Drug Development for Alzheimer's and Parkinson's Disease: Where Do We Go Now?

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.

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.

Research Models to Study Ferroptosis's Impact in Neurodegenerative Diseases.

Ferroptosis is a type of regulated cell death promoted by the appearance of oxidative perturbations in the intracellular microenvironment constitutively controlled by glutathione peroxidase 4 (GPX4). It is characterized by increased production of reactive oxygen species, intracellular iron accumulation, lipid peroxidation, inhibition of system Xc-, glutathione depletion, and decreased GPX4 activity. Several pieces of evidence support the involvement of ferroptosis in distinct neurodegenerative diseases. In vitro and in vivo models allow a reliable transition to clinical studies. Several in vitro models, including differentiated SH-SY5Y and PC12 cells, among others, have been used to investigate the pathophysiological mechanisms of distinct neurodegenerative diseases, including ferroptosis. In addition, they can be useful in the development of potential ferroptosis inhibitors that can be used as disease-modifying drugs for the treatment of such diseases. On the other hand, in vivo models based on the manipulation of rodents and invertebrate animals, such as Drosophila melanogaster, Caenorhabditis elegans, and zebrafish, have been increasingly used for research in neurodegeneration. This work provides an up-to-date review of the main in vitro and in vivo models that can be used to evaluate ferroptosis in the most prevalent neurodegenerative diseases, and to explore potential new drug targets and novel drug candidates for effective disease-modifying therapies.

Molecular mechanisms of ferroptosis and their involvement in brain diseases.

Ferroptosis is a type of regulated cell death characterized by intracellular accumulation of iron and reactive oxygen species, inhibition of system Xc-, glutathione depletion, nicotinamide adenine dinucleotide phosphate oxidation and lipid peroxidation. Since its discovery and characterization in 2012, many efforts have been made to reveal the underlying mechanisms, modulating compounds, and its involvement in disease pathways. Ferroptosis inducers include erastin, sorafenib, sulfasalazine and glutamate, which, by inhibiting system Xc-, prevent the import of cysteine into the cells. RSL3, statins, Ml162 and Ml210 induce ferroptosis by inhibiting glutathione peroxidase 4 (GPX4), which is responsible for preventing the formation of lipid peroxides, and FIN56 and withaferin trigger GPX4 degradation. On the other side, ferroptosis inhibitors include ferrostatin-1, liproxstatin-1, α-tocopherol, zileuton, FSP1, CoQ10 and BH4, which interrupt the lipid peroxidation cascade. Additionally, deferoxamine, deferiprone and N-acetylcysteine, by targeting other cellular pathways, have also been classified as ferroptosis inhibitors. Increased evidence has established the involvement of ferroptosis in distinct brain diseases, including Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, multiple sclerosis, and Friedreich's ataxia. Thus, a deep understanding of how ferroptosis contributes to these diseases, and how it can be modulated, can open a new window of opportunities for novel therapeutic strategies and targets. Other studies have shown a sensitivity of cancer cells with mutated RAS to ferroptosis induction and that chemotherapeutic agents and ferroptosis inducers synergize in tumor treatment. Thus, it is tempting to consider that ferroptosis may arise as a target mechanistic pathway for the treatment of brain tumors. Therefore, this work provides an up-to-date review on the molecular and cellular mechanisms of ferroptosis and their involvement in brain diseases. In addition, information on the main ferroptosis inducers and inhibitors and their molecular targets is also provided.

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.

Boosting caffeic acid performance as antioxidant and monoamine oxidase B/catechol-O-methyltransferase inhibitor.

Increased oxidative stress (OS) and depletion of nigrostriatal dopamine (DA) are closely linked to the neurodegeneration observed in Parkinson's Disease (PD). Caffeic acid (CA)-based antioxidants were developed, and their inhibitory activities towards monoamine oxidases (MAOs) and catechol O-methyltransferases (COMT) were screened. The results showed that the incorporation of an extra double bond maintained or even boosted the antioxidant properties of CA. α-CN derivatives displayed redox potentials (Ep) similar to CA (1) and inhibited hMAO-B with low µM IC50 values. Moreover, catechol amides acted as MB-COMT inhibitors, showing IC50 values within the low µM range. In general, CA derivatives presented safe cytotoxicity profiles at concentrations up to 10 µM. The formation of reactive oxygen species (ROS) induced by CA derivatives may be underlying the cytotoxic effects observed at higher concentrations. Catechol amides 3-6, 8-11 at 10 µM protected cells against oxidative damage. Compounds 3 and 8 were predicted to cross the blood-brain barrier (BBB) by passive diffusion. In summary, we report for the first time BBB-permeant CA-based multitarget lead compounds that may restore DAergic neurotransmission (dual hMAO-B/MB-COMT inhibition) and prevent oxidative damage. The data represents a groundbreaking advancement towards the discovery of the next generation of new drugs for PD.

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.

In Vitro Effects of Mitochondria-Targeted Antioxidants in a Small-Cell Carcinoma of the Ovary of Hypercalcemic Type and in Type 1 and Type 2 Endometrial Cancer.

Small-cell carcinoma of the ovary of hypercalcemic type (SCCOHT) and endometrial cancer from type 1 and type 2 are gynecological tumors that affect women worldwide. The treatment encompasses the use of cytotoxic drugs that are nonspecific and inefficient. "Mitocans", a family of drugs that specifically target tumor cells' mitochondria, might be a solution, as they conjugate compounds, such as antioxidants, with carriers, such as lipophilic cations, that direct them to the mitochondria. In this study, caffeic acid was conjugated with triphenylphosphonium (TPP), 4-picolinium, or isoquinolinium, forming 3 new compounds (Mito6_TPP, Mito6_picol., and Mito6_isoq.) that were tested on ovarian (COV434) and endometrial (Hec50co and Ishikawa) cancer cells. The results of MTT and neutral red assays suggested a time- and concentration-dependent decrease in cell viability in all tumor cell lines. The presence of apoptosis was indicated by the Giemsa and Höechst staining and by the decrease in mitochondrial membrane potential. The measurement of intracellular reactive oxygen species demonstrated the antioxidant properties of these compounds, which might be related to cell death. Generally, Mito6_TPP was more active at lower concentrations than Mito6_picol. or Mito6_isoq., but was accompanied by more cytotoxic effects, as shown by the lactate dehydrogenase release. Non-tumorous cells (HFF-1) showed no changes after treatment. This study assessed the potential of these compounds as anticancer agents, although further investigation is needed.

Molecular Modeling and Experimental Evaluation of Non-Chiral Components of Bergamot Essential Oil with Inhibitory Activity against Human Monoamine Oxidases.

Human monoamine oxidases (hMAOs) are well-established targets for the treatment of neurological disorders such as depression, Parkinson's disease and Alzheimer's disease. Despite the efforts carried out over the years, few selective and reversible MAO inhibitors are on the market. Thus, a continuous search for new compounds is needed. Herein, MAO inhibitors were searched among the non-chiral constituents of Bergamot Essential Oil (BEO) with the aid of computational tools. Accordingly, molecular modeling simulations were carried out on both hMAO-A and hMAO-B for the selected constituents. The theoretically predicted target recognition was then used to select the most promising compounds. Among the screened compounds, Bergamottin, a furocoumarin, showed selective hMAO-B inhibitory activity, fitting its active site well. Molecular dynamics simulations were used to deeply analyze the target recognition and to rationalize the selectivity preference. In agreement with the computational results, experimental studies confirmed both the hMAO inhibition properties of Bergamottin and its preference for the isoform B.

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.

Cytotoxicity and Mitochondrial Effects of Phenolic and Quinone-Based Mitochondria-Targeted and Untargeted Antioxidants on Human Neuronal and Hepatic Cell Lines: A Comparative Analysis.

Mitochondriotropic antioxidants (MC3, MC6.2, MC4 and MC7.2) based on dietary antioxidants and analogs (caffeic, hydrocaffeic, trihydroxyphenylpropanoic and trihydroxycinnamic acids) were developed. In this study, we evaluate and compare the cytotoxicity profile of novel mitochondria-targeted molecules (generally known as MitoCINs) on human HepG2 and differentiated SH-SY5Y cells with the quinone-based mitochondria-targeted antioxidants MitoQ and SkQ1 and with two non-targeted antioxidants, resveratrol and coenzyme Q10 (CoQ10). We further evaluate their effects on mitochondrial membrane potential, cellular oxygen consumption and extracellular acidification rates. Overall, MitoCINs derivatives reduced cell viability at concentrations about six times higher than those observed with MitoQ and SkQ1. A toxicity ranking for both cell lines was produced: MC4 < MC7.2 < MC3 < MC6.2. These results suggest that C-6 carbon linker and the presence of a pyrogallol group result in lower cytotoxicity. MC3 and MC6.2 affected the mitochondrial function more significantly relative to MitoQ, SkQ1, resveratrol and CoQ10, while MC4 and MC7.2 displayed around 100-1000 times less cytotoxicity than SkQ1 and MitoQ. Based on the mitochondrial and cytotoxicity cellular data, MC4 and MC7.2 are proposed as leads that can be optimized to develop safe drug candidates with therapeutic application in mitochondrial oxidative stress-related diseases.

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.

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.

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.

Exploring the Multi-Target Performance of Mitochondriotropic Antioxidants against the Pivotal Alzheimer's Disease Pathophysiological Hallmarks.

Alzheimer disease (AD) is the most common neurodegenerative disease featuring progressive and degenerative neurological impairments resulting in memory loss and cognitive decline. The specific mechanisms underlying AD are still poorly understood, but it is suggested that a deficiency in the brain neurotransmitter acetylcholine, the deposition of insoluble aggregates of fibrillar ß-amyloid 1-42 (Aß42), and iron and glutamate accumulation play an important role in the disease progress. Despite the existence of approved cholinergic drugs, none of them demonstrated effectiveness in modifying disease progression. Accordingly, the development of new chemical entities acting on more than one target is attracting progressively more attention as they can tackle intricate network targets and modulate their effects. Within this endeavor, a series of mitochondriotropic antioxidants inspired on hydroxycinnamic (HCA's) scaffold were synthesized, screened toward cholinesterases and evaluated as neuroprotectors in a differentiated human SH-SY5Y cell line. From the series, compounds 7 and 11 with a 10-carbon chain can be viewed as multi-target leads for the treatment of AD, as they act as dual and bifunctional cholinesterase inhibitors and prevent the neuronal damage caused by diverse aggressors related to protein misfolding and aggregation, iron accumulation and excitotoxicity.