Publications

Abstract Neurodegenerative diseases (NDs) are a set of progressive, chronic, and incurable diseases characterized by the gradual loss of neurons, culminating in the decline of cognitive and/or motor functions. Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common NDs and represent an enormous burden both in terms of human suffering and economic cost. The available therapies for AD and PD only provide symptomatic and palliative relief for a limited period and are unable to modify the diseases' progression. Over the last decades, research efforts have been focused on developing new pharmacological treatments for these NDs. However, to date, no breakthrough treatment has been discovered. Hence, the development of disease-modifying drugs able to halt or reverse the progression of NDs remains an unmet clinical need. This review summarizes the major hallmarks of AD and PD and the drugs available for pharmacological treatment. It also sheds light on potential directions that can be pursued to develop new, disease-modifying drugs to treat AD and PD, describing as representative examples some advances in the development of drug candidates targeting oxidative stress and adenosine A2A receptors.

Abstract We report here the first dual inhibitors of brain carbonic anhydrases (CAs) and monoamine oxidase-B (MAO-B) for the management of Alzheimer's disease. Classical CA inhibitors (CAIs) such as methazolamide prevent amyloid-beta-peptide (A beta)-induced overproduction of reactive oxygen species (ROS) and mitochondrial dysfunction. MAO-B is also implicated in ROS production, cholinergic system disruption, and amyloid plaque formation. In this work, we combined a reversible MAO-B inhibitor of the coumarin and chromone type with benzenesulfonamide fragments as highly effective CAIs. A hit-to-lead optimization led to a significant set of derivatives showing potent low nanomolar inhibition of the target brain CAs (K(I)s in the range of 0.1-90.0 nM) and MAO-B (IC50 in the range of 6.7-32.6 nM). Computational studies were conducted to elucidate the structure-activity relationship and predict ADMET properties. The most effective multitarget compounds totally prevented A beta-related toxicity, reverted ROS formation, and restored the mitochondrial functionality in an SH-SY5Y cell model surpassing the efficacy of single-target drugs.

Abstract The increasing interest in utilizing psychedelics for therapeutic purposes demands the development of tools capable of efficiently monitoring and accurately identifying these substances, thereby supporting medical interventions. 4-Bromo-2,5-dimethoxyphenethylamine (2C-B) has gained significant popularity as one of the most widely used psychedelic compounds in non-medical settings. In this study, we aimed to create a material with selective recognition of 2C-B by synthesizing a series of molecularly imprinted polymers (MIP) using 2C-B as the template and varying ratios of methacrylic acid (MAA) as the functional monomer (1:2, 1:3, and 1:4). Both thermal and microwave-assisted polymerization processes were employed. The molar ratio between the template molecule (2C-B) and functional monomer (MAA) was 1:4, utilizing a microwave-assisted polymerization process. Isotherm studies revealed a Langmuir's maximum absorption capacity (Bmax) value of 115.6 mu mol center dot mg-1 and Kd values of 26.7 mu M for this material. An imprint factor of 4.2 was determined for this material, against the corresponding non-imprinted polymer. The good selectivity against 14 other new psychoactive substances highlighted the material's potential for applications requiring selective recognition. These findings can contribute to the development of tailored materials for the detection and analysis of 2C-B, supporting advancements in non-medical use monitoring and potential therapeutic models involving psychedelics.

Abstract Tolcapone is an orally active catechol-O-methyltransferase (COMT) inhibitor used as adjuvant therapy in Parkinson's disease. However, it has a highly hepatotoxic profile, as recognized by the U.S. Food and Drug Administration. As a possible solution, nanoscience brought us several tools in the development of new functional nanomaterials with tunable physicochemical properties, which can be part of a solution to solve several drawbacks, including drug's short half-life and toxicity. This work aims to use PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a stable carrier with lower hydrodynamic size and polydispersity to encapsulate tolcapone in order to overcome its therapeutic drawbacks. Using the nanoprecipitation method, tolcapone-loaded nanoparticles with a DLC% of 5.7% were obtained (EE% of 47.0%) and subjected to a lyophilization optimization process to obtain a final shelf-stable formulation. Six different cryoprotectants in concentrations up to 10% (w/v) were tested. A formulation of PLGA nanoparticles with 3% hydroxypropyl-beta-cyclodextrin (HP beta CD) as a cryoprotectant (PLGA-HP@Tolc), presenting sub-200 nm sizes and low polydispersity (PdI < 0.200) was selected. Cytotoxicity assays, namely, MTT and SRB, were used to study the metabolic activity and cell density of tolcapone and PLGA-HP@Tolc-treated cells. In both assays, a hepatocarcinoma cell line (HepG2) growing in glucose or glucose-free media (galactose-supplemented medium) was used. The results demonstrated that the treatment with the PLGA-HP@Tolc formulation led to a decrease in cytotoxicity in comparison to free tolcapone-treated cells in both media tested. Moreover, the elected formulation also counteracted ATP-depletion and excessive ROS production induced by tolcapone. The results suggest that HP beta CD might have a dual function in the formulation: cryoprotectant and anticytotoxic agent, protecting cells from tolcapone-induced damage. Using an in vitro COMT inhibition assay, the PLGA-HP@Tolc formulation demonstrated to inhibit COMT as efficiently as free tolcapone. Overall, the results suggest that tolcapone-loaded PLGA NPs could be an interesting alternative to free tolcapone, demonstrating the same in vitro efficacy in inhibiting COMT but with a safer cytotoxic profile.

Abstract Substituted phenethylamines including 2C (2,5-dimethoxyphenethylamines) and NBOMe (N-(2-methoxybenzyl)phenethylamines) drugs are potent psychoactive substances with little to no knowledge available on their toxicity. In the present in vitro study, we explored the mechanisms underlying the neurotoxicity of six substituted phenethylamines: 2C-T-2, 2C-T-4, 2C-T-7 and their corresponding NBOMes. These drugs were synthesized and chemically characterized, and their cytotoxicity (0-1000 mu M) was evaluated in differentiated SH-SY5Y cells and primary rat cortical cultures, by the NR uptake and MTT reduction assays. In differentiated SH-SY5Y cells, mitochondrial membrane potential, intracellular ATP and calcium levels, reactive oxygen species production, and intracellular total glutathione levels were also evaluated. All the tested drugs exhibited concentration-dependent cytotoxic effects towards differentiated SH-SY5Y cells and primary rat cortical cultures. The NBOMe drugs presented higher cytotoxicity than their counterparts, which correlates with the drug's lipophilicity. These cytotoxic effects were associated with mitochondrial dysfunction, evident through mitochondrial membrane depolarization and lowered intracellular ATP levels. Intracellular calcium imbalance was observed for 2C-T-7 and 25T7-NBOMe, implying a disrupted calcium regulation. Although reactive species levels remained unchanged, a reduction in intracellular total GSH content was observed. Overall, these findings contribute to a deeper understanding of these drugs, shedding light on the mechanisms underpinning their neurotoxicity.

Abstract Quorum sensing (QS) has a central role in biofilm lifestyle and antimicrobial resistance, and disrupting these signaling pathways is a promising strategy to control bacterial pathogenicity and virulence. In this study, the efficacy of three structurally related benzaldehydes (4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde)) in disrupting the las and pqs systems of Pseudomonas aeruginosa was investigated using bioreporter strains and computational simulations. Additionally, these benzaldehydes were combined with tobramycin and ciprofloxacin antibiotics to evaluate their ability to increase antibiotic efficacy in preventing and eradicating P. aeruginosa biofilms. To this end, the total biomass, metabolic activity and culturability of the biofilm cells were determined. In vitro assays results indicated that the aromatic aldehydes have potential to inhibit the las and pqs systems by > 80 %. Molecular docking studies supported these findings, revealing the aldehydes binding in the same pocket as the natural ligands or receptor proteins (LasR, PQSA, PQSE, PQSR). Benzaldehydes were shown to act as virulence factor attenuators, with vanillin achieving a 48 % reduction in pyocyanin production. The benzaldehyde-tobramycin combination led not only to a 60 % reduction in biomass production but also to a 90 % reduction in the metabolic activity of established biofilms. A similar result was observed when benzaldehydes were combined with ciprofloxacin. 4-Hydroxybenzaldehyde demonstrated relevant action in increasing biofilm susceptibility to ciprofloxacin, resulting in a 65 % reduction in biomass. This study discloses, for the first time, that the benzaldehydes studied are potent QS inhibitors and also enhancers of antibiotics antibiofilm activity against P. aeruginosa.

Abstract Lewy body dementia (LBD) represents the second most common neurodegenerative dementia but is a quite underexplored therapeutic area. Nepflamapimod (1) is a brain-penetrant selective inhibitor of the alpha isoform of the mitogen-activated serine/threonine protein kinase (MAPK) p38 alpha, recently repurposed for LBD due to its remarkable antineuroinflammatory properties. Neuroprotective propargylamines are another class of molecules with a therapeutical potential against LBD. Herein, we sought to combine the antineuroinflammatory core of 1 and the neuroprotective propargylamine moiety into a single molecule. Particularly, we inserted a propargylamine moiety in position 4 of the 2,6-dichlorophenyl ring of 1, generating neflamapimod-propargylamine hybrids 3 and 4. These hybrids were evaluated using several cell models, aiming to recapitulate the complexity of LBD pathology through different molecular mechanisms. The N-methyl-N-propargyl derivative 4 showed a nanomolar p38 alpha-MAPK inhibitory activity (IC50 = 98.7 nM), which is only 2.6-fold lower compared to that of the parent compound 1, while displaying no hepato- and neurotoxicity up to 25 mu M concentration. It also retained a similar immunomodulatory profile against the N9 microglial cell line. Gratifyingly, at 5 mu M concentration, 4 demonstrated a neuroprotective effect against dexamethasone-induced reactive oxygen species production in neuronal cells that was higher than that of 1.

Abstract Antimicrobial resistance (AMR) poses a global threat to human health, as exemplified by the devastating impact of ESKAPE pathogens, reducing treatment options, increasing disease burden, and elevating death rates due to treatment failure. This looming health threat has rekindled interest in the development of new antimicrobial therapies. In this study, a library of 49 structurally related quaternary heteronium salts (QHSs), such as quaternary ammonium and phosphonium compounds, was screened at a concentration of 32 mu g/mL against five ESKAPE pathogens (methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli), as well as the yeast -like fungus Candida albicans. The preliminary high -throughput screening (HTS) data revealed that QHSs with longer alkyl chain lengths (>= 12 carbons) exhibited broad-spectrum antimicrobial activity, with the C14-C16 homologous demonstrating the highest potency. The alkyl-triphenylphosphonium salts 1a-g exhibited the most significant antimicrobial activity among all tested compound series. The compounds identified as active in the initial HTS underwent confirmation assays to determine minimum inhibitory concentration (MIC) and cytotoxicity, following established protocols. Confirmatory assays identified 33 hits (MIC <= 16 mu g/mL), with 78% effective at very low concentrations (<= 0.25 mu g/mL). Seven hits showed safety profiles against HEK-293 cells and red blood cells at concentrations below 32 mu g/mL. Based on a comparative analysis of their potency against pathogenic microorganisms and cell toxicity, the salts of triphenylphosphonium and quinolinium with linear C8 hydrocarbon substituents (compounds 1b and 5b, respectively), and isoquinolinium, methylpyridinium and triethylammonium with linear C18 hydrocarbon substituents (compounds 3g, 4g, and 7g, respectively) have emerged as the most promising candidates for microbial growth control.

Abstract Nitrocatechols are the standard pharmacophore to develop potent tight-binding inhibitors of catechol O-methyltransferase (COMT), which can be used as coadjuvant drugs to manage Parkinson's disease. Tolcapone is the most potent drug of this class, but it has raised safety concerns due to its potential to induce liver damage. Tolcapone-induced hepatotoxicity has been attributed to the nitrocatechol moiety; however, other nitrocatechol-based COMT inhibitors, such as entacapone, are safe and do not damage the liver. There is a knowledge gap concerning which mechanisms and chemical properties govern the toxicity of nitrocatechol-based COMT inhibitors. Using a vast array of cell-based assays, we found that tolcapone-induced toxicity is caused by direct interference with mitochondria that does not depend on bioactivation by P450. Our findings also suggest that (a) lipophilicity is a key property in the toxic potential of nitrocatechols; (b) the presence of a carbonyl group directly attached to the nitrocatechol ring seems to increase the reactivity of the molecule, and (c) the presence of cyano moiety in double bond stabilizes the reactivity decreasing the cytotoxicity. Altogether, the fine balance between lipophilicity and the chemical nature of the C1 substituents of the nitrocatechol ring may explain the difference in the toxicological behavior observed between tolcapone and entacapone.

Abstract Carbon electrodes, especially the glassy carbon electrodes (GCE) are widely accepted as very versatile sensing platforms. However, correlating the behaviour of the ferri/ferrocyanide redox couple ([Fe(CN) 6 ] 3-/4 ) with the GCE ' s surface modification is challenging. The surface modification can be achieved by applying a preconditioning electrochemical activation procedure. Hence, we report the investigation performed in order to provide further insights into the electrochemical behaviour of the commonly used redox probe in aqueous solutions. To that aim we took advantage of powerful and complementary electrochemical analytical techniques, like cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Thus, this work highlights the critical role of the GCE initial conditions for optimizing the charge transfer processes and therefore to improve the [Fe(CN) 6 ] 3-/4- performance in sensing applications. The best results were obtained in phosphate buffer saline solution with previous electrochemical activation by fast potential cycling between [-0.5 and +1.8] V (vs. Ag| AgCl (KCl sat.)). Finally, one can consider this an eco-friendly and simple procedure to be carried out in the lab, however, its use must be carefully optimized when exploring other systems, as highlighted herein.