Primary Antioxidant Power and Mpro SARS-CoV-2 Non-Covalent Inhibition Capabilities of Miquelianin.

The antioxidant power of quercetin-3-O-glucuronide (miquelianin) has been studied, at the density functional level of theory, in both lipid-like and aqueous environments. In the aqueous phase, the computed pKa equilibria allowed the identification of the neutral and charged species present in solution that can react with the ⋅OOH radical. The Hydrogen Atom Transfer (HAT), Single Electron Transfer (SET) and Radical Adduct Formation (RAF) mechanisms were considered, and the individual, total and fraction corrected rate constants were obtained. Potential non-covalent inhibition of Mpro from SARS-CoV-2 by miquelianin has been also evaluated.

On the antibacterial photodynamic inactivation mechanism of Emodin and Dermocybin natural photosensitizers: A theoretical investigation.

A DFT and TDDFT study has been carried out on monomeric anthraquinones Emodin and Dermocybin (Em, Derm) recently proposed as natural antibacterial photosensitizers able to act also against gram-negative microbes. The computational study has been performed considering the relative amount of neutral and ionic forms of each compound in water, with the variation of pH. The occurrence of both Type I and Type II photoreactions has been explored computing the absorption properties of each species, the spin-orbit coupling constants (SOC), the vertical ionization potentials and the vertical electron affinities. The most plausible deactivation channels leading to the population of excited triplet states have been proposed. Our data indicate Emodin as more active than Dermocybin in antimicrobial photodynamic therapy throughout the Type II mechanism. Our data support a dual TypeI/II activity of the monomeric anthraquinones Emodin and Dermccybin in water, in all the considered protonation states.

Viral RNA Replication Suppression of SARS-CoV-2: Atomistic Insights into Inhibition Mechanisms of RdRp Machinery by ddhCTP.

The nonstructural protein 12, known as RNA-dependent RNA polymerase (RdRp), is essential for both replication and repair of the viral genome. The RdRp of SARS-CoV-2 has been used as a promising candidate for drug development since the inception of the COVID-19 spread. In this work, we performed an in silico investigation on the insertion of the naturally modified pyrimidine nucleobase ddhCTP into the SARS-CoV-2 RdRp active site, in a comparative analysis with the natural one (CTP). The modification in ddhCTP involves the removal of the 3'-hydroxyl group that prevents the addition of subsequent nucleotides into the nascent strand, acting as an RNA chain terminator inhibitor. Quantum mechanical investigations helped to shed light on the mechanistic source of RdRp activity on the selected nucleobases, and comprehensive all-atom simulations provided insights about the structural rearrangements occurring in the active-site region when inorganic pyrophosphate (PPi) is formed. Subsequently, the intricate pathways for the release of PPi, the catalytic product of RdRp, were investigated using Umbrella Sampling simulations. The results are in line with the available experimental data and contribute to a more comprehensive point of view on such an important viral enzyme.

Degradation of Penicillinic Antibiotics and ß-Lactamase Enzymatic Catalysis in a Biomimetic Zn-Based Metal-Organic Framework.

Invited for the cover of this issue are Jesús Ferrando-Soria, Donatella Armentano, Antonio Leyva-Pérez, Emilio Pardo and co-workers at University of Valencia, Technical University of Valencia and University of Calabria. The image depicts the crystal structure of a novel ZnII biological metal-organic framework that mimics ß-lactamase enzymes. Read the full text of the article at 10.1002/chem.202301325.

On the Role of Temperature in the Depolymerization of PET by FAST-PETase: An Atomistic Point of View on Possible Active Site Pre-Organization and Substrate-Destabilization Effects.

Enzyme FAST-PETase, recently obtained by a machine learning approach, can depolymerize poly(ethylene terephthalate) (PET), a synthetic resin employed in plastics and in clothing fibers. Therefore it represents a promising solution for the recycling of PET-based materials. In this study, a model of PET was adopted to describe the substrate, and all-atoms classical molecular dynamics (MD) simulations on apo- and substrate-bound FAST-PETase were carried out at 30 and 50 °C to provide atomistic details on the binding step of the catalytic cycle. Comparative analysis shed light on the interactions occurring between the FAST-PETase and 4PET at 50 °C, the optimal working conditions of the enzyme. Pre-organization of the enzyme active and binding sites has been highlighted, while MD simulations of FAST-PETase:4PET pointed out the occurrence of solvent-inaccessible conformations of the substrate promoted by the enzyme. Indeed, neither of these conformations was observed during MD simulations of the substrate alone in solution performed at 30, 50 and 150 °C. The analysis led us to propose that, at 50 °C, the FAST-PETase is pre-organized to bind the PET and that the interactions occurring in the binding site can promote a more reactive conformation of PET substrate, thus enhancing the catalytic activity of the enzyme.

Degradation of Penicillinic Antibiotics and ß-Lactamase Enzymatic Catalysis in a Biomimetic Zn-Based Metal-Organic Framework.

ß-Lactam antibiotics are one of the most commonly prescribed drugs to treat bacterial infections. However, their use has been somehow limited given the emergence of bacteria with resistance mechanisms, such as ß-lactamases, which inactivate them by degrading their four-membered ß-lactam rings. So, a total knowledge of the mechanisms governing the catalytic activity of ß-lactamases is required. Here, we report a novel Zn-based metal-organic framework (MOF, 1), possessing functional channels capable to accommodate and interact with antibiotics, which catalyze the selective hydrolysis of the penicillinic antibiotics amoxicillin and ceftriaxone. In particular, MOF 1 degrades, very efficiently, the four-membered ß-lactam ring of amoxicillin, acting as a ß-lactamase mimic, and expands the very limited number of MOFs capable to mimic catalytic enzymatic processes. Combined single-crystal X-ray diffraction (SCXRD) studies and density functional (DFT) calculations offer unique snapshots on the host-guest interactions established between amoxicillin and the functional channels of 1. This allows to propose a degradation mechanism based on the activation of a water molecule, promoted by a Zn-bridging hydroxyl group, concertedly to the nucleophilic attack to the carbonyl moiety and the cleaving of C-N bond of the lactam ring.

Incorporation of N7-Platinated Guanines into Thermus Aquaticus (Taq) DNA Polymerase: Atomistic Insights from Molecular Dynamics Simulations.

In this work, we elucidated some key aspects of the mechanism of action of the cisplatin anticancer drug, cis-[Pt(NH3)2Cl2], involving direct interactions with free nucleotides. A comprehensive in silico molecular modeling analysis was conducted to compare the interactions of Thermus aquaticus (Taq) DNA polymerase with three distinct N7-platinated deoxyguanosine triphosphates: [Pt(dien)(N7-dGTP)] (1), cis-[Pt(NH3)2Cl(N7-dGTP)] (2), and cis-[Pt(NH3)2(H2O)(N7-dGTP)] (3) {dien = diethylenetriamine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate}, using canonical dGTP as a reference, in the presence of DNA. The goal was to elucidate the binding site interactions between Taq DNA polymerase and the tested nucleotide derivatives, providing valuable atomistic insights. Unbiased molecular dynamics simulations (200 ns for each complex) with explicit water molecules were performed on the four ternary complexes, yielding significant findings that contribute to a better understanding of experimental results. The molecular modeling highlighted the crucial role of a specific α-helix (O-helix) within the fingers subdomain, which facilitates the proper geometry for functional contacts between the incoming nucleotide and the DNA template needed for incorporation into the polymerase. The analysis revealed that complex 1 exhibits a much lower affinity for Taq DNA polymerase than complexes 2-3. The affinities of cisplatin metabolites 2-3 for Taq DNA polymerase were found to be quite similar to those of natural dGTP, resulting in a lower incorporation rate for complex 1 compared to complexes 2-3. These findings could have significant implications for the cisplatin mechanism of action, as the high intracellular availability of free nucleobases might promote the competitive incorporation of platinated nucleotides over direct cisplatin attachment to DNA. The study's insights into the incorporation of platinated nucleotides into the Taq DNA polymerase active site suggest that the role of platinated nucleotides in the cisplatin mechanism of action may have been previously underestimated.

Periodicity of the Affinity of Lanmodulin for Trivalent Lanthanides and Actinides: Structural and Electronic Insights from Quantum Chemical Calculations.

Lanmodulin (LanM) is the first identified macrochelator that has naturally evolved to sequester ions of rare earth elements (REEs) such as Y and all lanthanides, reversibly. This natural protein showed a 106 times better affinity for lanthanide cations than for Ca, which is a naturally abundant and biologically relevant element. Recent experiments have shown that its metal ion binding activity can be further extended to some actinides, like Np, Pu, and Am. For this reason, it was thought that LanM could be adopted for the separation of REE ions and actinides, thus increasing the interest in its potential use for industry-oriented applications. In this work, a systematic study of the affinity of LanM for lanthanides and actinides has been carried out, taking into account all trivalent ions belonging to the 4f (from La to Lu) and 5f (from Ac to Lr) series, starting from their chemistry in solution. On the basis of a recently published nuclear magnetic resonance structure, a model of the LanM-binding site was built and a detailed structural and electronic description of initial aquo- and LanM-metal ion complexes was provided. The obtained binding energies are in agreement with the available experimental data. A possible reason that could explain the origin of the affinity of LanM for these metal ions is also discussed.

Sulphur- and Selenium-for-Oxygen Replacement as a Strategy to Obtain Dual Type I/Type II Photosensitizers for Photodynamic Therapy.

The effect on the photophysical properties of sulfur- and selenium-for-oxygen replacement in the skeleton of the oxo-4-dimethylaminonaphthalimide molecule (DMNP) has been explored at the density functional (DFT) level of theory. Structural parameters, excitation energies, singlet-triplet energy gaps (ΔES-T), and spin-orbit coupling constants (SOC) have been computed. The determined SOCs indicate an enhanced probability of intersystem crossing (ISC) in both the thio- and seleno-derivatives (SDMNP and SeDMNP, respectively) and, consequently, an enhancement of the singlet oxygen quantum yields. Inspection of Type I reactions reveals that the electron transfer mechanisms leading to the generation of superoxide is feasible for all the compounds, suggesting a dual Type I/Type II activity.

On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV-2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3'-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.

Structural Characterization of Peripolin and Study of Antioxidant Activity of HMG Flavonoids from Bergamot Fruit.

The structural characterization of a new flavonoid from bergamot fruit (Citrus bergamia Risso) carrying the 3-hydroxy-3-methyl glutaryl (HMG) ester moiety has been accomplished, and its antioxidant ability was tested from a chemical point of view. The peculiarity of the new molecule, named peripolin, relies on the presence of the HMG chemical group linked to the sugar portion of neoeriocitrin; the structure was elucidated using both high-resolution mass spectrometry and nuclear magnetic resonance experiments performed on the purified molecule extracted from the fruit. The antioxidant ability of the new molecule was tested by classical chemical approaches, such as DPPH, ABTS and FRAP assays, and from a theoretical point of view. 1H and 13C NMR experiments and HR-ESI-MS/MS experiments show unequivocally that the HMG moiety is linked to the primary position of the glucose unit of neohesperidose, while the chemical tests and the computational results show that peripolin possesses strong antioxidant behavior, similar to that of neoeriocitrin and remarkably higher respect to the other flavonoids present in the fruit. Furthermore, the quantitative assays carried out by UPLC-MS/MS showed that its amount in the fruit is similar to that of the other main flavonoids. Furthermore, molecular dynamics simulations allowed us to investigate the possible conformations adopted by the antioxidants in the presence of water molecules. In particular, the switch of open-closed conformations of HMG-containing species was evidenced. As far as the reaction with DPPH, the calculation of ΔGrea supported the experimental outcomes regarding the peripolin and neoeriocitrin activity. In conclusion, bergamot fruit, already known for its potential to lower the level of blood cholesterol, has been proven to contain molecules such as neoeriocitrin and the newly characterized peripolin, which could have important in-vivo antioxidant characteristics.

Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations.

Human PAICS is a bifunctional enzyme that is involved in the de novo purine biosynthesis, catalyzing the conversion of aminoimidazole ribonucleotide (AIR) into N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). It comprises two distinct active sites, AIR carboxylase (AIRc) where the AIR is initially converted to carboxyaminoimidazole ribonucleotide (CAIR) by reaction with CO2 and SAICAR synthetase (SAICARs) in which CAIR then reacts with an aspartate to form SAICAR, in an ATP-dependent reaction. Human PAICS is a promising target for the treatment of various types of cancer, and it is therefore of high interest to develop a detailed understanding of its reaction mechanism. In the present work, density functional theory calculations are employed to investigate the PAICS reaction mechanism. Starting from the available crystal structures, two large models of the AIRc and SAICARs active sites are built and different mechanistic proposals for the carboxylation and phosphorylation-condensation mechanisms are examined. For the carboxylation reaction, it is demonstrated that it takes place in a two-step mechanism, involving a C-C bond formation followed by a deprotonation of the formed tetrahedral intermediate (known as isoCAIR) assisted by an active site histidine residue. For the phosphorylation-condensation reaction, it is shown that the phosphorylation of CAIR takes place before the condensation reaction with the aspartate. It is further demonstrated that the three active site magnesium ions are involved in binding the substrates and stabilizing the transition states and intermediates of the reaction. The calculated barriers are in good agreement with available experimental data.

Inhibition of the carnitine acylcarnitine carrier by carbon monoxide reveals a novel mechanism of action with non-metal-containing proteins.

Both toxic and physiological effects of CO are mostly caused by well described interactions with heme-groups of proteins. Interactions of CO with non-heme proteins have also been unveiled. Besides interaction of CO with mitochondrial heme containing respiratory complexes, a BK channel and the phosphate carrier which do not contain metal cofactors, have been identified as CO targets. However, the molecular mechanisms of interaction with non-metal-containing proteins are not understood. We show in this work the effect of CO on the mitochondrial carnitine carrier (SLC25A20) using CORM-3, a widely recognized CO releasing compound. CO exerts an inhibitory effect at the micromolar concentration on the transport function of the transporter extracted from treated mitochondria. The effect is due to a single Cys residue, C136 as revealed by mass spectrometry analysis. A computational approach predicted the need for vicinal Asp and Lys residues for the C136 carbonylation to occur. These data demonstrate a novel mechanism of interaction of CO with a protein not containing metal atoms and will enable the prediction of CO targets.

Antioxidant and copper-chelating power of new molecules suggested as multiple target agents against Alzheimer's disease. A theoretical comparative study.

In this study, the scavenging activity against OOH radicals and the copper-chelating ability of two new synthesized molecules (named L1 and L2) that can act as multiple target agents against Alzheimer's disease have been investigated at the density functional theory level. The pKa and molar fractions at physiological pH have been predicted. The main antioxidant reaction mechanisms in lipid-like and water environments have been considered and the relative rate constants determined. The copper-chelating ability of the two compounds has also been explored at different coordination sites and computing the complexation kinetic constants. Results show the L1 compound is a more effective radical scavenging and copper-chelating agent than L2.

On the Scavenging Ability of Scutellarein against the OOH Radical in Water and Lipid-like Environments: A Theoretical Study.

The antioxidant capability of scutellarein, a flavonoid extracted from different plants of the Scutellaria family, was computationally predicted by considering its reaction with the OOH radical in both lipid-like and water environments. The pKa and equilibrium behavior in the aqueous phase were also calculated. Different reaction mechanisms involving the most populated species were considered. The work was performed by using the density functional level of theory. The individual, total, and fraction-corrected total rate constants were obtained. The results show that scutellarein has scavenging power against the hydroperoxyl radical similar to that of Trolox, which is generally used as a reference antioxidant.

Computational Study of Mechanism and Enantioselectivity of Imine Reductase from Amycolatopsis orientalis.

Imine reductases (IREDs) are NADPH-dependent enzymes (NADPH=nicotinamide adenine dinucleotide phosphate) that catalyze the reduction of imines to amines. They exhibit high enantioselectivity for a broad range of substrates, making them of interest for biocatalytic applications. In this work, we have employed density functional theory (DFT) calculations to elucidate the reaction mechanism and the origins of enantioselectivity of IRED from Amycolatopsis orientalis. Two substrates are considered, namely 1-methyl-3,4-dihydroisoquinoline and 2-propyl-piperideine. A model of the active site is built on the basis of the available crystal structure. For both substrates, different binding modes are first evaluated, followed by calculation of the hydride transfer transition states from each complex. We have also investigated the effect of mutations of certain important active site residues (Tyr179Ala and Asn241Ala) on the enantioselectivity. The calculated energies are consistent with the experimental observations and the analysis of transition states geometries provides insights into the origins of enantioselectivity of this enzyme.

Computational Study Reveals the Role of Water Molecules in the Inhibition Mechanism of LAT1 by 1,2,3-Dithiazoles.

The L-type amino acid transporter LAT1, involved in many biological processes including the overexpression of some tumors, is considered a potential pharmacological target. The 1,2,3-Dithiazole scaffold was predicted to inhibit LAT1 by the formation of an intermolecular disulfide bond with the thiolate group of cysteine(s). As a result of the identification of these irreversible covalent inhibitors, we decided to deeply investigate the recognition stage and the covalent interaction, characterizing the chemical structures of the selected ligands. With the aim to provide new insights into the access of the ligands to the binding pocket and to reveal the residues involved in the inhibition, we performed docking, molecular dynamics simulations, and density functional theory-based investigation of three 1,2,3-dithiazoles against LAT1. Our computational analysis further highlighted the crucial role played by water molecules in the inhibition mechanism. The results here presented are consistent with experimental observations and provide insights that can be helpful for the rational design of new-to-come LAT1's inhibitors.

Exploring the Structure-Performance Relationship of Sulfonated Polysulfone Proton Exchange Membrane by a Combined Computational and Experimental Approach.

Sulfonated Polysulfone (sPSU) is emerging as a concrete alternative to Nafion ionomer for the development of proton exchange electrolytic membranes for low cost, environmentally friendly and high-performance PEM fuel cells. This ionomer has recently gained great consideration since it can effectively combine large availability on the market, excellent film-forming ability and remarkable thermo-mechanical resistance with interesting proton conductive properties. Despite the great potential, however, the morphological architecture of hydrated sPSU is still unknown. In this study, computational and experimental advanced tools are combined to preliminary describe the relationship between the microstructure of highly sulfonated sPSU (DS = 80%) and its physico-chemical, mechanical and electrochemical features. Computer simulations allowed for describing the architecture and to estimate the structural parameters of the sPSU membrane. Molecular dynamics revealed an interconnected lamellar-like structure for hydrated sPSU, with ionic clusters of about 14-18 Å in diameter corresponding to the hydrophilic sulfonic-acid-containing phase. Water dynamics were investigated by 1H Pulsed Field Gradient (PFG) NMR spectroscopy in a wide temperature range (20-120 °C) and the self-diffusion coefficients data were analyzed by a "two-sites" model. It allows to estimate the hydration number in excellent agreement with the theoretical simulation (e.g., about 8 mol H2O/mol SO3- @ 80 °C). The PEM performance was assessed in terms of dimensional, thermo-mechanical and electrochemical properties by swelling tests, DMA and EIS, respectively. The peculiar microstructure of sPSU provides a wider thermo-mechanical stability in comparison to Nafion, but lower dimensional and conductive features. Nonetheless, the single H2/O2 fuel cell assembled with sPSU exhibited better features than any earlier published hydrocarbon ionomers, thus opening interesting perspectives toward the design and preparation of high-performing sPSU-based PEMs.

Capsaicin, a Powerful •OH-Inactivating Ligand.

Oxidative conditions are frequently enhanced by the presence of redox metal ions. In this study, the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide, CAP) in copper-induced oxidative stress was investigated using density functional theory simulations. It was found that CAP has the capability to chelate Cu(II), leading to complexes that are harder to reduce than free Cu(II). CAP fully turns off the Cu(II) reduction by Asc-, and slows down the reduction in this cation by O2•-. Therefore, CAP is proposed as an •OH-inactivating ligand by impeding the reduction in metal ions (OIL-1), hindering the production of •OH via Fenton-like reactions, at physiological pH. CAP is also predicted to be an excellent antioxidant as a scavenger of •OH, yielded through Fenton-like reactions (OIL-2). The reactions between CAP-Cu(II) chelates and •OH were estimated to be diffusion-limited. Thus, these chelates are capable of deactivating this dangerous radical immediately after being formed by Fenton-like reactions.

On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study.

Recently major advances were gained on the designed proteins aimed to generate biomolecular mimics of proteases. Although such enzyme-like catalysts must still suffer refinements for improving the catalytic activity, at the moment, they represent a good example of artificial enzymes to be tested in different fields. Herein, a de novo designed homo-heptameric peptide assembly (CC-Hept) where the esterase activity towards p-nitro-phenylacetate was obtained for introduction of the catalytic triad (Cys-His-Glu) into the hydrophobic matrix, is the object of the present combined molecular dynamics and quantum mechanics/molecular mechanics investigation. Constant pH Molecular Dynamics simulations on the apoform of CC-Hept suggested that the Cys residues are present in the protonated form. Molecular dynamics (MD) simulations of the enzyme-substrate complex evidenced the attitude of the enzyme-like system to retain water molecules, necessary in the hydrolytic reaction, in correspondence of the active site, represented by the Cys-His-Glu triad on each of the seven chains, without significant structural perturbations. A detailed reaction mechanism of esterase activity of CC-Hept-Cys-His-Glu was investigated on the basis of the quantum mechanics/molecular mechanics calculations employing a large quantum mechanical (QM) region of the active site. The proposed mechanism is consistent with available esterases kinetics and structural data. The roles of the active site residues were also evaluated. The deacylation phase emerged as the rate-determining step, in agreement with esterase activity of other natural proteases.