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.

Toxicity of organochalcogens in human leukocytes is associated, but not directly related with reactive species production, apoptosis and changes in antioxidant gene expression.

Selenium (Se) containing organic compounds, such as ebselen (Ebs) and diphenyl diselenide [(PhSe)2], have been used as pharmacological agents due to their antioxidant properties. Tellurium (Te) does not have any biological function in mammals, but Te-containing organic compounds, such as diphenyl ditelluride [(PhTe)2], has been used both as an antioxidant or neurotoxic agent. At high concentrations, these compounds cause toxicity by oxidising thiol and selenol groups of proteins. Here, we analysed whether these compounds could modulate reactive species (RS) production, apoptosis and antioxidant gene expression profile of some selenoproteins and antioxidant enzymes or transcription factors in leukocytes isolated from human blood. Since no data is available about their accumulation in isolated leukocytes, we determine their concentration in the cells by CG-MS. Apoptosis (propidium iodide) and RS production (dichloro fluorescein) were determined by flow cytometry. The expression of CAT, SOD1, GPX3, GPX4, TRXR1, and NFLE2L2 genes were analysed by RT-PCR. (PhTe)2 was the only compound able to increase apoptosis rate. (PhSe)2 altered the expression of CAT and SOD1, and this was associated with a high RS production. All compounds decreased the expression of GPX3 but did not alter GPX4 and TRXR1 expression. All compounds decreased NFE2L2 expression (Ebs > (PhTe)2> (PhSe)2). We hypothesise that the toxicity induced by these organochalcogens is not directly related to their ability of inducing RS production.

Virtual screening of acetylcholinesterase inhibitors using the Lipinski's rule of five and ZINC databank.

Alzheimer's disease (AD) is a progressive and neurodegenerative pathology that can affect people over 65 years of age. It causes several complications, such as behavioral changes, language deficits, depression, and memory impairments. One of the methods used to treat AD is the increase of acetylcholine (ACh) in the brain by using acetylcholinesterase inhibitors (AChEIs). In this study, we used the ZINC databank and the Lipinski's rule of five to perform a virtual screening and a molecular docking (using Auto Dock Vina 1.1.1) aiming to select possible compounds that have quaternary ammonium atom able to inhibit acetylcholinesterase (AChE) activity. The molecules were obtained by screening and further in vitro assays were performed to analyze the most potent inhibitors through the IC50 value and also to describe the interaction models between inhibitors and enzyme by molecular docking. The results showed that compound D inhibited AChE activity from different vertebrate sources and butyrylcholinesterase (BChE) from Equus ferus (EfBChE), with IC50 ranging from 1.69 ± 0.46 to 5.64 ± 2.47 µM. Compound D interacted with the peripheral anionic subsite in both enzymes, blocking substrate entrance to the active site. In contrast, compound C had higher specificity as inhibitor of EfBChE. In conclusion, the screening was effective in finding inhibitors of AChE and BuChE from different organisms.

Cytotoxicity and genotoxicity evaluation of organochalcogens in human leucocytes: a comparative study between ebselen, diphenyl diselenide, and diphenyl ditelluride.

Organochalcogens, particularly ebselen, have been used in experimental and clinical trials with borderline efficacy. (PhSe)2 and (PhTe)2 are the simplest of the diaryl dichalcogenides and share with ebselen pharmacological properties. In view of the concerns with the use of mammals in studies and the great number of new organochalcogens with potential pharmacological properties that have been synthesized, it becomes important to develop screening protocols to select compounds that are worth to be tested in vivo. This study investigated the possible use of isolated human white cells as a preliminary model to test organochalcogen toxicity. Human leucocytes were exposed to 5-50 µM of ebselen, (PhSe)2, or (PhTe)2. All compounds were cytotoxic (Trypan's Blue exclusion) at the highest concentration tested, and Ebselen was the most toxic. Ebselen and (PhSe)2 were genotoxic (Comet Assay) only at 50 µM, and (PhTe)2 at 5-50 µM. Here, the acute cytotoxicity did not correspond with in vivo toxicity of the compounds. But the genotoxicity was in the same order of the in vivo toxicity to mice. These results indicate that in vitro genotoxicity in white blood cells should be considered as an early step in the investigation of potential toxicity of organochalcogens.

Effect of different oximes on rat and human cholinesterases inhibited by methamidophos: a comparative in vitro and in silico study.

Methamidophos is one of the most toxic organophosphorus (OP) compounds. It acts via phosphorylation of a serine residue in the active site of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), leading to enzyme inactivation. Different oximes have been developed to reverse this inhibition. Thus, our work aimed to test the protective or reactivation capability of pralidoxime and obidoxime, as well as two new oximes synthesised in our laboratory, on human and rat cholinesterases inhibited by methamidophos. In addition, we performed molecular docking studies in non-aged methamidophos-inhibited AChE to understand the mechanisms involved. Our results suggested that pralidoxime protected and reactivated methamidophos-inhibited rat brain AChE. Regarding human erythrocyte AChE, all oximes tested protected and reactivated the enzyme, with the best reactivation index observed at the concentration of 50 µM. Concerning BChE, butane-2,3-dionethiosemicarbazone oxime (oxime 1) was able to protect and reactivate the methamidophos-inhibited BChE by 45% at 50 µM, whereas 2(3-(phenylhydrazono)butan-2-one oxime (oxime 2) reactivated 28% of BChE activity at 100 µM. The two classical oximes failed to reactivate BChE. The molecular docking study demonstrated that pralidoxime appears to be better positioned in the active site to attack the O-P moiety of the inhibited enzyme, being near the oxyanion hole, whereas our new oximes were stably positioned in the active site in a manner similar to that of obidoxime. In conclusion, our work demonstrated that the newly synthesised oximes were able to reactivate not only human erythrocyte AChE but also human plasma BChE, which could represent an advantage in the treatment of OP compounds poisoning.