCs) fraction was evaluated by the response to their natural ligand (B7-H6) using an acridine orange (AO)-based assay. The successful selection of NK cells from PBMCs by our nanoplatform was assessed by the photoluminescent properties of AO. This easy and straightforward ZnO-mAb nanoplatform paves the way for the design of biosensors for clinic diagnosis, and, due to its inherent biocompatibility, for the initial selection of NK cells for autotransplantation immunotherapies.

NKp30 - A prospective target for new cancer immunotherapy strategies.

Natural killer (NK) cells are an important arm of the innate immune system. They constitutively express the NKp30 receptor. NKp30-mediated responses are triggered by the binding of specific ligands e.g. tumour cell-derived B7-H6 and involve the secretion of cytotoxic mediators including TNF-α, IFN-γ, perforins and granzymes. The latter two constitute a target cell-directed response that is critical in the process of immunosurveillance. The structure of NKp30 is presented, focusing on the ligand-binding site, on the ligand-induced structural changes and on the experimental data available correlating structure and binding affinity. The translation of NKp30 structural changes to disease progression is also reviewed. NKp30 role in immunotherapy has been explored in chimeric antigen receptor T-cell (CAR-T) therapy. However, antibodies or small ligands targeting NKp30 have not yet been developed. The data reviewed herein unveil the key structural aspects that must be considered for drug design in order to develop novel immunotherapy approaches.

Synthesis, Crystal Structure, and Biological Evaluation of Fused Thiazolo[3,2-a]Pyrimidines as New Acetylcholinesterase Inhibitors.

A new series of thiazolo[3,2-a]pyrimidine bromide salt derivatives 7a-d were synthesized from 3,4-dihydropyrimidinethione precursors. The target compounds were fully characterized by 1D- and 2D-NMR, high resolution ESI-MS/MS and single crystal X-ray diffraction analysis, which confirmed a regioselective 5H cyclization of the dihydropyrimidinethiones. All target compounds were evaluated in vitro as human acetylcholinesterase (hAChE) inhibitors via an Ellman-based colorimetric assay and showed good inhibition activities (better than 70% at 10 µM and IC50 values in the 1 µM range). Molecular docking simulations for all target products into hAChE were performed and confirmed strong binding to the enzyme. These results provide a promising and new starting point to improve acetylcholinesterase inhibitors and explore novel treatment options against Alzheimer's disease.

Antibodies against HDL Components in Ischaemic Stroke and Coronary Artery Disease.

Quantitative and qualitative defects of high-density lipoprotein (HDL) are important in atherogenesis. In this study, we investigated whether antibodies against HDL components had additional value to conventional cardiovascular risk factors for the diagnosis of ischaemic stroke (IS) and coronary artery disease (CAD). Cross-sectional study was conducted on 53 patients with IS, 51 with CAD and 55 healthy controls, and in vitro studies to validate findings of the clinical study. We determined serum immunoglobulin G (IgG) antibodies against HDL (aHDL), apolipoproteins (aApoA-I, aApoA-II and aApoC-I) and paraoxonase-1 (aPON1) as well as PON1 activity (PON1a), total antioxidant capacity and biomarkers of endothelial activation (serum nitric oxide metabolites, 3-nitrotyrosine, VCAM-1 and ICAM-1); in vitro assays tested the capacity of IgG aHDL purified from high titer patients to inhibit PON1a and to reverse protective effect of HDL on endothelial cells. IgG aHDL, aApoA-I and aPON1 were higher in IS and CAD than controls (p < 0.001), predicted negatively PON1a and positively VCAM-1 and ICAM-1. By adding IgG aHDL and aApoA-I to a traditional cardiovascular risk factors model for IS and by adding IgG aHDL in a similar model for CAD, we obtained better discrimination of IS and CAD from healthy controls. IgG aHDL purified from IS and CAD inhibited PON1a by 38% (p < 0.01) and abrogated the protective effect of HDL on VCAM-1 expression by 126% compared with non-specific human IgG (p < 0.001). IgG against HDL components interfere with the antioxidant and anti-inflammatory properties of HDL and may represent novel biomarkers for vascular disease that need to be investigated in prospective studies.

Selenium-containing chrysin and quercetin derivatives: attractive scaffolds for cancer therapy.

Selenium-containing chrysin (SeChry) and 3,7,3',4'-tetramethylquercetin (SePQue) derivatives were synthesized by a microwave-based methodology. In addition to their improvement in terms of DPPH scavenging and potential GPx-like activities, when tested in a panel of cancer cell lines both selenium-derivatives revealed consistently to be more cytotoxic when compared with their oxo and thio-analogues, evidencing the key role of selenocabonyl moiety for these activities. In particular, SeChry elicited a noteworthy cytotoxic activity with mean IC50 values 18- and 3-fold lower than those observed for chrysin and cisplatin, respectively. Additionally, these seleno-derivatives evidenced an ability to overcome cisplatin and multidrug resistance. Notably, a differential behavior toward malignant and nonmalignant cells was observed for SeChry and SePQue, exhibiting higher selectivity indexes when compared with the chalcogen-derivatives and cisplatin. Our preliminary investigation on the mechanism of cytotoxicity of SeChry and SePQue in MCF-7 human mammary cancer cells demonstrated their capacity to efficiently suppress the clonal expansion along with their ability to hamper TrxR activity leading to apoptotic cell death.

Electronic communication in linear oligo(azobenzene) radical anions.

The radical anions of five bis(azobenzene) and one tris(azobenzene) compounds were studied by optical and electron paramagnetic resonance (EPR) spectroscopies in polar aprotic solvents. The radicals with planar or almost-planar bridges are charge-delocalized mixed-valence species. Localization of charge occurs only on radicals with highly twisted biphenyl bridges. The electronic coupling between the azobenzene charge-bearing units, calculated as half the energy of the intervalence band for the charge-delocalized and by the Hush equation for the charge-localized radicals, decreases with the distance and torsion angle between azobenzene units. These radicals have smaller electronic couplings between charge-bearing units than other mixed-valence organic radicals with similar bridges. However, the application of a three-stage model to the tris(azobenzene) radical anion intervalence band yields an electronic coupling between consecutive azobenzenes that is higher than in any of the bis(azobenzene) radicals studied.

Binding of V(IV)O²âº to the Fe binding sites of human serum transferrin. A theoretical study.

The binding of V(IV)O²âº to human serum transferrin (hTF) at the FeIII binding sites is addressed. Geometry optimization calculations were performed for the binding of V(IV)O²âº to the N-terminal lobe of hTF (hTFN), and indicate that in the presence of CO3²â» or HCO3 −, V(IV) is bound to five atoms in a distorted geometry. The structures of V(IV)O-hTFN species optimized at the semiempirical level were also used to calculate the 5¹V and ¹4N A tensors by density functional theory methods, and were compared with the reported experimental values. Globally, of all the calculated V(IV)O-hTF structures, the one that yields the lowest calculated heats of formation and minimum deviations from the experimental values of the 5¹V and ¹4N A tensor components is the structure that includes CO3²â» as a synergistic anion. In this structure the V=O bond length is approximately 1.6 Å, and the vanadium atom is also coordinated to the phenolate oxygen atom of Tyr188 (at approximately 1.9 Å), the aspartate oxygen atom of Asp63 (at approximately 1.9 Å), the His249 Nτ atom (at approximately 2.1 Å), and a carbonate oxygen atom (at approximately 1.8 Å). The Tyr95 phenolic ocygen atom is approximately 3.3 Å from the metal center, and thus is very weakly bound to V(IV). All of these oxygen atoms are able to establish dipolar interactions with groups of the protein.

Demethylation of theophylline (1,3-dimethylxanthine) to 1-methylxanthine: the first step of an antioxidising cascade.

The reaction of theophylline with HO(*) radical, produced by photolytic methods at pH 7, was studied in aqueous solution and the products characterised by HPLC and GC-MS. In addition to the expected 1,3-dimethyluric acid, the formation of 1-methylxanthine and, to a lesser extent, of 3-methylxanthine was observed. Theoretical calculations confirmed the preferred formation of the former compound. Both demethylated products were also observed upon reaction of theophylline with O(*-) radical anion at pH approximately 13, and 1-methylxanthine was consumed faster than 3-methylxanthine after its formation. Molecular oxygen had no significant effect on the formation of the mono-methylxanthine derivatives. A reaction mechanism for the demethylation of theophylline by oxidising radicals is proposed. This demethylation reaction can play an important role in the protection of biological targets against oxidative stress as the first step of an antioxidising cascade.

Amino acid adduct formation by the nevirapine metabolite, 12-hydroxynevirapine--a possible factor in nevirapine toxicity.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child transmission of the virus in developing countries. However, reports of severe NVP-induced hepatotoxicity and serious adverse cutaneous effects have raised concerns about its use. NVP metabolism involves oxidation of the 4-methyl substituent to 4-hydroxymethyl-NVP (12-hydroxy-NVP) and the formation of phenolic derivatives. Further metabolism, through either oxidation to quinoid derivatives or phase II esterification, may produce electrophilic derivatives capable of reacting with bionucleophiles to yield covalent adducts. These adducts could potentially be involved in the initiation of toxic responses. To gain insight into potentially reactive sites in proteins and prepare reliable and fully characterized NVP-amino acid adduct standards for subsequent assessment as biomarkers of NVP toxicity, we have used the model electrophile, 12-mesyloxy-NVP, as a synthetic surrogate for the NVP metabolite, 12-sulfoxy-NVP. Reactions of this model ester were conducted with glutathione and the nucleophilic amino acids arginine, cysteine, histidine, and tryptophan. Moreover, because adducts through the N-terminal valine of hemoglobin are convenient biomarkers of exposure to electrophilic toxicants, we also investigated the reaction with valine. We obtained very efficient (>80%) binding through the sulfur of both glutathione and N-acetylcysteine and moderate yields (10-14%) for binding through C2 of the indole ring of tryptophan and N1 of the imidazole ring of histidine. Reaction with arginine occurred through the alpha-amino group, possibly due to the high basicity of the guanidino group in the side chain. Reaction at the alpha-amino group of valine occurred to a significant extent (33%); the resulting adduct was converted to a thiohydantoin derivative, to obtain a standard useful for prospective biomonitoring studies. All adducts were characterized by a combination of (1)H and (13)C NMR spectroscopy and mass spectrometry techniques. The NVP conjugates with glutathione and N-acetylcysteine identified in this work were previously reported to be formed in vivo, although the corresponding structures were not fully characterized. Our results support the validity of 12-mesyloxy-NVP as a surrogate for 12-sulfoxy-NVP and suggest that NVP metabolism to 12-hydroxy-NVP, and subsequent esterification, could potentially be a factor in NVP toxicity. They further imply that multiple sites in proteins may be targets for modification by 12-hydroxy-NVP-derived electrophiles in vivo. Additionally, we obtained reliable, fully characterized standards for the assessment of protein modification by NVP in vivo, which should help clarify the potential role of metabolism in NVP-induced toxicity.

Antioxidant mechanisms of Quercetin and Myricetin in the gas phase and in solution--a comparison and validation of semi-empirical methods.

Flavonoids have long been recognized for their general health-promoting properties, of which their antioxidant activity may play an important role. In this work we have studied the properties of two flavonols, quercetin and myricetin, using semi-empirical methods in order to validate the application of the recent Parametric Model 6 and to understand the fundamental difference between the two molecules. Their geometries have been optimized and important molecular properties have been calculated. The energetic of the possible antioxidant mechanisms have also been analyzed. The two studied flavonols do not differ significantly in their molecular properties, but the antioxidant mechanisms by which they may act in solution can be rather different. Moreover, we also show that the Parametric Model 6 can produce reliable information for this type of compounds.

Structure and antioxidant activity of brominated flavonols and flavanones.

Hypobromous acid (HOBr) produced by both eosinophil peroxidase (EPO) and myeloperoxidase (MPO) is a stronger oxidant than HOCl, and is also essential for optimal and efficient microbial killing. Considering the potential cytotoxic effect of HOBr, if it is formed outside the phagosome, it should be useful to scavenge it in order to protect the nearby tissues. In this study the ability of selected flavonoids to protect against HOBr mediated oxidation reactions was performed through a competitive reaction, and the resulting products identified by high performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectrometry(ESI-MS/MS). Several structural features were found to be important to confer high antioxidant activity to flavonoids towards HOBr: the C2=C3 double bond and the 3-OH group in the C-ring, and the presence of both 5-OH and 7-OH groups in the A-ring. The MS results showed that flavonoids are dibrominated in the A-ring, suggesting that (except for fisetin) bromination occurs at C6 and C8 positions, through an electrophilic aromatic substitution reaction. The chemical modifications achieved by bromination of flavonoids have changed their biological properties, presenting their brominated derivatives higher antioxidant activity, as radical scavengers, and higher lipophilicity, than the parent flavonoids. Brominated flavonoids may then diffuse easily through membranes increasing the intracellular concentration of the compounds. These locally formed metabolites may also interact with signaling cascades involving cytokines and regulatory transcription factors, thus playing a role in inflammation and in the regulation of immune response.

Electrospray ionization tandem mass spectrometry fragmentation of protonated flavone and flavonol aglycones: a re-examination.

Flavonoids are important phytochemicals which have been intensively studied in the last decades in view of their antioxidant activity, which is of particular importance in the case of flavones and flavonols, that differ in a single 3-OH group. Mass spectrometry has been used to elucidate the structures of many types of flavonoids and their metabolites. The work we present here is focused on the electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of flavone and flavonols aglycones. Their fragmentation mechanisms in the positive ion mode are described and compared with previously reported mechanisms. We analyzed flavonoid derivatives produced by reaction of the flavonoids with chemically synthesized hypohalous acids (HOCl, HOBr and HOI) and peroxynitrite, reactive species involved in the inflammatory response. All the proposed pathways have been analyzed using computational chemistry methods in order to seek for possible variations and establish the most plausible ones. We observed that the losses of one and two CO molecules can be useful in terms of antioxidant activity prediction. Losses of one and two C(2)H(2)O groups are also informative in terms of structure and activity predictions. The retro-Diels-Alder fragmentations, and subsequent neutral losses, were reviewed and, according to our calculations, the most plausible structures for the product ions were established. These fingerprints will be of great value for differentiating flavonoids from other compounds in complex biological mixtures and for a thorough structural identification of flavonoid aglycones and their in vivo metabolites.