Mitochondria dysfunction induced by decyl-TPP mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6 sensitizes cisplatin lung anticancer therapy due to a remodeling of energy metabolism.

The pharmacological interest in mitochondria is very relevant since these crucial organelles are involved in the pathogenesis of multiple diseases, such as cancer. In order to modulate cellular redox/oxidative balance and enhance mitochondrial function, numerous polyphenolic derivatives targeting mitochondria have been developed. Still, due to the drug resistance emergence in several cancer therapies, significant efforts are being made to develop drugs that combine the induction of mitochondrial metabolic reprogramming with the ability to generate reactive oxygen species, taking into consideration the varying metabolic profiles of different cell types. We previously developed a mitochondria-targeted antioxidant (AntiOxCIN6) by linking caffeic acid to lipophilic triphenylphosphonium cation through a 10-carbon aliphatic chain. The antioxidant activity of AntiOxCIN6 has been documented but how the mitochondriotropic compound impact energy metabolism of both normal and cancer cells remains unknown. We demonstrated that AntiOxCIN6 increased antioxidant defense system in HepG2 cells, although ROS clearance was ineffective. Consequently, AntiOxCIN6 significantly decreased mitochondrial function and morphology, culminating in a decreased capacity in complex I-driven ATP production without affecting cell viability. These alterations were accompanied by an increase in glycolytic fluxes. Additionally, we demonstrate that AntiOxCIN6 sensitized A549 adenocarcinoma cells for CIS-induced apoptotic cell death, while AntiOxCIN6 appears to cause metabolic changes or a redox pre-conditioning on lung MRC-5 fibroblasts, conferring protection against cisplatin. We propose that length and hydrophobicity of the C10-TPP+ alkyl linker play a significant role in inducing mitochondrial and cellular toxicity, while the presence of the antioxidant caffeic acid appears to be responsible for activating cytoprotective pathways.

Unraveling the In Vitro Toxicity Profile of Psychedelic 2C Phenethylamines and Their N-Benzylphenethylamine (NBOMe) Analogues.

Mescaline derivative (2C phenethylamines) drugs have been modified by the introduction of a N-2-methoxybenzyl group to originate a new series of compounds with recognized and potent psychedelic effects, the NBOMe-drugs. Although they are prevalent in unregulated drug markets, their toxicity profile is still poorly understood, despite several reports highlighting cases of acute intoxication, with brain and liver toxicity. Thus, in this study, mescaline, 2C-N (insertion of a nitro in the para position of the 2C phenethylamines aromatic ring) and 2C-B (insertion of a bromide in the para position of the 2C phenethylamines aromatic ring) and their corresponding NBOMe counterparts, mescaline-NBOMe, 25N-NBOMe and 25B-NBOMe, were synthetized and the in vitro neuro- and hepatocytotoxicity evaluated in differentiated SH-SY5Y and HepG2 cell lines, respectively. Cytotoxicity, oxidative stress, metabolic and energetic studies were performed to evaluate the main pathways involved in their toxicity. Our results demonstrated that the presence of the N-2-methoxybenzyl group significantly increased the in vitro cytotoxicity of 2C phenethylamines drugs in both cell lines, with the NBOMe drugs presenting lower EC50 values when compared to their counterparts. Consistently, our data showed a correlation between the drug's lipophilicity and the EC50 values, except for 2C-B. The 2C-B presented higher cytotoxic effects in both cell lines than mescaline-NBOMe, a result that can be explained by its higher passive permeability. All the NBOMe derivatives were able to cross the blood-brain barrier. Considering metabolic studies, the cytotoxicity of these drugs was shown to be influenced by inhibition of cytochrome P450 (CYP), which suggests a potential role of this enzyme complex, especially CYP3A4 and CYP2D6 isoenzymes in SH-SY5Y cells, in their detoxification or bioactivation. Furthermore, in differentiated SH-SY5Y cells, the drugs were able to induce mitochondrial membrane depolarization, and to disrupt GSH and ATP intracellular levels, these effects being concentration dependent and more pronounced for the NBOMe derivatives. No ROS overproduction was detected for any of the drugs in the tested experimental conditions. A correlation between a drug's lipophilicity and the EC50 values in both cell lines, except for 2C-B, was also obtained. In summary, the introduction of a NBOMe moiety to the parent drugs significantly increases their lipophilicity, brain permeability and cytotoxic effects, with GSH and ATP homeostasis disruption. The inhibition of CYP3A4 and CYP2D6 emphasized that CYP-mediated metabolism impacts the toxicity of these drugs.

Phytochemicals and quaternary phosphonium ionic liquids: Connecting the dots to develop a new class of antimicrobial agents.

INTRODUCTION: The infections by multidrug-resistant bacteria are a growing threat to human health, and the efficacy of the available antibiotics is gradually decreasing. As such, new antibiotic classes are urgently needed. OBJECTIVES: This study aims to evaluate the antimicrobial activity, safety and mechanism of action of phytochemical-based triphenylphosphonium (TPP+) conjugates. METHODS: A library of phytochemical-based TPP+ conjugates was repositioned and extended, and its antimicrobial activity was evaluated against a panel of Gram-positive (methicillin-resistant Staphylococcus aureus - MRSA) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii) and fungi (Candida albicans, Cryptococcus neoformans var. grubii). The compounds' cytotoxicity and haemolytic profile were also evaluated. To unravel the mechanism of action of the best compounds, the alterations in the surface charge, bacterial membrane integrity, and cytoplasmic leakage were assessed. RESULTS: Structure-activity-toxicity data revealed the contributions of the different structural components (phenolic ring, carbon-based spacers, carboxamide group, alkyl linker) to the compounds' bioactivity and safety. Dihydrocinnamic derivatives 5 m and 5n stood out as safe, potent and selective antibacterial agents against S. aureus (MIC < 0.25 µg/mL; CC50 > 32 µg/mL; HC10 > 32 µg/mL). Mechanistic studies suggest that the antibacterial activity of compounds 5 m and 5n may result from interactions with the bacterial cell wall and membrane. CONCLUSIONS: Collectively, these studies demonstrate the potential of phytochemical-based TPP+ conjugates as a new class of antibiotics.

Modulating Cytotoxicity with Lego-like Chemistry: Upgrading Mitochondriotropic Antioxidants with Prototypical Cationic Carrier Bricks.

Although the lipophilic triphenylphosphonium (TPP+) cation is widely used to target antioxidants to mitochondria, TPP+-based derivatives have shown cytotoxicity in several biological in vitro models. We confirmed that Mito.TPP is cytotoxic to both human neuronal (SH-SY5Y) and hepatic (HepG2) cells, decreasing intracellular adenosine triphosphate (ATP) levels, leading to mitochondrial membrane depolarization and reduced mitochondrial mass after 24 h. We surpassed this concern using nitrogen-derived cationic carriers (Mito.PICO, Mito.ISOQ, and Mito.IMIDZ). As opposed to Mito.TPP, these novel compounds were not cytotoxic to SH-SY5Y and HepG2 cells up to 50 µM and after 24 h of incubation. All of the cationic derivatives accumulated inside the mitochondrial matrix and acted as neuroprotective agents against iron(III), hydrogen peroxide, and tert-butyl hydroperoxide insults. The overall data showed that nitrogen-based cationic carriers can modulate the biological performance of mitochondria-directed antioxidants and are an alternative to the TPP cation.

Efficacy of Novel Quaternary Ammonium and Phosphonium Salts Differing in Cation Type and Alkyl Chain Length against Antibiotic-Resistant Staphylococcus aureus.

Antibacterial resistance poses a critical public health threat, challenging the prevention and treatment of bacterial infections. The search for innovative antibacterial agents has spurred significant interest in quaternary heteronium salts (QHSs), such as quaternary ammonium and phosphonium compounds as potential candidates. In this study, a library of 49 structurally related QHSs was synthesized, varying the cation type and alkyl chain length. Their antibacterial activities against Staphylococcus aureus, including antibiotic-resistant strains, were evaluated by determining minimum inhibitory/bactericidal concentrations (MIC/MBC) ≤ 64 µg/mL. Structure-activity relationship analyses highlighted alkyl-triphenylphosphonium and alkyl-methylimidazolium salts as the most effective against S. aureus CECT 976. The length of the alkyl side chain significantly influenced the antibacterial activity, with optimal chain lengths observed between C10 and C14. Dose-response relationships were assessed for selected QHSs, showing dose-dependent antibacterial activity following a non-linear pattern. Survival curves indicated effective eradication of S. aureus CECT 976 by QHSs at low concentrations, particularly compounds 1e, 3e, and 5e. Moreover, in vitro human cellular data indicated that compounds 2e, 4e, and 5e showed favourable safety profiles at concentrations ≤ 2 µg/mL. These findings highlight the potential of these QHSs as effective agents against susceptible and resistant bacterial strains, providing valuable insights for the rational design of bioactive QHSs.

Targeting Hydroxybenzoic Acids to Mitochondria as a Strategy to Delay Skin Ageing: An In Vitro Approach.

Targeting antioxidants to mitochondria is considered a promising strategy to prevent cellular senescence and skin ageing. In this study, we investigate whether four hydroxybenzoic acid-based mitochondria-targeted antioxidants (MitoBENs, MB1-4) could be used as potential active ingredients to prevent senescence in skin cells. Firstly, we evaluated the chemical stability, cytotoxicity, genotoxicity and mitochondrial toxicity of all compounds. We followed this by testing the antioxidant protective capacity of the two less toxic compounds on human skin fibroblasts. We then assessed the effects of the best hit on senescence, inflammation and mitochondrial remodeling on a 3D skin cell model, while also testing its mutagenic potential. Cytotoxicity and mitochondrial toxicity rankings were produced: MB3 < MB4 ≃ MB1 < MB2 and MB3 < MB1 < MB4 < MB2, respectively. These results suggest that pyrogallol-based compounds (MB2 and MB4) have lower cytotoxicity. The pyrogallol derivative, MB2, containing a 6-carbon spacer, showed a more potent antioxidant protective activity against hydrogen peroxide cytotoxicity. In a 3D skin cell model, MB2 also decreased transcripts related to senescence. In sum, MB2's biological safety profile, good chemical stability and lack of mutagenicity, combined with its anti-senescence effect, converts MB2 into a good candidate for further development as an active ingredient for skin anti-ageing products.

Mitochondria-targeted anti-oxidant AntiOxCIN4 improved liver steatosis in Western diet-fed mice by preventing lipid accumulation due to upregulation of fatty acid oxidation, quality control mechanism and antioxidant defense systems.

Non-alcoholic fatty liver disease (NAFLD) is a health concern affecting 24% of the population worldwide. Although the pathophysiologic mechanisms underlying disease are not fully clarified, mitochondrial dysfunction and oxidative stress are key players in disease progression. Consequently, efforts to develop more efficient pharmacologic strategies targeting mitochondria for NAFLD prevention/treatment are underway. The conjugation of caffeic acid anti-oxidant moiety with an alkyl linker and a triphenylphosphonium cation (TPP+), guided by structure-activity relationships, led to the development of a mitochondria-targeted anti-oxidant (AntiOxCIN4) with remarkable anti-oxidant properties. Recently, we described that AntiOxCIN4 improved mitochondrial function, upregulated anti-oxidant defense systems, and cellular quality control mechanisms (mitophagy/autophagy) via activation of the Nrf2/Keap1 pathway, preventing fatty acid-induced cell damage. Despite the data obtained, AntiOxCIN4 effects on cellular and mitochondrial energy metabolism in vivo were not studied. In the present work, we proposed that AntiOxCIN4 (2.5 mg/day/animal) may prevent non-alcoholic fatty liver (NAFL) phenotype development in a C57BL/6J mice fed with 30% high-fat, 30% high-sucrose diet for 16 weeks. HepG2 cells treated with AntiOxCIN4 (100 µM, 48 h) before the exposure to supraphysiologic free fatty acids (FFAs) (250 µM, 24 h) were used for complementary studies. AntiOxCIN4 decreased body (by 43%), liver weight (by 39%), and plasma hepatocyte damage markers in WD-fed mice. Hepatic-related parameters associated with a reduction of fat liver accumulation (by 600%) and the remodeling of fatty acyl chain composition compared with the WD-fed group were improved. Data from human HepG2 cells confirmed that a reduction of lipid droplets size and number can be a result from AntiOxCIN4-induced stimulation of fatty acid oxidation and mitochondrial OXPHOS remodeling. In WD-fed mice, AntiOxCIN4 also induced a hepatic metabolism remodeling by upregulating mitochondrial OXPHOS, anti-oxidant defense system and phospholipid membrane composition, which is mediated by the PGC-1α-SIRT3 axis. AntiOxCIN4 prevented lipid accumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity. AntiOxCIN4 improved NAFL phenotype of WD-fed mice, via three main mechanisms: a) increase mitochondrial function (fatty acid oxidation); b) stimulation anti-oxidant defense system (enzymatic and non-enzymatic) and; c) prevent the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/treatment of NAFLD.

Design and synthesis of chromone-based monoamine oxidase B inhibitors with improved drug-like properties.

The absence of disease modifying drugs in Parkinson's disease therapy urges for new chemical entities acting on relevant PD-associated biological targets. As a result, developing selective and reversible inhibitors targeting MAO-B is still a desirable line of therapeutic research. Within this framework, a small library of chromone derivatives was synthesized and screened towards human monoamine oxidases. Structural modifications on the chromone 3-phenylcarboxamide resulted in potent MAO-B inhibitors with an improved drug-like profile, and for the first time we obtained potent and selective chromone 2-phenylcarboxamides acting in the low nanomolar range. Compounds 5-hydroxy-4-oxo-N-phenyl-4H-chromene-3-carboxamide (38) (IC50 = 13.0 nM) and N-(4-chlorophenyl)-5-hydroxy-4-oxo-4H-chromene-3-carboxamide (41) (IC50 = 8.3 nM) stood out as reversible, potent, selective and non-cytotoxic MAO-B inhibitors bearing a favourable drug-like profile. Both compounds displayed cytoprotective effects towards iron(III) oxidative stressor.

Effect of a Novel Hydroxybenzoic Acid Based Mitochondria Directed Antioxidant Molecule on Bovine Sperm Function and Embryo Production.

Sperm cells are particularly vulnerable to reactive oxygen species (ROS), impairing their fertilizing ability. Our objective was to study the effect of a novel mitochondrial-directed antioxidant, AntiOxBEN2, on bovine sperm function. This antioxidant was added to the semen capacitation medium (CAP), during the swim-up process, and to the fertilization medium (FERT) during the co-incubation of matured oocytes and capacitated spermatozoa, in concentrations of 0 (control), 1, and 10 µM. After the swim-up, sperm motility (CASA and visual analysis), vitality (eosin-nigrosin), mitochondrial membrane potential (JC1), intracellular ROS, adenosine triphosphate (ATP) levels, and basal metabolism (Seahorse Xfe96) were evaluated. Embryo development and quality were also assessed. Higher cleavage rates were obtained when 1 µM AntiOxBEN2 were added to CAP and FERT media (compared to control, p < 0.04). A positive effect of AntiOxBEN2 on intracellular ROS reduction (p = 0.01), on the increment of mitochondrial membrane potential (p ≤ 0.003) and, consequently, on the sperm quality was identified. However, the highest dose impaired progressive motility, ATP production, and the number of produced embryos. The results demonstrate a beneficial effect of AntiOxBEN2 (1 µM) on sperm capacitation and fertilization processes, thus improving embryonic development. This may constitute a putative novel therapeutic strategy to improve the outcomes of assisted reproductive techniques (ART).

Mitochondriotropic antioxidant based on caffeic acid AntiOxCIN4 activates Nrf2-dependent antioxidant defenses and quality control mechanisms to antagonize oxidative stress-induced cell damage.

Mitochondria are key organelles involved in cellular survival, differentiation, and death induction. In this regard, mitochondrial morphology and/or function alterations are involved in stress-induced adaptive pathways, priming mitochondria for mitophagy or apoptosis induction. We have previously shown that the mitochondriotropic antioxidant AntiOxCIN4 (100 µM; 48 h) presented significant cytoprotective effect without affecting the viability of human hepatoma-derived (HepG2) cells. Moreover, AntiOxCIN4 (12.5 µM; 72 h) caused a mild increase of reactive oxygen species (ROS) levels without toxicity to primary human skin fibroblasts (PHSF). As Nrf2 is a master regulator of the oxidative stress response inducing antioxidant-encoding gene expression, we hypothesized that AntiOxCIN4 could increase the resistance of human hepatoma-derived HepG2 to oxidative stress by Nrf2-dependent mechanisms, in a process mediated by mitochondrial ROS (mtROS). Here we showed that after an initial decrease in oxygen consumption paralleled by a moderate increase in superoxide anion levels, AntiOxCIN4 led to a time-dependent Nrf2 translocation to the nucleus. This was followed later by a 1.5-fold increase in basal respiration and a 1.2-fold increase in extracellular acidification. AntiOxCIN4 treatment enhanced mitochondrial quality by triggering the clearance of defective organelles by autophagy and/or mitophagy, coupled with increased mitochondrial biogenesis. AntiOxCIN4 also up-regulated the cellular antioxidant defense system. AntiOxCIN4 seems to have the ability to maintain hepatocyte redox homeostasis, regulating the electrophilic/nucleophilic tone, and preserve cellular physiological functions. The obtained data open a new avenue to explore the effects of AntiOxCIN4 in the context of preserving hepatic mitochondrial function in disorders, such as NASH/NAFLD and type II diabetes.

A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients.

Parkinson's Disease (PD) is a neurodegenerative disorder affecting more than 10 million people worldwide. Currently, PD has no cure and no early diagnostics methods exist. Mitochondrial dysfunction is presented in the early stages of PD, and it is considered an important pathophysiology component. We have previously developed mitochondria-targeted hydroxycinnamic acid derivatives, presenting antioxidant and iron-chelating properties, and preventing oxidative stress in several biological models of disease. We have also demonstrated that skin fibroblasts from male sporadic PD patients (sPD) presented cellular and mitochondrial alterations, including increased oxidative stress, hyperpolarized and elongated mitochondria and decreased respiration and ATP levels. We also showed that forcing mitochondrial oxidative phosphorylation (OXPHOS) in sPD fibroblasts uncovers metabolic defects that were otherwise hidden. In this work, we tested the hypothesis that a lead mitochondria-targeted hydroxycinnamic acid derivative would revert the phenotype found in skin fibroblasts from sPD patients. Our results demonstrated that treating human skin fibroblasts from sPD patients with non-toxic concentrations of AntiOxCIN4 restored mitochondrial membrane potential and mitochondrial fission, decreased autophagic flux, and enhanced cellular responses to stress by improving the cellular redox state and decreasing reactive oxygen species (ROS) levels. Besides, fibroblasts from sPD patients treated with AntiOxCIN4 showed increased maximal respiration and metabolic activity, converting sPD fibroblasts physiologically more similar to their sex- and age-matched healthy controls. The positive compound effect was reinforced using a supervised machine learning model, confirming that AntiOxCIN4 treatment converted treated fibroblasts from sPD patients closer to the phenotype of control fibroblasts. Our data points out a possible mechanism of AntiOxCIN4 action contributing to a deeper understanding of how the use of mitochondria-targeted antioxidants based on a polyphenol scaffold can be used as potential drug candidates for delaying PD progression, validating the use of fibroblasts from sPD patients with more active OXPHOS as platforms for mitochondria-based drug development.

Bridging the Gap Between Nature and Antioxidant Setbacks: Delivering Gallic Acid to Mitochondria.

Research on mitochondria-targeted active molecules became a hot topic in the past decade. Development of mitochondria permeability transition pore (mPTP )-targeting agents with clinical applications is needed not only because of the importance of the target in several diseases but also due to the fact that the current developed molecules have shown poor clinical success. In fact, only a reduced percentage reach mitochondria , effectively preventing pathological mPTP opening. The mitochondrial-targeting strategies should be a promising solution to increase the selectivity of compounds to the mPTP , reducing also their potential side effects. Chemical conjugation of bioactive molecules with a lipophilic cation such as the triphenylphosphonium (TPP +) has been established as a robust strategy to specifically target mitochondria . Phytochemicals such as hydroxybenzoic acids are normal constituents of the human diet. These molecules display beneficial healthy effects, ranging from antioxidant action through diverse mechanisms to modulation of mitochondrial-related apoptotic system, although their therapeutic application is limited due to pharmacokinetic drawbacks. Accordingly, the development of a new antioxidant based on the dietary benzoic acid-gallic acid -is described as well as the demonstration of its mitochondriotropic characteristics.

Fine-Tuning the Biological Profile of Multitarget Mitochondriotropic Antioxidants for Neurodegenerative Diseases.

Neurotransmitter depletion and mitochondrial dysfunction are among the multiple pathological events that lead to neurodegeneration. Following our previous studies related with the development of multitarget mitochondriotropic antioxidants, this study aims to evaluate whether the π-system extension on the chemical scaffolds of AntiOXCIN2 and AntiOXCIN3 affects their bioactivity and safety profiles. After the synthesis of four triphenylphosphonium (TPP+) conjugates (compounds 2-5), we evaluated their antioxidant properties and their effect on neurotransmitter-metabolizing enzymes. All compounds were potent equine butyrylcholinesterase (eqBChE) and moderate electric eel acetylcholinesterase (eeAChE) inhibitors, with catechols 4 and 5 presenting lower IC50 values than AntiOXCIN2 and AntiOXCIN3, respectively. However, differences in the inhibition potency and selectivity of compounds 2-5 towards non-human and human cholinesterases (ChEs) were observed. Co-crystallization studies with compounds 2-5 in complex with human ChEs (hChEs) showed that these compounds exhibit different binging modes to hAChE and hBChE. Unlike AntiOXCINs, compounds 2-5 displayed moderate human monoamine oxidase (hMAO) inhibitory activity. Moreover, compounds 4 and 5 presented higher ORAC-FL indexes and lower oxidation potential values than the corresponding AntiOXCINs. Catechols 4 and 5 exhibited broader safety windows in differentiated neuroblastoma cells than benzodioxole derivatives 2 and 3. Compound 4 is highlighted as a safe mitochondria-targeted antioxidant with dual ChE/MAO inhibitory activity. Overall, this work is a contribution for the development of dual therapeutic agents addressing both mitochondrial oxidative stress and neurotransmitter depletion.

Design and characterization of Nanostructured lipid carriers (NLC) and Nanostructured lipid carrier-based hydrogels containing Passiflora edulis seeds oil.

This study aims to design and characterize Nanostructured lipid carriers (NLC) and Nanostructured lipid carrier-based hydrogels with Passiflora edulis seeds oil, a by-product from Madeira Island food industry. NLC were prepared by the ultrasonication technique, using passion fruit seeds oil as a liquid lipid and glyceryl distearate as a solid lipid. These NLC were then gelled with Poly (acrylic acid). Long-term stability studies were conducted with NLC and NLC-based hydrogels stored for 12 months. The following tests were performed: morphology, encapsulation efficiency, particle size analysis, polydispersity index analysis, zeta potential, pH measurement, color analysis, viscosity studies, texture analysis, in vitro occlusion test, ex vivo skin penetration study, tyrosinase inhibition activity, in vitro skin permeation experiments and in vitro cytotoxicity studies. The developed NLC had spherical shape and narrow particle sizes distribution with mean sizes in the range of 150 nm and PDI below 0.3, Zeta potential values around -30 mV and high Encapsulation efficiency. The tyrosinase inhibitory activity and skin retention of the nanoparticles was superior to that of the non-encapsulated oil. The developed formulations did not show cytotoxicity towards HaCat cells and presented suitable viscosity and texture properties for skin application, proving to be good candidates as depigmenting agent.

Biofilm control by ionic liquids.

Ionic liquids (ILs) are remarkable chemical compounds with applications in many areas of modern science. They are increasingly recognized as promising compounds to fight microorganisms in both planktonic and biofilm states, contributing to reinvent the antimicrobial pipeline. Biofilm-related infections are particularly challenging given that the scientific community has not yet identified a reliable control strategy. Understanding of the action of ILs in biofilm control is is still in a very early stage. However, given the highly tunable nature and exceptional properties of ILs, they are excellent candidates for biofilm control. Here, we review the major advances in, and challenges tothe use of ILs for effective biofilm control.

Lipid Nanosystems and Serum Protein as Biomimetic Interfaces: Predicting the Biodistribution of a Caffeic Acid-Based Antioxidant.

PURPOSE: AntiOxCIN3 is a novel mitochondriotropic antioxidant developed to minimize the effects of oxidative stress on neurodegenerative diseases. Prior to an investment in pre-clinical in vivo studies, it is important to apply in silico and biophysical cell-free in vitro studies to predict AntiOxCIN3 biodistribution profile, respecting the need to preserve animal health in accordance with the EU principles (Directive 2010/63/EU). Accordingly, we propose an innovative toolbox of biophysical studies and mimetic models of biological interfaces, such as nanosystems with different compositions mimicking distinct membrane barriers and human serum albumin (HSA). METHODS: Intestinal and cell membrane permeation of AntiOxCIN3 was predicted using derivative spectrophotometry. AntiOxCIN3 -HSA binding was evaluated by intrinsic fluorescence quenching, synchronous fluorescence, and dynamic/electrophoretic light scattering. Steady-state and time-resolved fluorescence quenching was used to predict AntiOxCIN3-membrane orientation. Fluorescence anisotropy, synchrotron small- and wide-angle X-ray scattering were used to predict lipid membrane biophysical impairment caused by AntiOxCIN3 distribution. RESULTS AND DISCUSSION: We found that AntiOxCIN3 has the potential to permeate the gastrointestinal tract. However, its biodistribution and elimination from the body might be affected by its affinity to HSA (>90%) and by its steady-state volume of distribution (VDSS =1.89± 0.48 L∙Kg-1). AntiOxCIN3 is expected to locate parallel to the membrane phospholipids, causing a bilayer stiffness effect. AntiOxCIN3 is also predicted to permeate through blood-brain barrier and reach its therapeutic target - the brain. CONCLUSION: Drug interactions with biological interfaces may be evaluated using membrane model systems and serum proteins. This knowledge is important for the characterization of drug partitioning, positioning and orientation of drugs in membranes, their effect on membrane biophysical properties and the study of serum protein binding. The analysis of these interactions makes it possible to collect valuable knowledge on the transport, distribution, accumulation and, eventually, therapeutic impact of drugs which may aid the drug development process.

Mitochondria-targeted phenolic antioxidants induce ROS-protective pathways in primary human skin fibroblasts.

Phytochemical antioxidants like gallic and caffeic acid are constituents of the normal human diet that display beneficial health effects, potentially via activating stress response pathways. Using primary human skin fibroblasts (PHSFs) as a model, we here investigated whether such pathways were induced by novel mitochondria-targeted variants of gallic acid (AntiOxBEN2) and caffeic acid (AntiOxCIN4). Both molecules reduced cell viability with similar kinetics and potency (72 h incubation, IC50 ~23 µM). At a relatively high but non-toxic concentration (12.5 µM), AntiOxBEN2 and AntiOxCIN4 increased ROS levels (at 24 h), followed by a decline (at 72 h). Further analysis at the 72 h timepoint demonstrated that AntiOxBEN2 and AntiOxCIN4 did not alter mitochondrial membrane potential (Δψ), but increased cellular glutathione (GSH) levels, mitochondrial NAD(P)H autofluorescence, and mitochondrial superoxide dismutase 2 (SOD2) protein levels. In contrast, cytosolic SOD1 protein levels were not affected. AntiOxBEN2 and AntiOxCIN4 both stimulated the gene expression of Nuclear factor erythroid 2-related factor 2 (NRF2; a master regulator of the cellular antioxidant response toward oxidative stress). AntiOxBEN2 and ANtiOxCIN4 differentially affected the gene expression of the antioxidants Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1). Both antioxidants did not protect from cell death induced by GSH depletion and AntiOxBEN2 (but not AntiOxCIN4) antagonized hydrogen peroxide-induced cell death. We conclude that AntiOxBEN2 and AntiOxCIN4 increase ROS levels, which stimulates NRF2 expression and, as a consequence, SOD2 and GSH levels. This highlights that AntiOxBEN2 and AntiOxCIN4 can act as prooxidants thereby activating endogenous ROS-protective pathways.

Surface Wiping Test to Study Biocide -Cinnamaldehyde Combination to Improve Efficiency in Surface Disinfection.

Disinfection is crucial to control and prevent microbial pathogens on surfaces. Nonetheless, disinfectants misuse in routine disinfection has increased the concern on their impact on bacterial resistance and cross-resistance. This work aims to develop a formulation for surface disinfection based on the combination of a natural product, cinnamaldehyde, and a widely used biocide, cetyltrimethylammonium bromide. The wiping method was based on the Wiperator test (ASTM E2967-15) and the efficacy evaluation of surface disinfection wipes test (EN 16615:2015). After formulation optimization, the wiping of a contaminated surface with 6.24 log10 colony-forming units (CFU) of Escherichia coli or 7.10 log10 CFU of Staphylococcus aureus led to a reduction of 4.35 log10 CFU and 4.27 log10 CFU when the wipe was impregnated with the formulation in comparison with 2.45 log10 CFU and 1.50 log10 CFU as a result of mechanical action only for E. coli and S. aureus, respectively. Furthermore, the formulation prevented the transfer of bacteria to clean surfaces. The work presented highlights the potential of a combinatorial approach of a classic biocide with a phytochemical for the development of disinfectant formulations, with the advantage of reducing the concentration of synthetic biocides, which reduces the potentially negative environmental and public health impacts from their routine use.

Optimizing the Synthetic Route of Chromone-2-carboxylic Acids: A Step forward to Speed-Up the Discovery of Chromone-Based Multitarget-Directed Ligands.

6-Bromochromone-2-carboxylic acid (3) was synthesized by a microwave-assisted process. The optimization of the reaction was performed varying parameters, such as type of base/number of reagent equivalents, solvent, temperature and reaction time. The yield of the reaction was improved to 87%. The new synthetic route is versatile as several chromone-2-carboxylic acids (compounds 4B-10B) were obtained with good yields (54-93%). Only in the case of the nitro substituent (compound 11B), an ester was obtained instead of the desired carboxylic acid. Following this synthetic route chromone carboxylic acids can be attained with a high degree of purity, without the need of the tedious and expensive purification processes through column chromatography. The reaction is safe, cost-effective, fast and robust, and can be used in the development of concise and diversity-oriented libraries based on chromone scaffold. The overall study can be looked as a step forward to speed-up the discovery of chromone-based multitarget-directed ligands.