Publications

Abstract We report herein the design, synthesis and biological evaluation of new antioxidant and neuroprotective multitarget directed ligands (MTDLs) able to block Ca2+ channels. New dialkyl 2,6-dimethyl-4-(4-(prop-2-yn-1-yloxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate MTDLs 3a-t, resulting from the juxtaposition of nimodipine, a Ca2+ channel antagonist, and rasagiline, a known MAO inhibitor, have been obtained from appropriate and commercially available precursors using a Hantzsch reaction. Pertinent biological analysis has prompted us to identify the MTDL 3,5-dimethyl-2,6-dimethyl-4-[4-(prop-2-yn-1-yloxy)phenyl]-1,4-dihydro- pyridine- 3,5-dicarboxylate (3a), as an attractive antioxidant (1.75 TE), Ca2+ channel antagonist (46.95% at 10 mu M), showing significant neuroprotection (38%) against H2O2 at 10 mu M, being considered thus a hit-compound for further investigation in our search for anti-Alzheimer's disease agents.

Abstract Background: Half of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanolderived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1. Methods and results: By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced. Conclusions: SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53. General Significance: This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

Abstract Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin-chalcone hybrids were synthesized (compounds 1-8) and screened in radioligand binding (hA(1), hA(2A) and hA(3)) and adenylyl cyclase (hA(2B)) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin-chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA(1) or hA(3) subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA(1), while the methoxy counterparts were selective for the hA(3). The most potent hA(1) ligand was compound 7 (K-i = 17.7 mu M), whereas compound 4 was the most potent ligand for hA(3) (K-i = 2.49 mu M). In addition, docking studies with hA(1) and hA(3) homology models were established to analyze the structure-function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.

Abstract The importance of isothiazole and of compounds containing the isothiazole nucleus has been growing over the last few years. Isothiazolinones are used in cosmetic and as chemical additives for occupational and industrial usage due to their bacteriostatic and fungiostatic activity. Despite their effectiveness as biocides, isothiazolinones are strong sensitizers, producing skin irritations and allergies and may pose ecotoxicological hazards. Therefore, their use is restricted by EU legislation. Considering the relevance and importance of isothiazolinone biocides, the present review describes the state-of-the-art knowledge regarding their synthesis, antibacterial components, toxicity (including structure-activity-toxicity relationships) outlines, and (photo)chemical stability. Due to the increasing prevalence and impact of isothiazolinones in consumer's health, analytical methods for the identification and determination of this type of biocides were also discussed.

Abstract An in-situ approach based in multiple spectroscopic techniques and benchmarked with DSC was used to characterise beta-Lg thermally-induced transitions. The methodology applied overcomes previously reported limitations by ensuring similar experimental conditions in different determinations, non-aggregation conditions and allowing differentiation between fluorescent variations due to collisional quenching and structural modifications. These experimental improvements along with the correlation of complementary data from the assessment of several unfolding-related events, allowed a real time, precise and detailed description of the unfolding/refolding pathways of beta-Lg. The existence of a complex multi-step unfolding mechanism was confirmed, with a focus on the reversible conformational changes. The elusive unfolding intermediates were characterised in terms of structural swelling, hydrophobic sites accessibility and tryptophan exposure. This approach allowed establishing a clear order of events during thermally-induced structural changes, representing a step forward in the understanding of protein stability and interactions, useful, e.g., when establishing heat treatments of dairy products.

Abstract The effects of light-absorbing atmospheric particles on climate forcing have been integrated into climate models, but the absence of brown carbon (BrC) in these models has led to differences between model predictions and measured data. Herein, we have used density functional theory (DFT) to generate models for the atmospheric absorption of polycyclic aromatic hydrocarbons (PAHs), major contributors to BrC light absorption, found over Seoul (South Korea), considering their seasonal and yearly variation. Winter was found to be the most problematic season, with significant absorption from the PAHs, while the absorption was more moderate in the autumn and in the spring. In the summer, the absorption is relatively quite weak. This is in line with the higher concentration of PAHs during winter, followed by autumn and spring, while being lower during summer. Moreover, these models showed that PAHs absorb strongly in the UVA and UVB regions of the UV spectrum, followed by moderate absorption in the UVC region and weak absorption in the visible region. Nevertheless, only absorption at the UVA and the visible region should be relevant for climate forcing. Finally, fluoranthene and benzo[a]anthracene are the most relevant contributors for UVB absorption, while benzo[a]pyrene, benzo[g,h,i]perylene, indeno[1,2,3-cd]pyrene and benzo[k]fluoranthene are the main responsible for absorption in the visible region. Thus, our modelling approach allowed us to identify which should be the most relevant PAHs for climate forcing on this region of the globe.

Abstract Persistent infections, usually associated with biofilm-producing bacteria, are challenging for both medical and scientific communities. The potential interest in drug repurposing for biofilm control is growing due to both disinvestment in antibiotic R&D and reduced efficacy of the available panel of antibiotics. In the present study, the antibacterial and antibiofilm activities of four non-steroidal anti-inflammatory drugs (NSAIDs), piroxicam (PXC), diclofenac sodium (DCF), acetylsalicylic acid (ASA) and naproxen sodium (NPX) were evaluated againstEscherichia coliandStaphylococcus aureus. The minimum inhibitory/bactericidal concentrations (MICs and MBCs) and the dose-response curves from exposure to the selected NSAIDs were determined. MICs were found for PXC (800 mu g/mL) and ASA (1750 mu g/mL) againstE. coli, and for DCF (2000 mu g/mL) and ASA (2000 mu g/mL) againstS. aureus. No MBCs were found (>2000 mu g/mL). The potential of NSAIDs to eradicate preformed biofilms was characterized in terms of biofilm mass, metabolic activity and cell culturability. Additionally, the NSAIDs were tested in combination with kanamycin (KAN) and tetracycline (TET). ASA, DCF and PXC promoted significant reductions in metabolic activity and culturability. However, only PXC promoted biofilm mass removal. Additive interactions were obtained for most of the combinations between NSAIDs and KAN or TET. In general, NSAIDs appear to be a promising strategy to control biofilms as they demonstrated to be more effective than conventional antibiotics.

Abstract alpha-Synuclein (alpha syn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an alpha-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on alpha-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on alpha syn secondary-structure formation, using human-, elephant- and whale -alpha syn. Our results confirm that negatively charged lipids induce alpha syn folding in h-alpha syn and e-alpha syn but not in w-alpha syn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 degrees C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying alpha syn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as alpha syn into nontoxic alpha-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and alpha-helix formation by alpha syn, paramount to the proposal of new methods to avoid protein aggregation and disease.

Abstract The selective fluorescence sensing of hypochlorite (ClO-) was achieved at pH 7.4 by a simple analytical procedure through the fluorescence quenching of autoclave synthesized carbon dots (CDs), which used as precursor an adduct formed between 3-aminophenylboronic acid (APBA) and alizarin red S (ARS). The use of this adduct allowed the preparation of CDs with a red shifted emission (560 nm) and excitation in the visible range (490 nm). Quantification of hypochlorite was achieved at physiological pH (pH 7.4) in aqueous solutions by fluorescence quenching with a linearity range of 0-200 mu M (limit of detection of 4.47 mu M, and limit of quantification of 13.41 mu M). The selectivity of hypochlorite sensing was confirmed by comparison with other potential analytes, such as glucose, fructose and hydrogen peroxide. Finally, the validity of the proposed assay was further demonstrated by performing recovery assays in different matrices. Thus, this CDs allows the fluorescent sensing of ClO- with spectral properties more suitable for in vitro/in viva applications.