Enzyme-ligand Complexes Research Articles

Valproic acid as a potential inhibitor of Plasmodium falciparum histone deacetylase 1 (PfHDAC1): an in silico approach.

A new Plasmodium falciparum histone deacetylase1 (PfHDAC1) homology model was built based on the highest sequence identity available template human histone deacetylase 2 structure. The generated model was carefully evaluated for stereochemical accuracy, folding correctness and overall structure quality. All evaluations were acceptable and consistent. Docking a group of hydroxamic acid histone deacetylase inhibitors and valproic acid has shown binding poses that agree well with inhibitor-bound histone deacetylase-solved structural interactions. Docking affinity dG scores were in agreement with available experimental binding affinities. Further, enzyme-ligand complex stability and reliability were investigated by running 5-nanosecond molecular dynamics simulations. Thorough analysis of the simulation trajectories has shown that enzyme-ligand complexes were stable during the simulation period. Interestingly, the calculated theoretical binding energies of the docked hydroxamic acid inhibitors have shown that the model can discriminate between strong and weaker inhibitors and agrees well with the experimental affinities reported in the literature. The model and the docking methodology can be used in screening virtual libraries for PfHDAC1 inhibitors, since the docking scores have ranked ligands in accordance with experimental binding affinities. Valproic acid calculated theoretical binding energy suggests that it may inhibit PfHDAC1.

DRUG-RECEPTOR INTERACTIONS:IN SILICOAPPROACHES APPLIED TO EXPERIMENTAL CLASSES REGARDING THE EVOLUTION OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS

Teaching classes and events regarding the molecular aspects of drug-receptor interactions is not an easy task. The ligand stereochemistry and the spatial arrangement of the macromolecular targets highly increase the complexity of the process. In this context, the use of alternative and more playful approaches could allow students to gain a more thorough understanding of this important topic in medicinal chemistry. Herein, we describe a practical teaching approach that uses computational strategies as a tool for drug-receptor interaction studies performed for angiotencsin converting enzyme inhibitors (ACEi). Firstly, the students learn how to find the crystallographic structure (enzyme-ligand complex). Then, they proceed to the treatment of crude crystallographic data. Thereafter, they learn how to analyze the positioning of the drug on the active site of the enzyme, looking for regions related to the molecular recognition. At the end of the study, students can summarize the molecular requirements for the interaction and the structure-activity relationships of the studied drugs.

Effects of Molecular Crowding on the Binding Affinity of Dihydrofolate for Dihydrofolate Reductase

The reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) by the enzyme dihydrofolate reductase (DHFR), using NADPH as a cofactor, is an essential part of the folate cycle. The inhibition of DHFR leads to interruption of DNA synthesis and consequently cell death, making this enzyme a crucial target in the treatment of cancer and other diseases. Previous studies examined the effects of small molecule osmolytes on the substrate interactions with two non-homologous DHFRs, E. coli chromosomal DHFR (EcDHFR) and R67 DHFR, with vastly different active site structures. The results indicated that DHF weakly interacts with the osmolytes in solution, shifting the binding equilibrium from DHF bound to DHFR to unbound DHF. It is hypothesized that similar weak, nonspecific interactions may also occur between cellular proteins and DHF. Weak interactions between cellular proteins and DHF would have consequences in vivo, where the concentration of the cellular milieu is approximately 300 g/L. Under the crowded conditions in the cell, there is a higher propensity for intermolecular interaction.Crowding effects of macromolecules in concentrations similar to those in vivo were examined. Isothermal titration calorimetry (ITC) and enzyme kinetic assays were used to detect effects of molecular crowders by monitoring activity of the (DHFR)-NADPH or DHF complex and the ternary DHFR-DHF-NADPH complex in the presence of these crowders. To recreate the conditions of molecular crowding in vivo, the binding of the enzyme-ligand complexes in the presence of molecular weight crowding agents (lysozyme or casein) was examined. Analysis of the Kd's and Km's indicated a correlation between increased molecular crowding in the solution and weakened binding of the DHFR-substrate complexes. These findings indicate an importance of molecular crowding on EcDHFR activity in vivo.

Evaluation of 11 scoring functions performance on matrix metalloproteinases.

Matrix metalloproteinases (MMPs) have distinctive roles in various physiological and pathological processes such as inflammatory diseases and cancer. This study explored the performance of eleven scoring functions (D-Score, G-Score, ChemScore, F-Score, PMF-Score, PoseScore, RankScore, DSX, and X-Score and scoring functions of AutoDock4.1 and AutoDockVina). Their performance was judged by calculation of their correlations to experimental binding affinities of 3D ligand-enzyme complexes of MMP family. Furthermore, they were evaluated for their ability in reranking virtual screening study results performed on a member of MMP family (MMP-12). Enrichment factor at different levels and receiver operating characteristics (ROC) curves were used to assess their performance. Finally, we have developed a PCA model from the best functions. Of the scoring functions evaluated, F-Score, DSX, and ChemScore were the best overall performers in prediction of MMPs-inhibitors binding affinities while ChemScore, Autodock, and DSX had the best discriminative power in virtual screening against the MMP-12 target. Consensus scorings did not show statistically significant superiority over the other scorings methods in correlation study while PCA model which consists of ChemScore, Autodock, and DSX improved overall enrichment. Outcome of this study could be useful for the setting up of a suitable scoring protocol, resulting in enrichment of MMPs inhibitors.

Discovery of novel covalent proteasome inhibitors through a combination of pharmacophore screening, covalent docking, and molecular dynamics simulations.

The ubiquitin-proteasome pathway plays a pivotal role in the regulation of cellular protein processing and degradation. Proteasome inhibitors (PIs) have enormous potential to treat multiple myeloma, solid tumors, parasites, inflammation, and immune diseases, which is spurring the development of new types of PIs with enhanced efficacy, fewer side effects, and reduced drug resistance. Nevertheless, virtual screening for covalent PIs has rarely been reported because calculating the covalent binding energy is a challenging task. The aim of this study was to discover new covalent inhibitors of the 20S proteasome. The structures of PIs were manually divided into two parts: a noncovalent binding part resulting from virtual screening, and an epoxyketone group that was pre-selected as a covalent binding part. The SPECS database was screened by noncovalent docking and a pharmacophore model built with the 20S proteasome. After validating the covalent conjugation, 88 hits with epoxyketone were covalently docked into the 20S proteasome to analyze the intermolecular interactions. Four compounds were selected after multiple filtration and validations. Molecular dynamics simulations were performed to check the stability of the noncovalent and covalent docked ligand-enzyme complexes and investigate the interaction patterns of the screened inhibitors. Finally, two compounds with novel aromatic backbones, reasonable interactions, and stable covalent binding modes were retained. These compounds can serve as potential hits for further biological evaluation.

Mis-sesnse mutations in Tafazzin (TAZ) that escort to mild clinical symptoms of Barth syndrome is owed to the minimal inhibitory effect of the mutations on the enzyme function: In-silico evidence

Tafazzin (EC 2.3.1.23) is a Phospholipid Transacylase involved in Cardiolipin remodeling on mitochondrial membrane and coded by TAZ gene (Cytogenetic Location: Xq28) in human. Its mutations cause Barth syndrome (MIM ID: #302060)/3-Methyl Glutaconyl Aciduria Type II, an inborn error of metabolism often leading to foetal or infantile fatality. Nevertheless, some mis-sense mutations result in mild clinical symptoms. To evaluate the rationale of mild symptoms and for an insight of Tafazzin active site, sequence based and structure based ramifications of wild and mutant Tafazzins were compared in-silico. Sequence based domain predictions, surface accessibilities on substitution & conserved catalytic sites with statistical drifts, as well as thermal stability changes for the mutations and the interaction analysis of Tafazzin were performed. Crystal structure of Tafazzin is not yet resolved experimentally, therefore 3D coordinates of Tafazzin and its mutants were spawned through homology modeling. Energetically minimized and structurally validated models were used for comparative docking simulations. We analyzed active site geometry of the models in addition to calculating overall substrate binding efficiencies for each of the enzyme-ligand complex deduced from binding energies instead of comparing only the docking scores. Also, individual binding energies of catalytic residues on conserved HX4D motif of Acyltransferase superfamily present in Tafazzins were estimated. This work elucidates the basis of mild symptoms in patients with mis-sense mutations, identifies the most pathogenic mutant among others in the study and also divulges the critical role of HX4D domain towards successful transacylation by Taffazin. The in-silico observations are in complete agreement with clinical findings reported for the patients with mutations.

Computational study on substrate specificity of a novel cysteine protease 1 precursor from Zea mays.

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.

Design of new and potent diethyl thiobarbiturates as urease inhibitors: a computational approach.

Urease is an important enzyme both in agriculture and medicine research. Strategies based on urease inhibition is critically considered as the first line treatment of infections caused by urease producing bacteria. Since, urease possess agro-chemical and medicinal importance, thus, it is necessary to search for the novel compounds capable of inhibiting this enzyme. Several computational methods were employed to design novel and potent urease inhibitors in this work. First docking simulations of known compounds consists of a set of arylidine barbiturates (termed as reference) were performed on the Bacillus pasteurii (BP) urease. Subsequently, two fold strategies were used to design new compounds against urease. Stage 1 comprised of the energy minimization of enzyme-ligand complexes of reference compounds and the accurate prediction of the molecular mechanics generalized born (MMGB) interaction energies. In the second stage, new urease inhibitors were then designed by the substitution of different groups consecutively in the aryl ring of the thiobarbiturates and N, N-diethyl thiobarbiturates of the reference ligands.. The enzyme-ligand complexes with lowest interaction energies or energies close to the calculated interaction energies of the reference molecules, were selected for the consequent chemical manipulation. This was followed by the substitution of different groups on the 2 and 5 positions of the aryl ring. As a result, several new and potent diethyl thiobarbiturates were predicted as urease inhibitors. This approach reflects a logical progression for early stage drug discovery that can be exploited to successfully identify potential drug candidates.

Structure and dynamics studies of sterol 24-C-methyltransferase with mechanism based inactivators for the disruption of ergosterol biosynthesis

The enzyme sterol 24-C-methyltransferase (SMT) belongs to the family of transferases, specifically to the one-carbon transferring methyltransferases. SMT has been found playing a major role during the production of steroids, especially for the biosynthesis of ergosterol, which is the major membrane sterol in leishmania parasites, causing leishmaniasis. However, SMT and ergosterol are not found in mammals, so, an extensive study has been carried out over the susceptible SMT protein, which is found to be highly conserved among all the Leishmania species and holds a significant anti-leishmanial drug target. To date, there is no computational data available for SMT, due to its highly unexplored profile. In this work, a complete set of structural attributes have been examined through the available computational procedures, along with an attempt to characterize the most capable modeling server available. The exploration ranges from physicochemical characterization, pairwise alignment, secondary structure prediction, to active site detection. With this information, a docking study was carried out to find the compound that best binds into the active site. Moreover, molecular dynamics simulation was conducted to examine the stability of the homology modeled protein and the ligand-enzyme complex. The results indicate that the ligand-enzyme complex is more stable.

Elucidating Substrate Promiscuity in the Human Cytochrome 3A4

The human cytochrome P450 enzymes (CYPs) are heme-protein monooxygenases, which catalyze oxidative reactions of a broad spectrum of substrates. Consequently, they play a critical role in the metabolism of xenobiotics, such as drugs and carcinogens, and the catabolism of endogenous lipophilic factors. Bioavailability and toxicity, both of which can be related to CYPs, continue to pose problems in the development of new drugs. The isoform which metabolizes over one-third of drugs, CYP 3A4, was investigated employing ensemble-docking experiments of a 195-substrate library with induced fit and GOLD docking algorithms and a number of scoring functions. Enzyme conformations included three currently available CYP 3A4 crystal structures. All docking experiments were performed in duplicates with and without inclusion of crystallographic waters. Resultant poses were assessed based on accuracy of site of metabolism prediction. Analyses of the docked solutions pertaining to ranking efficacy, ligand molecular properties, stabilizing residues in the ligand-enzyme complexes, and metabolic reactions are discussed. Our analyses suggest that certain residues make favorable interactions with the bound substrates. Employing multiple receptor conformations enhances the accuracy of catalytic prediction, while ligand size and flexibility impact docking performance. The presence of waters observed in crystal complexes does not necessarily lead to improved performance.

Promotion of Enzyme Flexibility by Dephosphorylation and Coupling to the Catalytic Mechanism of a Phosphohexomutase

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.

Conformational Change and Ligand Binding in the Aristolochene Synthase Catalytic Cycle

Terpene synthases are potentially useful biocatalysts for the synthesis of valuable compounds, such as anticancer drugs and antibiotics. The design of altered activities requires better knowledge of their mechanisms, for example, an understanding of the complex conformational changes that are part of their catalytic cycle, how they are coordinated, and what drives them. Crystallographic studies of the sesquiterpene synthase artistolochene synthase have led to a proposed sequence of ligand binding and conformational change but have provided only indirect insight. Here, we have performed extensive molecular dynamics simulations of multiple enzyme-ligand complexes (over 2 μs in total). The simulations provide clear evidence of what drives the conformational changes required for reaction. They support a picture in which the substrate farnesyl diphosphate binds first, followed by three magnesium ions in sequence, and, after reaction, the release of aristolochene and two magnesium ions followed by the final magnesium ion and diphosphate. Binding of farnesyl diphosphate leads to an increased level of sampling of open conformations, allowing the first two magnesium ions to bind. The closed enzyme conformation is maintained with a diphosphate moiety and two magnesium ions bound. The open-to-closed transition reduces flexibility around the active site entrance, partly through a lid closing over it. The simulations with all three magnesium ions and farnesyl diphosphate bound provide, for the first time, a realistic model of the Michaelis complex involved in reaction, which is inaccessible to experimental structural studies. These insights could help with the design of altered activities in a range of terpene synthases.

Identification of irreversible protein splicing inhibitors as potential anti-TB drugs: insight from hybrid non-covalent/covalent docking virtual screening and molecular dynamics simulations

Tuberculosis is responsible for ~3 million deaths annually and is one of the most prevalent infectious diseases known to mankind. Despite ongoing developments in medicine, the emergence of drug resistant Mycobacterium tuberculosis remains of great interest, specifically in developing countries where medical treatment is not readily available. The aim of this study is to identify and explore the binding affinities of novel potential inhibitors that can irreversibly inhibit the intein protein via a covalent bond formation with its active site cysteine. Our search for new leads as potential protein splicing inhibitors is based on Michael acceptor-like structures since they are strong electrophiles which react covalently with the nucleophilic cysteine SH group in the enzyme active site. Structure-based virtual screening using a hybrid non-covalent/covalent docking was performed. Furthermore, molecular dynamic simulations (MD) and extensive post-dynamic analysis were performed in order to ensure the stability of the docked ligand-enzyme complexes and provide insight into the binding affinities and interaction patterns of the screened inhibitors. Interestingly, three novel hits have shown better binding affinity in comparison to experimentally determined compounds with known protein splicing inhibitory activity. MD simulations also revealed that the docked compounds are fairly stable in the protein active site. Per-residue interaction analysis has highlighted the most important active site residues contributing to the inhibitor binding.

Optimization of N-Benzoylindazole Derivatives as Inhibitors of Human Neutrophil Elastase

Human neutrophil elastase (HNE) is an important therapeutic target for treatment of pulmonary diseases. Previously, we identified novel N-benzoylindazole derivatives as potent, competitive, and pseudoirreversible HNE inhibitors. Here, we report further development of these inhibitors with improved potency, protease selectivity, and stability compared to our previous leads. Introduction of a variety of substituents at position 5 of the indazole resulted in the potent inhibitor 20f (IC50 ∼10 nM) and modifications at position 3 resulted the most potent compound in this series, the 3-CN derivative 5b (IC50 = 7 nM); both derivatives demonstrated good stability and specificity for HNE versus other serine proteases. Molecular docking of selected N-benzoylindazoles into the HNE binding domain suggested that inhibitory activity depended on geometry of the ligand-enzyme complexes. Indeed, the ability of a ligand to form a Michaelis complex and favorable conditions for proton transfer between Hys57, Asp102, and Ser195 both affected activity.

Interaction between Trehalose and MTHase from Sulfolobus solfataricus Studied by Theoretical Computation and Site-Directed Mutagenesis

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose. Computer simulation using the hydrogen bond analysis, free energy decomposition, and computational alanine scanning were employed to investigate the interaction between maltooligosyltrehalose and the enzyme. The same residues that were chosen for theoretical investigation were also studied by site-directed mutagenesis and enzyme kinetic analysis. The importance of residues determined either experimentally or computed theoretically were in good accord with each other. It was found that residues Y155, D156, and W218 of subsites -2 and -3 of the enzyme might play an important role in interacting with the ligand. The theoretically constructed structure of the enzyme-ligand complex was further validated through an ab initio quantum chemical calculation using the Gaussian09 package. The activation energy computed from this latter study was very similar to those reported in literatures for the same type of hydrolysis reactions.

Structural and thermodynamic basis of (+)-α-pinene binding to human cytochrome P450 2B6.

Despite recent advances in atomic-level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-α-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-α-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-α-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-α-pinene complex has a significant enthalpic component. A 2.0 Å resolution crystal structure of this enzyme-ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility.

In-silico analysis of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK): a potential drug target against Salmonella enterica serovar typhi

Salmonella enterica serovar typhi is a gram-negative pathogenic bacterium that inhabits the lymphatic tissues of the small intestine, liver, spleen and bloodstream of infected humans. It spreads mainly through contaminated food and drinks. It is estimated that nearly 16–33 million infections occur annually and cause 216,000 deaths in endemic areas. Its incidence is high in children and young adults between 5 and 19 years of age. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase is one of the key enzymes that play a vital role in folate metabolism which is important for bacterial growth and survival. The absence of this enzyme in higher eukaryotes, including mammals, makes the enzyme an excellent target for designing antimicrobial agents. As the structure of this enzyme has not been experimentally determined, a 3-dimensional model of this target enzyme was generated by comparative modelling programme Modeller. Since the magnesium ions are needed for enzyme activity, Mg2+ ions were complexed with the predicted protein model and molecular docking studies were carried out with the possible inhibitors using Schrodinger’s Glide. From this study, it has been shown that the lead molecules chosen bind with the receptor molecule with better binding energy. The top scoring enzyme–lead complex having a Glide energy of −171.98259 was further validated through simulation studies using Gromacs simulation suite. The simulation study confirms a stable enzyme–ligand complex.

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC50=150nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC50=25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC50=7.5nM) and TNF-α production in mouse splenocytes (IC50=9.8nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID50=18mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand–enzyme complex.

Study of binding of pyridoacridine alkaloids on topoisomerase II using in silico tools

In this research, the pyridoacridines alkaloids have been docked computationally to the active site of Topoisomerase II using four PDB structures (1PVG, 1QZR, 1AJ6 and 1ZXM). iGEMDOCK 2.1, AutoDock Vina 1.1.2 and AutoDock 4.2.1 were employed to perform the automated molecular docking. The results of docking studies generated docking scores and IC50 values. Moreover, 3D pictures of ligand enzyme complexes afforded valuable data regarding the binding orientation of each inhibitor in the active site of Topoisomerase II.

Homology modeling, docking and molecular dynamics of the Leishmania mexicana arginase: A description of the catalytic site useful for drug design

The crystallographic structure of the Leishmania mexicana arginase, an attractive target for the design of leishmanicidal agents, is still unknown. For this reason, we report a computer-assisted homology study conducted to build its three-dimensional structure based on the known sequence of amino acids of this enzyme. In this study, the amino acid sequence in the arginase of the parasite was compared with the sequence of the amino acids in the crystallographic structure of rat and human liver arginases. The best similarity was found with the rat liver arginase. The catalytic site of the three-dimensional arginase structure built for L. mexicana has important structural differences as compared with that of the human liver arginase with regard to reasonable design selective compounds against L. mexicana. With this information, a docking study was conducted to find the inhibitors of this enzyme. 1439 molecules were docked and 18 were selective to the L. mexicana arginase. Moreover, molecular dynamics were carried out to study the stability of the homologue protein (including manganeses) and the ligand–enzyme complex. The results indicated that the manganese remains inside the protein throughout the simulation. Besides, hydrogen bonds interactions between the ligand and the arginase were analyzed. These results provide important information for the design of new inhibitors.