Novel Probucol Analogue, 4,4'-Diselanediylbis (2,6-Di-tert-Butylphenol), Prevents Oxidative Glutamate Neurotoxicity In Vitro and Confers Neuroprotection in a Rodent Model of Ischemic Stroke.

Oxidative glutamate toxicity is regarded as one of the injurious mechanisms associated with ischemic stroke, which represents a major health problem and requires improved pharmacological treatments. We designed and synthesized two new probucol analogues [2,6-di-tert-butyl-4-selenocyanatophenol (C1) and 4,4'-diselanediylbis (2,6-di-tert-butylphenol) (C2)] and investigated their effects against glutamate-induced neuronal oxidative toxicity in vitro in cultured HT22 cells, compared with their parental compound (probucol). In addition, C2, which exhibited the lowest toxicity, was investigated in an in vivo rodent model of ischemic stroke. Glutamate caused concentration- and time-dependent cytotoxicity in HT22 neuronal cells, which was preceded by increased levels of oxidants and depletion of the antioxidant glutathione. The analogues (C1 and C2), but not probucol, significantly decreased the levels of oxidants (including mitochondrial superoxide anion and lipid reactive oxygen species (ROS)) and protected against glutamate-induced cytotoxicity. In the in vivo model of ischemic stroke, which was based on central injections of the vasoconstrictor agent endothelin-1 (800 pmol/site), C2 (20 or 50 mg/kg/day, intraperitoneally, for 4 consecutive days after stroke) displayed significant beneficial effects against ischemic injury in vivo, improving rats' motor-related behavioral skills and decreasing stroke-related striatal gliosis. This is the first study to design, synthesize, and present a probucol analogue (C2) with in vivo beneficial effects against ischemic stroke. This novel compound, which was able to mitigate glutamate-induced oxidative toxicity in vitro, represents a promising neuroprotective drug.

Dihydropyrimidinone-derived selenoesters efficacy and safety in an in vivo model of Aß aggregation.

In a previous in vitro study, dihydropyrimidinone-derived selenoesteres demonstrated antioxidant properties, metal chelators and inhibitory acetylcholinesterase (AChE) activity, making these compounds promising candidates for Alzheimer's Disease (AD) treatment. However, these effects have yet to be demonstrated in an in vivo animal model; therefore, this study aimed to evaluate the safety and efficacy of eight selenoester compounds in a Caenorhabditis elegans model using transgenic strains for amyloid-beta peptide (Aß) aggregation. The L1 stage worms were acutely exposed (30 min) to the compounds at concentrations ranging from 5 to 200 µM and after 48 h the maintenance temperature was increased to 25 ° C for Aß expression and aggregation. After 48 h, several parameters related to phenotypic manifestations of Aß toxicity and mechanistic elucidation were analyzed. At the concentrations tested no significant toxicity of the compounds was found. The selenoester compound FA90 significantly reduced the rate of paralyzed worms and increased the number of swimming movements compared to the untreated worms. In addition, FA90 and FA130 improved egg-laying induced by levamisole and positively modulated HSP-6 and HSP-4 expression, thereby increasing reticular and mitochondrial protein folding response in C. elegans, which could attenuate Aß aggregation in early exposure. Therefore, our initial screening using an alternative model demonstrated that FA90, among the eight selenoesters evaluated, was the most promising compound for AD evaluation screening in more complex animals.

Prevention of ferroptosis in acute scenarios: an in vitro study with classic and novel anti-ferroptotic compounds.

Ferroptosis, an iron-dependent form of cell death, has critical roles in diverse pathologies. Data on the temporal events mediating the prevention of ferroptosis are lacking. Focused on temporal aspects of cytotoxicity/protection, we investigated the effects of classic (Fer-1) and novel [2,6-di-tert-butyl-4-(2-thienylthio)phenol (C1) and 2,6-di-tert-butyl-4-(2-thienylselano)phenol (C2)] anti-ferroptotic agents against RSL3-, BSO- or glutamate-induced ferroptosis in cultured HT22 neuronal cell line, comparing their effects with those of the antioxidants trolox, ebselen and probucol. Glutamate (5 mM), BSO (25 µM) and RSL3 (50 nM) decreased approximately 40% of cell viability at 24 h. At these concentrations, none of these agents changed cell viability at 6 h after treatments; RSL3 increased lipoperoxidation from 6 h, although BSO and glutamate only did so at 12 h after treatments. At similar conditions, BSO and glutamate (but not RSL3) decreased GSH levels at 6 h after treatments. Fer-1, C1 and C2 exhibited similar protective effects against glutamate-, BSO- and RSL3-cytotoxicity, but this protection was limited when the protective agents were delivered to cells at time-points characterized by increased lipoperoxidation (but not glutathione depletion). Compared to Fer-1, C1 and C2, the anti-ferroptotic effects of trolox, ebselen and probucol were minor. Cytoprotective effects were not associated with direct antioxidant efficacies. These results indicate that the temporal window is central in affecting the efficacies of anti-ferroptotic drugs in acute scenarios; ferroptosis prevention is improbable when significant rates of lipoperoxidation were already achieved. C1 and C2 displayed remarkable cytoprotective effects, representing a promising new class of compounds to treat ferroptosis-related pathologies.

New Probucol Analogues Inhibit Ferroptosis, Improve Mitochondrial Parameters, and Induce Glutathione Peroxidase in HT22 Cells.

Probucol, a hypocholesterolemic compound, is neuroprotective in several models of neurodegenerative diseases but has serious adverse effects in vivo. We now describe the design and synthesis of two new probucol analogues that protect against glutamate-induced oxidative cell death, also known as ferroptosis, in cultured mouse hippocampal (HT22) cells and in primary cortical neurons, while probucol did not show any protective effect. Treatment with both compounds did not affect glutathione depletion but still significantly decreased glutamate-induced production of oxidants, mitochondrial superoxide generation, and mitochondrial hyperpolarization in HT22 cells. Both compounds increase glutathione peroxidase (GPx) 1 levels and GPx activity, also exhibiting protection against RSL3, a GPx4 inactivator. These two compounds are therefore potent activators of GPx activity making further studies of their neuroprotective activity in vivo worthwhile.

Design, Synthesis, and In Vitro Evaluation of a Novel Probucol Derivative: Protective Activity in Neuronal Cells Through GPx Upregulation.

Recent studies have shown that probucol (PB), a hipocholesterolemic agent with antioxidant and anti-inflammatory properties, presents neuroprotective properties. On the other hand, adverse effects have limited PB's clinical application. Thus, the search for PB derivatives with no or less adverse effects has been a topic of research. In this study, we present a novel organoselenium PB derivative (RC513) and investigate its potential protective activity in an in vitro experimental model of oxidative toxicity induced by tert-butyl hydroperoxide (tBuOOH) in HT22 neuronal cells, as well as exploit potential protective mechanisms. tBuOOH exposure caused a significant decrease in the cell viability, which was preceded by (i) increased reactive species generation and (ii) decreased mitochondrial maximum oxygen consumption rate. RC513 pretreatment (48 h) significantly prevented the tBuOOH-induced decrease of cell viability, RS generation, and mitochondrial dysfunction. Of note, RC513 significantly increased glutathione peroxidase (GPx) activity and mRNA expression of GPx1, a key enzyme involved in peroxide detoxification. The use of mercaptosuccinic acid, an inhibitor of GPx, significantly decreased the protective activity of RC513 against tBuOOH-induced cytotoxicity in HT22 cells, highlighting the importance of GPx upregulation in the observed protection. In summary, the results showed a significant protective activity of a novel PB derivative against tBuOOH-induced oxidative stress and mitochondrial dysfunction, which was related to the upregulation of GPx. Our results point to RC513 as a promising neuroprotective molecule, even though studies concerning potential beneficial effects and safety aspects of RC513 under in vivo conditions are well warranted.