A Quantum Mechanics / Molecular Mechanics Study of the Protein-Ligand Interaction of two potent inhibitors of human O-GlcNAcase: PUGNAc and NAG-thiazoline

O-glycoprotein 2-acetamino-2-deoxy-β-D-glucopyranosidase (O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-β-D-glucopyranose (O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer’s disease, and type II diabetes. In this work, we have carried out molecular dynamics (MD) simulations using a hybrid Quantum Mechanics / Molecular Mechanics (QM/MM) approach, to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298 and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structur

Exploring the Catalytic Mechanism of the RNA Cap Modification by nsp16-nsp10 Complex of SARS-CoV-2 through a QM/MM Approach

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 50 -end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 20 -O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 20 -O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis

Experimental study and computational modelling of cruzain cysteine protease inhibition by dipeptidyl nitriles

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases

Obtenção E Caracterização De Complexo De Inclusão De ?-Ciclodextrina E Eugenol / Preparation And Characterization Of ?-Cyclodextrin Inclusion Complex Of Eugenol

O eugenol é um fenilpropanóide presente em óleos essenciais de diversas plantas, cabendo destaque ao Cravo-da-Índia. Esta substância possui considerável importância farmacológica devido às suas atividades antioxidantes, porém, sua alta volatilização e instabilidade físico-química a fatores ambientais (luz, oxigênio e calor) têm dificultado seu uso tal qual nas formulações farmacêuticas. Neste contexto, buscou-se no presente trabalho produzir complexos de inclusão de eugenol em ?-ciclodextrina a fim de amenizar tais limitações. A complexação foi realizada por meio de coprecipitação e evaporação do solvente, em proporção molar de eugenol:?-ciclodextrina (1:1). Os complexos foram caracterizados por Difratometria de Raios-X (DRX); Espectroscopia de Infravermelho por Transformada de Fourier (FTIR). Os resultados revelaram que é possível caracterizar a interação hóspede/hospedeiro e, portanto, o êxito no processo de complexação, a partir de dados difratométricos (DRX) e espectrais (FTIR), o que torna o uso destas duas técnicas adequado para pesquisas no desenvolvimento de compostos com interesse biológico

The catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi elucidated via the QM/MM approach

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been identified as a key enzyme involved in glycolysis processes for energy production in the Trypanosoma cruzi parasite. This enzyme catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate (G3P) in the presence of inorganic phosphate (Pi) and nicotinamide adenosine dinucleotide (NAD+). The catalytic mechanism used by GAPDH has been intensively investigated. However, the individual roles of Pi and the C3 phosphate of G3P (Ps) sites, as well as some residues such as His194 in the catalytic mechanism, remain unclear. In this study, we have employed Molecular Dynamics (MD) simulations within hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to obtain the Potential of Mean Force of the catalytic oxidative phosphorylation mechanism of the G3P substrate used by GAPDH. According to our results, the first stage of the reaction (oxidoreduction) takes place in the Pi site (energetically more favourable), with the formation of oxyanion thiohemiacetal and thioacylenzyme intermediates without acidbase assistance of His194. Analysis of the interaction energy by residues shows that Arg249 has an important role in the ability of the enzyme to bind the G3P substrate, which interacts with NAD+ and other important residues, such as Cys166, Glu109, Thr167, Ser247 and Thr226, in the GAPDH active site. Finally, the inhibition mechanism of the GAPDH enzyme by the 3-(p-nitrophenoxycarboxyl)-3-ethylene propyl dihydroxyphosphonate inhibitor was investigated in order to contribute to the design of new inhibitors of GAPDH from Trypanosoma cruzi

Computational study of the mechanism of half-reactions in class 1A dihydroorotate dehydrogenase from Trypanosoma cruzi

Chagas' disease is considered to be a health problem affecting millions of people in Latin America. This disease is caused by the parasite Trypanosoma cruzi. Recently dihydroorotate dehydrogenase class 1A from Trypanosoma cruzi (TcDHODA) was shown to be essential for the survival and growth of T. cruzi and proposed as a drug target against Chagas' disease. This enzyme catalyzes the oxidation of (S)-dihydroorotate to orotate, with a proposed catalytic cycle consisting of two half-reactions. In the first half-reaction dihydroorotate is oxidized to orotate, with the consequent reduction of the flavin mononucleotide cofactor. In the second half-reaction fumarate is reduced to succinate. The first oxidation half-reaction may occur via a concerted or a stepwise mechanism. Herein, the catalytic mechanism of TcDHODA has been studied using hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) Molecular Dynamics (MD) simulations. The free energy profiles derived from the bidimensional potential of mean force reveal more details for two half-reaction processes

Enzyme Molecular Mechanism as a Starting Point to Design New Inhibitors: A Theoretical Study of O-GlcNAcase

O-Glycoprotein 2-acetamino-2-deoxy-β-d-glucopyranosidase (O-GlcNAcase) hydrolyzes O-linked 2-acetamido-2-deoxy-β-d-glucopyranoside (O-GlcNAc) residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. The chemical process involves substrate-assisted catalysis, where two aspartate residues have been identified as the two key catalytic residues of O-GlcNAcase. In this report, the first step of the catalytic mechanism used by O-GlcNAcase involving substrate-assisted catalysis has been studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) Molecular Dynamics (MD) calculations. The free energy profile shows that the formation of the oxazoline intermediate in the O-GlcNAcase catalytic reaction takes place by means of a stepwise mechanism. The first step would be a cyclization of the acetomide group, which seems to be dependent on the proton transfer from a conserved aspartate, Asp298 in Clostridium perfringens O-GlcNAcase. From this new intermediate, a proton is transferred from the azoline ring to another conserved aspartate (Asp297) thus forming the oxazoline ion and departure of the aglycone. In addition, averaged values of protein–substrate interaction energy along the reaction path shows that, in fact, the transition states present the highest binding affinities. A deeper analysis of the binding contribution of the individual residues shows that Asp297, Asp298, and Asp401 are basically responsible of the stabilization of these complexes. These results would explain why O-(2-acetamido-2deoxy-d-glucopyranosylidene)amino-N-phenycarbamate (PUGNAc), 1,2-dideoxy-2′-methyl-α-d-glucopyranoso-[2,1-d]-Δ2′-thiazoline (NAG-thiazoline), and GlcNAcstatin derivatives are potent inhibitors of this enzyme, resembling the two transition states of the O-GlcNAcase catalytic reaction path. These results may be useful to rational design compounds with more interesting inhibitory activity

Computational Investigation of Bisphosphate Inhibitors of 3-Deoxy-d-manno-octulosonate 8-phosphate Synthase

The synthase, 3-deoxy-d-manno-octulosonate 8-phosphate (KDO8P), is a key enzyme for the lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria and a potential target for developing new antimicrobial agents. In this study, computational molecular modeling methods were used to determine the complete structure of the KDO8P synthase from Neisseria meningitidis and to investigate the molecular mechanism of its inhibition by three bisphosphate inhibitors: BPH1, BPH2, and BPH3. Our results showed that BPH1 presented a protein–ligand complex with the highest affinity, which is in agreement with experimental data. Furthermore, molecular dynamics (MD) simulations showed that BPH1 is more active due to the many effective interactions, most of which are derived from its phosphoenolpyruvate moiety. Conversely, BPH2 exhibited few hydrogen interactions during the MD simulations with key residues located at the active sites of the KDO8P synthase. In addition, we hydroxylated BPH2 to create the hypothetical molecule named BPH3, to investigate the influence of the hydroxyl groups on the affinity of the bisphosphate inhibitors toward the KDO8P synthase. Overall, we discuss the main interactions between the KDO8P synthase and the bisphosphate inhibitors that are potential starting points for the design of new molecules with significant antibiotic activities

Structural analysis of viral infectivity factor of HIV type 1 and its interaction with A3G, EloC and EloB.

The virion infectivity factor (Vif) is an accessory protein, which is essential for HIV replication in host cells. Vif neutralizes the antiviral host protein APOBEC3 through recruitment of the E3 ubiquitin ligase complex.Fifty thousand Vif models were generated using the ab initio relax protocol of the Rosetta algorithm from sets of three- and nine-residue fragments using the fragment Monte Carlo insertion-simulated annealing strategy, which favors protein-like features, followed by an all-atom refinement. In the protocol, a constraints archive was used to define the spatial relationship between the side chains from Cys/His residues and zinc ions that formed the zinc-finger motif that is essential for Vif function. We also performed centroids analysis and structural analysis with respect to the formation of the zinc-finger, and the residue disposal in the protein binding domains. Additionally, molecular docking was used to explore details of Vif-A3G and Vif-EloBC interactions. Furthermore, molecular dynamics simulation was used to evaluate the stability of the complexes Vif-EloBC-A3G and Vif-EloC.The zinc in the HCCH domain significantly alters the folding of Vif and changes the structural dynamics of the HCCH region. Ab initio modeling indicated that the Vif zinc-finger possibly displays tetrahedral geometry as suggested by Mehle et al. (2006). Our model also showed that the residues L146 and L149 of the BC-box motif bind to EloC by hydrophobic interactions, and the residue P162 of the PPLP motif is important to EloB binding.The model presented here is the first complete three-dimensional structure of the Vif. The interaction of Vif with the A3G protein and the EloBC complex is in agreement with empirical data that is currently available in the literature and could therefore provide valuable structural information for advances in rational drug design