Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

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.

Evaluation interaction of graphene oxide with heparin for antiviral blockade: a study of ab initio simulations, molecular docking, and experimental analysis.

CONTEXT: Heparin, one of the drugs reused in studies with antiviral activity, was chosen to investigate a possible blockade of the SARS-CoV-2 spike protein for viral entry through computational simulations and experimental analysis. Heparin was associated to graphene oxide to increase in the binding affinity in biological system. First, the electronic and chemical interaction between the molecules was analyzed through ab initio simulations. Later, we evaluate the biological compatibility of the nanosystems, in the target of the spike protein, through molecular docking. The results show that graphene oxide interacts with the heparin with an increase in the affinity energy with the spike protein, indicating a possible increment in the antiviral activity. Experimental analysis of synthesis and morphology of the nanostructures were carried out, indicating heparin absorption by graphene oxide, confirming the results of the first principle simulations. Experimental tests were conducted on the structure and surface of the nanomaterial, confirming the heparin aggregation on the synthesis with a size between the GO layers of 7.44 Å, indicating a C-O type bond, and exhibiting a hydrophilic surface characteristic (36.2°). METHODS: Computational simulations of the ab initio with SIESTA code, LDA approximations, and an energy shift of 0.05 eV. Molecular docking simulations were performed in the AutoDock Vina software integrated with the AMDock Tools Software using the AMBER force field. GO, GO@2.5Heparin, and GO@5Heparin were synthesized by Hummers and impregnation methods, respectively, and characterized by X-ray diffraction and surface contact angle.

Nitriles: an attractive approach to the development of covalent inhibitors.

Nitriles have broad applications in medicinal chemistry, with more than 60 small molecule drugs on the market containing the cyano functional group. In addition to the well-known noncovalent interactions that nitriles can perform with macromolecular targets, they are also known to improve drug candidates' pharmacokinetic profiles. Moreover, the cyano group can be used as an electrophilic warhead to covalently bind an inhibitor to a target of interest, forming a covalent adduct, a strategy that can present benefits over noncovalent inhibitors. This approach has gained much notoriety in recent years, mainly with diabetes and COVID-19-approved drugs. Nevertheless, the application of nitriles in covalent ligands is not restricted to it being the reactive center, as it can also be employed to convert irreversible inhibitors into reversible ones, a promising strategy for kinase inhibition and protein degradation. In this review, we introduce and discuss the roles of the cyano group in covalent inhibitors, how to tune its reactivity and the possibility of achieving selectivity only by replacing the warhead. Finally, we provide an overview of nitrile-based covalent compounds in approved drugs and inhibitors recently described in the literature.

Experimental and Computational Study of Aryl-thiosemicarbazones Inhibiting Cruzain Reveals Reversible Inhibition and a Stepwise Mechanism.

Trypanosoma cruzi is a parasite that infects about 6-7 million people worldwide, mostly in Latin America, causing Chagas disease. Cruzain, the main cysteine protease of T. cruzi, is a validated target for developing drug candidates for Chagas disease. Thiosemicarbazones are one of the most relevant warheads used in covalent inhibitors targeting cruzain. Despite its relevance, the mechanism of inhibition of cruzain by thiosemicarbazones is unknown. Here, we combined experiments and simulations to unveil the covalent inhibition mechanism of cruzain by a thiosemicarbazone-based inhibitor (compound 1). Additionally, we studied a semicarbazone (compound 2), which is structurally similar to compound 1 but does not inhibit cruzain. Assays confirmed the reversibility of inhibition by compound 1 and suggested a two-step mechanism of inhibition. The Ki was estimated to be 36.3 µM and Ki* to be 11.5 µM, suggesting the pre-covalent complex to be relevant for inhibition. Molecular dynamics simulations of compounds 1 and 2 with cruzain were used to propose putative binding modes for the ligands. One-dimensional (1D) quantum mechanics/molecular mechanics (QM/MM) potential of mean force (PMF) and gas-phase energies showed that the attack of Cys25-S- on the C═S or C═O bond yields a more stable intermediate than the attack on the C═N bond of the thiosemicarbazone/semicarbazone. Two-dimensional (2D) QM/MM PMF revealed a putative reaction mechanism for compound 1, involving the proton transfer to the ligand, followed by the Cys25-S- attack at C═S. The ΔG and energy barrier were estimated to be -1.4 and 11.7 kcal/mol, respectively. Overall, our results shed light on the inhibition mechanism of cruzain by thiosemicarbazones.

Assessment of Reversibility for Covalent Cysteine Protease Inhibitors Using Quantum Mechanics/Molecular Mechanics Free Energy Surfaces.

We have used molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials to investigate the reaction mechanism for covalent inhibition of cathepsin K and assess the reversibility of inhibition. The computed free energy profiles suggest that a nucleophilic attack by the catalytic cysteine on the inhibitor warhead and proton transfer from the catalytic histidine occur in a concerted manner. The results indicate that the reaction is more strongly exergonic for the alkyne-based inhibitors, which bind irreversibly to cathepsin K, than for the nitrile-based inhibitor odanacatib, which binds reversibly. Gas-phase energies were also calculated for the addition of methanethiol to structural prototypes for a number of warheads of interest in cysteine protease inhibitor design in order to assess electrophilicity. The approaches presented in this study are particularly applicable to assessment of novel warheads, and computed transition state geometries can be incorporated into molecular models for covalent docking.

Metabolic Processing of Selenium-Based Bioisosteres of meso-Diaminopimelic Acid in Live Bacteria.

A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is composed of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are targets of potent antibiotics as proper assembly of the PG is essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study the bacterial cell structure, growth, and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to access synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of m-DAP (selenolanthionine) and show that it is installed within the PG of live bacteria by the native cell wall crosslinking machinery in mycobacterial species. This PG probe has an orthogonal release mechanism that could be important for downstream proteomics studies. Finally, we describe a bead-based assay that is compatible with high-throughput screening of cell wall enzymes. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP-containing organisms.

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.

A patent review on cathepsin K inhibitors to treat osteoporosis (2011 - 2021).

INTRODUCTION: Cathepsin K (CatK) is a lysosomal cysteine protease and the predominant cathepsin expressed in osteoclasts, where it degrades the bone matrix. Hence, CatK is an attractive therapeutic target related to diseases characterized by bone resorption, like osteoporosis. AREAS COVERED: This review summarizes the patent literature from 2011 to 2021 on CatK inhibitors and their potential use as new treatments for osteoporosis. The inhibitors were classified by their warheads, with the most explored nitrile-based inhibitors. Promising in vivo results have also been disclosed. EXPERT OPINION: As one of the most potent lysosomal proteins whose primary function is to mediate bone resorption, cathepsin K remains an excellent target for therapeutic intervention. Nevertheless, there is no record of any approved drug that targets CatK. The most notable cases of drug candidates targeting CatK were balicatib and odanacatib, which reached Phase II and III clinical trials, respectively, but did not enter the market. Further developments include exploring new chemical entities beyond the nitrile-based chemical space, with improved ADME and safety profiles. In addition, CatK's role in cancer immunoexpression and its involvement in the pathophysiology of osteo- and rheumatoid arthritis have raised the race to develop activity-based probes with excellent potency and selectivity.

Diversity of bacteriocins in the microbiome of the Tucuruí Hydroelectric Power Plant water reservoir and three-dimensional structure prediction of a zoocin.

Bacteriocins are antimicrobial peptides expressed by bacteria through ribosomal activity. In this study, we analyzed the diversity of bacteriocin-like genes in the Tucuruí-HPP using a whole-metagenome shotgun sequencing approach. Three layers of the water column were analyzed (photic, aphotic and sediment). Detection of bacteriocin-like genes was performed with blastx using the BAGEL4 database as subject sequences. In order to calculate the abundance of bacteriocin-like genes we also determined the number of 16S rRNA genes using blastn. Taxonomic analysis was performed using RAST server and the metagenome was assembled using IDBA-UD in order to recover the full sequence of a zoocin which had its three-dimensional structure determined. The photic zone presented the highest number of reads affiliated to bacteriocins. The most abundant bacteriocins were sonorensin, Klebicin D , pyocin and colicin. The zoocin model was composed of eight anti-parallel ß-sheets and two α-helices with a Zn2+ ion in the active site. This model was considerably stable during 10 ns of molecular dynamics simulation. We observed a high diversity of bacteriocins in the Tucuruí-HPP, demonstrating that the environment is an inexhaustible source for prospecting these molecules. Finally, the zoocin model can be used for further studies of substrate binding and molecular mechanisms involving peptidoglycan degradation.

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.

Predicting the Relative Binding Affinity for Reversible Covalent Inhibitors by Free Energy Perturbation Calculations.

Covalent inhibitors are assuming central importance in drug discovery projects, especially in this pandemic scenario. Many research groups have focused their attention on inhibiting viral proteases or human proteases such as cathepsin L (hCatL). The inhibition of these critical enzymes may impair viral replication. However, molecular modeling of covalent ligands is challenging since covalent and noncovalent ligand-bound states must be considered in the binding process. In this work, we evaluated the suitability of free energy perturbation (FEP) calculations as a tool for predicting the binding affinity of reversible covalent inhibitors of hCatL. Our strategy relies on the relative free energy calculated for both covalent and noncovalent complexes and the free energy changes have been compared with experimental data for eight nitrile-based inhibitors, including three new inhibitors of hCatL. Our results demonstrate that the covalent complex can be employed to properly rank the inhibitors. Nevertheless, a comparison of the free energy changes in both noncovalent and covalent states is valuable to interpret the effect triggered by the formation of the covalent bond on the interactions played by functional groups distant from the warhead. Overall, FEP can be employed as a powerful predictor tool in developing and understanding the activity of reversible covalent inhibitors.

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.

Applications of Virtual Screening in Bioprospecting: Facts, Shifts, and Perspectives to Explore the Chemo-Structural Diversity of Natural Products.

Natural products are continually explored in the development of new bioactive compounds with industrial applications, attracting the attention of scientific research efforts due to their pharmacophore-like structures, pharmacokinetic properties, and unique chemical space. The systematic search for natural sources to obtain valuable molecules to develop products with commercial value and industrial purposes remains the most challenging task in bioprospecting. Virtual screening strategies have innovated the discovery of novel bioactive molecules assessing in silico large compound libraries, favoring the analysis of their chemical space, pharmacodynamics, and their pharmacokinetic properties, thus leading to the reduction of financial efforts, infrastructure, and time involved in the process of discovering new chemical entities. Herein, we discuss the computational approaches and methods developed to explore the chemo-structural diversity of natural products, focusing on the main paradigms involved in the discovery and screening of bioactive compounds from natural sources, placing particular emphasis on artificial intelligence, cheminformatics methods, and big data analyses.

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.