Dynamic Profiling of ß-Coronavirus 3CL Mpro Protease Ligand-Binding Sites.

ß-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across ß-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.

Fluoride enhances generation of reactive oxygen and nitrogen species, oxidizes hemoglobin, lowers antioxidant power and inhibits transmembrane electron transport in isolated human red blood cells.

Fluoride is a widespread environmental pollutant that at high levels exerts numerous deleterious effects on human health. The toxic effects of fluoride are a matter of serious concern since many countries have regions of endemic fluorosis. The main source of fluoride exposure for humans is intake of contaminated groundwater. Fluoride is absorbed from the gastrointestinal tract and enters the circulating blood, where the abundant red blood cells (RBC) are an early and major target of fluoride toxicity. Chronic fluoride exposure generates free radicals, reactive species which leads to redox imbalance, cytotoxicity and hematological damage. This study aimed to determine the effect of sodium fluoride (NaF) on human RBC under in vitro conditions. Isolated RBC were incubated with different concentrations of NaF (10-500 µM) for 8 h at 37 °C. Several biochemical parameters were determined in hemolysates or whole cells. Treatment of RBC with NaF enhanced the generation of reactive oxygen and nitrogen species. This increased the oxidation of hemoglobin to yield methemoglobin and oxoferrylhemoglobin, which are inactive in oxygen transport. NaF treatment increased the degradation of heme causing release of free iron from its porphyrin ring. Cellular antioxidant power was significantly decreased in NaF-treated RBC, lowering the metal reducing and free radical quenching ability of cells. The two pathways of glucose metabolism in RBC i.e. glycolysis and hexose monophosphate shunt, were inhibited. NaF also inhibited the plasma membrane redox system, and its associated ascorbate free radical reductase, to disrupt transmembrane electron transport. These results suggest that fluoride generates reactive species that cause extensive oxidative modifications in human RBC.

3,4-Dihydroxybenzaldehyde mitigates fluoride-induced cytotoxicity and oxidative damage in human RBC.

BACKGROUND: Fluoride is an essential micronutrient that is needed for mineralization of bones and formation of dental enamel. It is a widely dispersed environmental pollutant and chronic exposure to it is toxic, resulting in malignancies and hematological damage in humans. Blood is a major and early target of environmental pollutants and toxicants like fluoride. Fluoride generates reactive oxygen species and free radicals which induce oxidative stress in target cells and mediate its toxic effects. The aim of this study was to determine the mitigating effect of plant antioxidant 3,4-dihydroxybenzaldehyde (DHB) on sodium fluoride (NaF) induced oxidative damage and cytotoxicity in isolated human red blood cells (RBC) METHOD: Isolated human RBC were treated with 0.5 mM NaF, in absence or presence of different concentrations of DHB (0.1-2.5 mM). Several biochemical parameters were analyzed in cell lysates and whole cells. RESULTS: Treatment of RBC with NaF increased the formation of reactive oxygen and nitrogen species. It oxidized thiols, proteins and lipids and generated their peroxidative products. Methemoglobin level, heme degradation and lipid peroxidation were increased but cellular antioxidant status declined significantly in NaF alone treated RBC, compared to the control. NaF inhibited antioxidant, membrane bound and glycolytic enzymes in RBC. However, prior incubation of RBC with DHB significantly attenuated the NaF-induced alterations in all these parameters in a DHB concentration-dependent manner. CONCLUSION: These results show that DHB mitigates NaF-induced oxidative damage in human RBC, probably because of its antioxidant character.