LDH-Indomethacin Nanoparticles Antitumoral Action: A Possible Coadjuvant Drug for Cancer Therapy.

Indomethacin (INDO) has a mechanism of action based on inhibiting fatty acids cyclooxygenase activity within the inflammation process. The action mechanism could be correlated with possible anticancer activity, but its high toxicity in normal tissues has made therapy difficult. By the coprecipitation method, the drug carried in a layered double hydroxides (LDH) hybrid matrix would reduce its undesired effects by promoting chemotherapeutic redirection. Therefore, different samples containing INDO intercalated in LDH were synthesized at temperatures of 50, 70, and 90 °C and synthesis times of 8, 16, 24, and 48 h, seeking the best structural organization. X-ray diffraction (XRD), vibrational Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), spectrophotometric analysis in UV-VIS, and differential thermogravimetric analysis (TGA/DTA) were used for characterization. Our results indicate that higher temperatures and longer synthesis time through coprecipitation reduce the possibility of INDO intercalation. However, it was possible to establish a time of 16 h and a temperature of 50 °C as the best conditions for intercalation. In vitro results confirmed the cell viability potential and anticancer activity in the LDH-INDO sample (16 h and 50 °C) for gastric cancer (AGP01, ACP02, and ACP03), breast cancer (MDA-MB-231 and MCF-7), melanoma (SK-MEL-19), lung fibroblast (MRC-5), and non-neoplastic gastric tissue (MN01) by MTT assay. Cell proliferation was inhibited, demonstrating higher and lower toxicity against MDA-MB-231 and SK-MEL-19. Thus, a clinical redirection of INDO is suggested as an integral and adjunctive anticancer medication in chemotherapy treatment.

FT-MIR-ATR Associated with Chemometrics Methods: A Preliminary Analysis of Deterioration State of Brazil Nut Oil.

Brazil nut oil is highly valued in the food, cosmetic, chemical, and pharmaceutical industries, as well as other sectors of the economy. This work aims to use the Fourier transform infrared (FTIR) technique associated with partial least squares regression (PLSR) and principal component analysis (PCA) to demonstrate that these methods can be used in a prior and rapid analysis in quality control. Natural oils were extracted and stored for chemical analysis. PCA presented two groups regarding the state of degradation, subdivided into super-degraded and partially degraded groups in 99.88% of the explained variance. The applied PLS reported an acidity index (AI) prediction model with root mean square error of calibration (RMSEC) = 1.8564, root mean square error of cross-validation (REMSECV) = 4.2641, root mean square error of prediction (RMSEP) = 2.1491, R2cal (calibration correlation coefficient) equal to 0.9679, R2val (validation correlation coefficient) equal to 0.8474, and R2pred (prediction correlation coefficient) equal to 0, 8468. The peroxide index (PI) prediction model showed RMSEC = 0.0005, REMSECV = 0.0016, RMSEP = 0.00079, calibration R2 equal to 0.9670, cross-validation R2 equal to 0.7149, and R2 of prediction equal to 0.9099. The physical-chemical analyses identified that five samples fit in the food sector and the others fit in other sectors of the economy. In this way, the preliminary monitoring of the state of degradation was reported, and the prediction models of the peroxide and acidity indexes in Brazil nut oil for quality control were determined.

Caffeic Acid-Zinc Basic Salt/Chitosan Nanohybrid Possesses Controlled Release Properties and Exhibits In Vivo Anti-Inflammatory Activities.

Caffeic acid (CA) exhibits a myriad of biological activities including cardioprotective action, antioxidant, antitumor, anti-inflammatory, and antimicrobial properties. On the other hand, CA presents low water solubility and poor bioavailability, which have limited its use for therapeutic applications. The objective of this study was to develop a nanohybrid of zinc basic salts (ZBS) and chitosan (Ch) containing CA (ZBS-CA/Ch) and evaluate its anti-edematogenic and antioxidant activity in dextran and carrageenan-induced paw edema model. The samples were obtained by coprecipitation method and characterized by X-ray diffraction, Fourier transform infrared (FT-IR), scanning electron microscope (SEM) and UV-visible spectroscopy. The release of caffeate anions from ZBS-CA and ZBS-CA/Ch is pH-dependent and is explained by a pseudo-second order kinetics model, with a linear correlation coefficient of R2 ≥ 0.99 at pH 4.8 and 7.4. The in vivo pharmacological assays showed excellent anti-edematogenic and antioxidant action of the ZBS-CA/Ch nanoparticle with slowly releases of caffeate anions in the tissue, leading to a prolongation of CA-induced anti-edematogenic and anti-inflammatory activities, as well as improving its inhibition or sequestration antioxidant action toward reactive species. Overall, this study highlighted the importance of ZBS-CA/Ch as an optimal drug carrier.

Computational Analysis of Triazole-Based Kojic Acid Analogs as Tyrosinase Inhibitors by Molecular Dynamics and Free Energy Calculations.

Molecular docking, molecular dynamics (MD) simulations and the linear interaction energy (LIE) method were used here to predict binding modes and free energy for a set of 1,2,3-triazole-based KA analogs as potent inhibitors of Tyrosinase (TYR), a key metalloenzyme of the melanogenesis process. Initially, molecular docking calculations satisfactorily predicted the binding mode of evaluated KA analogs, where the KA part overlays the crystal conformation of the KA inhibitor into the catalytic site of TYR. The MD simulations were followed by the LIE method, which reproduced the experimental binding free energies for KA analogs with an r2 equal to 0.97, suggesting the robustness of our theoretical model. Moreover, the van der Waals contributions performed by some residues such as Phe197, Pro201, Arg209, Met215 and Val218 are responsible for the binding recognition of 1,2,3-triazole-based KA analogs in TYR catalytic site. Finally, our calculations provide suitable validation of the combination of molecular docking, MD, and LIE approaches as a powerful tool in the structure-based drug design of new and potent TYR inhibitors.

Assessment of mutations on RBD in the Spike protein of SARS-CoV-2 Alpha, Delta and Omicron variants.

The severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) variant Omicron spread more rapid than the other variants of SARS-CoV-2 virus. Mutations on the Spike (S) protein receptor-binding domain (RBD) are critical for the antibody resistance and infectivity of the SARS-CoV-2 variants. In this study, we have used accelerated molecular dynamics (aMD) simulations and free energy calculations to present a systematic analysis of the affinity and conformational dynamics along with the interactions that drive the binding between Spike protein RBD and human angiotensin-converting enzyme 2 (ACE2) receptor. We evaluate the impacts of the key mutation that occur in the RBDs Omicron and other variants in the binding with the human ACE2 receptor. The results show that S protein Omicron has stronger binding to the ACE2 than other variants. The evaluation of the decomposition energy per residue shows the mutations N440K, T478K, Q493R and Q498R observed in Spike protein of SARS-CoV-2 provided a stabilization effect for the interaction between the SARS-CoV-2 RBD and ACE2. Overall, the results demonstrate that faster spreading of SARS-CoV-2 Omicron may be correlated with binding affinity of S protein RBD to ACE2 and mutations of uncharged residues to positively charged residues such as Lys and Arg in key positions in the RBD.

Host-Guest Inclusion Complexes of Natural Products and Nanosystems: Applications in the Development of Repellents.

Repellents are compounds that prevent direct contact between the hosts and the arthropods that are vectors of diseases. Several studies have described the repellent activities of natural compounds obtained from essential oils. In addition, these chemical constituents have been pointed out as alternatives to conventional synthetic repellents due to their interesting residual protection and low toxicity to the environment. However, these compounds have been reported with short shelf life, in part, due to their volatile nature. Nanoencapsulation provides protection, stability, conservation, and controlled release for several compounds. Here, we review the most commonly used polymeric/lipid nanosystems applied in the encapsulation of small organic molecules obtained from essential oils that possess repellent activity, and we also explore the theoretical aspects related to the intermolecular interactions, thermal stability, and controlled release of the nanoencapsulated bioactive compounds.

Computational insights for predicting the binding and selectivity of peptidomimetic plasmepsin IV inhibitors against cathepsin D.

Plasmepsins (Plms) are aspartic proteases involved in the degradation of human hemoglobin by P. falciparum and are essential for the survival and growth of the parasite. Therefore, Plm enzymes are reported as an important antimalarial drug target. Herein, we have applied molecular docking, molecular dynamics (MD) simulations, and binding free energy with the Linear Interaction Energy (LIE) approach to investigate the binding of peptidomimetic PlmIV inhibitors with a particular focus on understanding their selectivity against the human Asp protease cathepsin D (CatD). The residual decomposition analysis results suggest that amino acid differences in the subsite S3 of PlmIV and CatD are responsible for the higher selectivity of the 5a inhibitor. These findings yield excellent agreement with experimental binding data and provide new details regarding van der Waals and electrostatic interactions of subsite residues as well as structural properties of the PlmIV and CatD systems.

Assessment of host-guest molecular encapsulation of eugenol using ß-cyclodextrin.

Eugenol is a natural compound with well-known repellent activity. However, its pharmaceutical and cosmetic applications are limited, since this compound is highly volatile and thermolabile. Nanoencapsulation provides protection, stability, conservation, and controlled release for several compounds. Here, eugenol was included in ß-cyclodextrin, and the complex was characterized through X-ray diffraction analysis (XRD) and Fourier-transform infrared spectroscopy (FTIR). Additionally, we used molecular dynamics simulations to explore the eugenol-ß-cyclodextrin complex stability with temperature increases. Our computational result demonstrates details of the molecular interactions and conformational changes of the eugenol-ß-cyclodextrin complex and explains its stability between temperatures 27°C and 48°C, allowing its use in formulations that are subjected to varied temperatures.

Structural, energetic and lipophilic analysis of SARS-CoV-2 non-structural protein 9 (NSP9).

In SARS-CoV-2 replication complex, the Non-structural protein 9 (Nsp9) is an important RNA binding subunit in the RNA-synthesizing machinery. The dimeric forms of coronavirus Nsp9 increase their nucleic acid binding affinity and the N-finger motif appears to play a critical role in dimerization. Here, we present a structural, lipophilic and energetic study about the Nsp9 dimer of SARS-CoV-2 through computational methods that complement hydrophobicity scales of amino acids with molecular dynamics simulations. Additionally, we presented a virtual N-finger mutation to investigate whether this motif contributes to dimer stability. The results reveal for the native dimer that the N-finger contributes favorably through hydrogen bond interactions and two amino acids bellowing to the hydrophobic region, Leu45 and Leu106, are crucial in the formation of the cavity for potential drug binding. On the other hand, Gly100 and Gly104, are responsible for stabilizing the α-helices and making the dimer interface remain stable in both, native and mutant (without N-finger motif) systems. Besides, clustering results for the native dimer showed accessible cavities to drugs. In addition, the energetic and lipophilic analysis reveal that the higher binding energy in the native dimer can be deduced since it is more lipophilic than the mutant one, increasing non-polar interactions, which is in line with the result of MM-GBSA and SIE approaches where the van der Waals energy term has the greatest weight in the stability of the native dimer. Overall, we provide a detailed study on the Nsp9 dimer of SARS-CoV-2 that may aid in the development of new strategies for the treatment and prevention of COVID-19.

A molecular model to study FosA enzyme inhibition.

Fosfomycin resistance protein (FosA) is a metalloenzyme known for catalyzing a nucleophilic addition reaction of glutathione to the epoxide ring of Fosfomycin, a broad-spectrum antibiotic used to combat Gram-positive pathogens. The reaction leads fosfomycin to lose its pharmacological effect, thus promotes antibiotic resistance. A small-molecule FosA inhibitor has been discovered. ANY1 (3-bromo-6-[3-(3-bromo-2-oxo-1H-pyrazolo[1,5-a]pyrimidin-6-yl)-4-nitro-1H-pyrazol-5-yl]-1H-pyrazolo[1,5-a]pyrimidin-2-one) is competitive with the antibiotic for binding the active site of the enzyme. Through Molecular Mechanics methods, using the AMBER force field, we carry out molecular dynamics simulations and binding free energy calculations to investigate the most important interactions between the enzyme and inhibitor. Our results were able to reproduce the trend of experimental data with R2 of 77.51%. Furthermore, we have shown that electrostatic and van der Waals interactions, as well as cavitation energies, are favorable for maintaining the enzyme-inhibitor complex, while reactive field energies and non-polar interactions act in an unfavorable way for interactions between FosA and ANY1.

Assessment of the PETase conformational changes induced by poly(ethylene terephthalate) binding.

Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the ß1-ß2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the ß7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems.

QM/MM Study of the Fosfomycin Resistance Mechanism Involving FosB Enzyme.

Multidrug-resistant organisms contain antibiotic-modifying enzymes that facilitate resistance to a variety of antimicrobial compounds. Particularly, the fosfomycin (FOF) drug can be structurally modified by several FOF-modifying enzymes before it reaches the biological target. Among them, FosB is an enzyme that utilizes l-cysteine or bacillithiol in the presence of a divalent metal to open the epoxide ring of FOF and, consequently, inactivate the drug. Here, we have used hybrid quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of the reaction involving FosB and FOF. The calculated free-energy profiles show that the cost to open the epoxide ring of FOF at the C2 atom is ∼3.0 kcal/mol higher than that at the C1 atom. Besides, our QM/MM MD results revealed the critical role of conformation change of Cys9 and Asn50 to release the drug from the active site. Overall, the present study provides insights into the mechanism of FOF-resistant proteins.

Unraveling the conformational dynamics of glycerol 3-phosphate dehydrogenase, a nicotinamide adenine dinucleotide-dependent enzyme of Leishmania mexicana.

Allosteric changes modulate the enzymatic activity, leading to activation or inhibition of the molecular target. Understanding the induced fit accommodation mechanism of a ligand in its lowest-free energy state and the subsequent conformational changes induced in the protein are important questions for drug design. In the present study, molecular dynamics (MD) simulations, binding free energy calculations, and principal component analysis (PCA) were applied to analyze the glycerol-3-phosphate dehydrogenase of Leishmania mexicana (LmGPDH) conformational changes induced by its cofactor and substrate binding. GPDH is a nicotinamide adenine dinucleotide (NAD)-dependent enzyme, which has been reported as an interesting target for drug discovery and development against leishmaniasis. Despite its relevance for glycolysis and pentose phosphate pathways, the structural flexibility and conformational motions of LmGPDH in complex with NADH and dihydroxyacetone phosphate (DHAP) remain unexplored. Here, we analyzed the conformational dynamics of the enzyme-NADH complex (cofactor), and the enzyme-NADH-DHAP complex (adduct), mapped the hydrogen-bond interactions for the complexes and pointed some structural determinants of the enzyme that emerge from these contacts to NADH and DHAP. Finally, we proposed a consistent mechanism for the conformational changes on the first step of the reversible redox conversion of dihydroxyacetone phosphate to glycerol 3-phosphate, indicating key residues and interactions that could be further explored in drug discovery.

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 5'-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 2'-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 2'-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.

Evaluating the Performance of a Non-Bonded Cu2+ Model Including Jahn-Teller Effect into the Binding of Tyrosinase Inhibitors.

Tyrosinase (TYR) is a metalloenzyme classified as a type-3 copper protein, which is involved in the synthesis of melanin through a catalytic process beginning with the conversion of the amino acid l-Tyrosine (l-Tyr) to l-3,4-dihydroxyphenylalanine (l-DOPA). It plays an important role in the mechanism of melanogenesis in various organisms including mammals, plants, and fungi. Herein, we used a combination of computational molecular modeling techniques including molecular dynamic (MD) simulations and the linear interaction energy (LIE) model to evaluate the binding free energy of a set of analogs of kojic acid (KA) in complex with TYR. For the MD simulations, we used a dummy model including the description of the Jahn-Teller effect for Cu2+ ions in the active site of this enzyme. Our results show that the LIE model predicts the TYR binding affinities of the inhibitor in close agreement to experimental results. Overall, we demonstrate that the classical model provides a suitable description of the main interactions between analogs of KA and Cu2+ ions in the active site of TYR.

Investigation of the target-site resistance of EPSP synthase mutants P106T and T102I/P106S against glyphosate.

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) catalyzes the reaction involved in the production of amino acids essential for plant growth and survival. Thus, EPSPS is the main target of various herbicides, including glyphosate, a broad-spectrum herbicide that acts as a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. However, punctual mutations in the EPSPS gene have led to glyphosate resistance in some plants. Here, we investigated the mechanism of EPSPS resistance to glyphosate in mutants of two weed species, Conyza sumatrensis (mutant, P106T) and Eleusine indica (mutant, T102I/P106S), both of which have an economic impact on industrial crops. Molecular dynamics (MD) simulations and binding free energy calculations revealed the influence of the mutations on the affinity of glyphosate in the PEP-binding site. The amino acid residues of the EPSPS protein in both species involved in glyphosate resistance were elucidated as well as other residues that could be useful for protein engineering. In addition, during MD simulations, we identified conformational changes in glyphosate when complexed with resistant EPSPS, related to loss of herbicide activity and binding affinity. Our computational findings are consistent with previous experimental results and clarify the inhibitory activity of glyphosate as well as the structural target-site resistance of EPSPS against glyphosate.

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.

Exploring the Potentiality of Natural Products from Essential Oils as Inhibitors of Odorant-Binding Proteins: A Structure- and Ligand-Based Virtual Screening Approach To Find Novel Mosquito Repellents.

Odorant-binding proteins (OBPs) are the main olfactory proteins of mosquitoes, and their structures have been widely explored to develop new repellents. In the present study, we combined ligand- and structure-based virtual screening approaches using as a starting point 1633 compounds from 71 botanical families obtained from the Essential Oil Database (EssOilDB). Using as reference the crystallographic structure of N,N-diethyl-meta-toluamide interacting with the OBP1 homodimer of Anopheles gambiae (AgamOBP1), we performed a structural and pharmacophoric similarity search to select potential natural products from the library. Thymol acetate, 4-(4-methyl phenyl)-pentanal, thymyl isovalerate, and p-cymen-8-yl demonstrated a favorable chemical correlation with DEET and also had high-affinity interactions with the OBP binding pocket that molecular dynamics simulations showed to be stable. To the best of our knowledge, this is the first study to evaluate on a large scale the potentiality of NPs from essential oils as inhibitors of the mosquito OBP1 using in silico approaches. Our results could facilitate the design of novel repellents with improved selectivity and affinity to the protein binding pocket and can shed light on the mechanism of action of these compounds against insect olfactory recognition.

Molecular description of α-keto-based inhibitors of cruzain with activity against Chagas disease combining 3D-QSAR studies and molecular dynamics.

In this work, a group of α-keto-based inhibitors of the cruzain enzyme with anti-chagas activity was selected for a three-dimensional quantitative structure-activity relationship study (3D-QSAR) combined with molecular dynamics (MD). Firstly, statistical models based on Partial Least Square (PLS) regression were developed employing comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) descriptors. Validation parameters (q2 and r2 )for the models were, respectively, 0.910 and 0.997 (CoMFA) and 0.913 and 0.992 (CoMSIA). In addition, external validation for the models using a test group revealed r2pred  = 0.728 (CoMFA) and 0.971 (CoMSIA). The most relevant aspect in this study was the generation of molecular fields in both favorable and unfavorable regions based on the models developed. These fields are important to interpret modifications necessary to enhance the biological activities of the inhibitors. This analysis was restricted considering the inhibitors in a fixed conformation, not interacting with their target, the cruzain enzyme. Then, MD was employed taking into account important variables such as time and temperature. MD helped describe the behavior of the inhibitors and their properties showed similar results as those generated by QSAR-3D study.

Unraveling the Addition-Elimination Mechanism of EPSP Synthase through Computer Modeling.

Enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the hydroxyl group of shikimate-5-OH-3-phosphate (S3P) is catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase in a reaction that involves breaking the C-O bond of PEP. Catalysis involves an addition-elimination mechanism with the formation of a tetrahedral intermediate (THI). Experiments have elucidated the mechanism of THI formation and breakdown. However, the catalytic action of EPSP synthase and the individual roles of catalytic residues Asp313 and Glu341 remains unclear. We have used a hybrid quantum mechanical/molecular mechanical (QM/MM) approach to explore the free energy surface in a reaction catalyzed by EPSP synthase. The Glu341 was the most favorable acid/base catalyst. Our results indicate that the protonation of PEP C3 precedes the nucleophilic attack on PEP C2 in the addition mechanism. Also, the breaking of the C-O bond of THI to form an EPSP cation intermediate must occur before proton transfer from PEP C3 to Glu341 in the elimination mechanism. Analysis of the FES supports cationic intermediate formation during the reaction catalyzed by EPSP synthase. Finally, the computational model indicates a proton transfer shift (Hammond shift) from Glu341 to C3 for an enzyme-based reaction with the shifted transition state, earlier than in the reference reaction in water.