Enzyme Substrate Research Articles

The effect of buffer pH on enzyme activity

This study investigates the effect of pH on enzyme-catalyzed activity, with an emphasis on the involvement of wheat germ acid phosphatase in dephosphorylation processes. Temperature, pH, and enzyme concentration all have a substantial impact on enzyme activity and substrate interaction. Wheat germ acid phosphatase was chosen for this work. The activity of the enzyme in dephosphorylating p-nitrophenyl phosphate (p-NPP) into p-nitrophenol (p-NP) was evaluated using a stopped assay approach. The reaction was stopped by introducing sodium hydroxide, which ionises p-NP and produces a yellow anion visible at 405 nm. The experiment measured product production in nmol per minute and included three sections: Created a standard for the amount of p-NP with its absorbance at 405 nm; Found the ideal incubation period for maximal product generation by wheat germ acid phosphatase; Evaluated the effect of pH on enzyme activity measuring the first reaction velocity in various pH settings. The results showed that when the buffer pH diverged from the ideal value of 5.5, catalysis rates reduced dramatically, implying that high pH levels may modify the protein structure of the enzyme and reduce its catalytic effectiveness. This work emphasises the importance of pH in enzymatic activity and its potential impacts on enzyme structural integrity.

Probe Substrate Dependencies in CYP3A4 Allosteric Inhibition: A Novel Molecular Mechanism Involving F-F' Loop Perturbations.

The biochemical basis for substrate dependences in apparent inhibition constant values (Ki) remains unknown. Our study aims to elucidate plausible structural determinants underpinning these observations. In vitro steady-state inhibition assays conducted using human recombinant CYP3A4 enzyme and testosterone substrate revealed that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9 μM, respectively. However, when rivaroxaban was adopted as the probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into this phenomenon, erdafitinib and pemigatinib were docked to allosteric sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations of apo- and holo-CYP3A4 were conducted to investigate the structural changes induced. Comparative structural analyses of representative MD frames extracted by hierarchical clustering revealed that the allosteric inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially modulate its active site characteristics. In contrast, we discovered that allosteric binding of the FGFR inhibitors reduces the structural flexibility of the F-F' loop region, an important gating mechanism to regulate access of the substrate to the catalytic heme. We surmised that the increased rigidity of the F-F' loop engenders a more constrained entrance to the CYP3A4 active site, which in turn impedes access to the larger rivaroxaban molecule to a greater extent than testosterone and culminates in more potent inhibition of its CYP3A4-mediated metabolism. Our findings suggest a potential mechanism to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of CYP3A4.

Assessing viability of Escherichia coli in mining-impacted surface waters

Multiple threats to surface water quality, including both fecal pollution and acid mine drainage (AMD) are frequently coincident in mining regions, such as parts of the Appalachian region of the U.S. While it has been established that AMD can have toxic effects to fecal bacteria in surface waters, there is also evidence that AMD may induce a viable but non-culturable (VBNC) state, leaving fecal bacteria undetectable by common standard methods. This study aims to better understand the occurrence of VBNC Escherichia coli in mining-impacted waters (MIW) through three objectives: (1) assess the relationship between MIWs and concentrations of culturable fecal bacteria, (2) compare standard methods for quantification of E. coli in MIW, and (3) investigate whether MIW can induce a VBNC state in E. coli. An analysis of historic data from the Deckers Creek and West Run Watersheds of West Virginia revealed a moderate correlation between pH and E. coli abundance determined via culture and enzyme-based methods. In samples collected from sites within the two watersheds impacted by historic mining activities, the E. coli uidA gene was measured via quantitative polymerase chain reaction (qPCR) and found to be significantly more abundant than E. coli concentrations measured via culture or enzyme substrate coliform test. However, this discrepancy between methods was greatest in samples with a low pH. Further, experiments in which an environmental strain of E. coli was dosed into samples of MIW diluted with saline at different concentrations confirmed that the culturability of E. coli significantly decreased with increasing MIW concentration, while the total number of intact cells, determined via "live/dead” staining and microscopy, remained high. This finding suggests that culture and enzyme methods may underestimate viable E. coli levels in AMD-impacted waters.

Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME). In colorectal liver metastasis (CLM), TAM morphology correlates with prognosis, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger TAMs (L-TAMs). However, the metabolic profile of in vivo human TAM populations remains unknown. Multiparametric flow cytometry was used to freshly isolate S- and L-TAMs from surgically resected CLM patients (n = 14S-, 14L-TAMs). Mass spectrometry-based metabolomics analyses were implemented for the metabolic characterization of TAM populations. Gene expression analysis and protein activity were used to support the biochemical effects of the enzyme–substrate link between riboflavin and (lysine-specific demethylase 1A, LSD1) with TAM morphologies. L-TAMs were characterized by a positive correlation and a strong association between riboflavin and TAM morphologies. Riboflavin in both L-TAMs and in-vitro M2 polarized macrophages modulates LSD1 protein expression and activity. The inflammatory stimuli promoted by TNFα induced the increased expression of riboflavin transporter SLC52A3 and LSD1 in M2 macrophages. The modulation of the riboflavin-LSD1 axis represents a potential target for reprogramming TAM subtypes, paving the way for promising anti-tumor therapeutic strategies.

Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes

In the last decades enormous advances have been made in characterizing the atomic and molecular structure of respiratory chain supercomplexes [1]. However, it still remains a challenge to stitch this refined spatial atomistic description with functional information provided by biochemical studies of isolated protein material. Development of functional assays that detect respiratory chain complexes in their native membrane environment contribute to address the open questions related to the role played by their association and interactions. We present a characterization assay in which a functionalized gold electrode is modified with mitochondrial membrane fragments that allows monitoring electrochemically the activity of different respiratory chain complexes immersed in the mitochondrial membrane. We measure the intensity of the reducing current of the electron mediator CoQ1 at the electrode surface and its variation upon addition of the corresponding enzymatic substrates. The activities of Complex I, Complex II and Complex III were monitored by the way in which they reduce the current, reflecting the amount of quinone reduced by the complexes in the presence of their substrates. We detect that CoQ1H2 produced by Complex I remains partially trapped within the membrane and is more easily oxidized by Complex III or the electrode than the quinone reduced by Complex II.

Substrate-Selective Catalysis Enabled Synthesis of Azaphilone Natural Products.

Achieving substrate-selectivity is a central element of nature's approach to synthesis. By relying on the ability of a catalyst to discriminate between components in a mixture, control can be exerted over which molecules will move forward in a synthesis. This approach can be powerful when realized but can be challenging to duplicate in the laboratory. In this work, substrate-selective catalysis is leveraged to discriminate between two intermediates that exist in equilibrium, subsequently directing the final cyclization to arrive at either the linear or angular tricyclic core common to subsets of azaphilone natural products. By using a flavin-dependent monooxygenase (FDMO) in sequence with an acyl transferase (AT), the conversion of several orcinaldehyde substrates directly to the corresponding linear tricyclic azaphilones in a single reaction vessel was achieved. Further, mechanistic studies support that a substrate equilibrium together with enzyme substrate selectivity play an import role in the selectivity of the final cyclization step. Using this strategy, five azaphilone natural products were synthesized for the first time as well as a number of unnatural derivatives thereof.

Discovering New Substrates of a UDP-Glycosyltransferase with a High-Throughput Method.

UDP-glycosyltransferases (UGTs) form a large enzyme family that is found in a wide range of organisms. These enzymes are known for accepting a wide variety of substrates, and they derivatize xenobiotics and metabolites for detoxification. However, most UGT homologs have not been well characterized, and their potential for biomedical and environmental applications is underexplored. In this work, we have used a fluorescent assay for screening substrates of a plant UGT homolog by monitoring the formation of UDP. We optimized the assay such that it could be used for high-throughput screening of substrates of the Medicago truncatula UGT enzyme, UGT71G1, and our results show that 34 of the 159 screened compound samples are potential substrates. With an LC-MS/MS method, we confirmed that three of these candidates indeed were glycosylated by UGT71G1, which includes bisphenol A (BPA) and 7-Ethyl-10-hydroxycamptothecin (SN-38); derivatization of these toxic compounds can lead to new environmental and medical applications. This work suggests that UGT homologs may recognize a substrate profile that is much broader than previously anticipated. Additionally, it demonstrates that this screening method provides a new means to study UDP-glycosyltransferases, facilitating the use of these enzymes to tackle a wide range of problems.

Light‐Up Sensing Citrate Using a Capture‐Selected DNA Aptamer

AbstractCitrate is a key metabolite and nutrient in humans. Its level is associated with many diseases from tumor growth to bone diseases. Detection of citrate has relied on its high negative charge, metal chelating properties and as an enzyme substrate. In this work, the capture‐selection method is used to isolate DNA aptamers for citrate. After 18 rounds of selection, a highly converged library is obtained and the first two sequences reached 99.6% of the library. Using the most abundant sequence named CA1, thioflavin T fluorescence spectroscopy and isothermal titration calorimetry show dissociation constants of 7.4 and 4.4 µm citrate, respectively. CA1 does not require sodium for binding but requires 1.0 mm magnesium. Among the tested carboxylate molecules, only citrate can bind to the aptamer. A light‐up fluorescence strand displacement biosensor is developed and it can detect citrate in simulated urine with a detection limit of 1.1 µm. This short 42‐nucleotide aptamer can be readily adapted to other types of sensing mechanisms for the detection of citrate.

RNA Interference in Insects: From a Natural Mechanism of Gene Expression Regulation to a Biotechnological Crop Protection Promise.

Insect pests rank among the major limiting factors in agricultural production worldwide. In addition to direct effect on crops, some phytophagous insects are efficient vectors for plant disease transmission. Large amounts of conventional insecticides are required to secure food production worldwide, with a high impact on the economy and environment, particularly when beneficial insects are also affected by chemicals that frequently lack the desired specificity. RNA interference (RNAi) is a natural mechanism gene expression regulation and protection against exogenous and endogenous genetic elements present in most eukaryotes, including insects. Molecules of double-stranded RNA (dsRNA) or highly structured RNA are the substrates of cellular enzymes to produce several types of small RNAs (sRNAs), which play a crucial role in targeting sequences for transcriptional or post-transcriptional gene silencing. The relatively simple rules that underlie RNAi regulation, mainly based in Watson-Crick complementarity, have facilitated biotechnological applications based on these cellular mechanisms. This includes the promise of using engineered dsRNA molecules, either endogenously produced in crop plants or exogenously synthesized and applied onto crops, as a new generation of highly specific, sustainable, and environmentally friendly insecticides. Fueled on this expectation, this article reviews current knowledge about the RNAi pathways in insects, and some other applied questions such as production and delivery of recombinant RNA, which are critical to establish RNAi as a reliable technology for insect control in crop plants.

Use of an inexpensive carbon source for the production of a cellulase enzyme complex from Penicillium ucsense S1M29 and enzymatic hydrolysis optimization

AbstractThe high cost of cellulolytic enzyme complexes (CECs) has been a significant impediment to the commercial production of bioproducts from lignocellulose biomass. This study aimed to develop a cost‐effective CEC derived from Penicillium ucsense (former Penicillium echinulatum), utilizing diverse forms of pretreated sugarcane bagasse as the primary carbon/inductor source. Among the different pretreatments used, the hydrothermal pretreatment followed by NaOH delignification (BHD) produced higher FPase and xylanase activities (4.5 FPU mL–1 and 120 IU mL–1) in bioreactor experiments at 20 g BHD L–1 initial concentration. A batch‐mode assay conducted across a range of initial carbon source (5 to 60 g L–1) confirmed the highest FPase activity (4.0 to 5.0 FPU mL–1 at 120 h), in the range of 20–40 g BHD L–1. During these assays the agitation rate, controlled by dissolved O2, tended to stabilize at lower levels, indicating substrate limitation. Conversely, higher initial carbon source concentrations led to an excess of glucose, likely triggering carbon catabolite repression and inhibiting cellulase production. This insight prompted the development of a controlled pulsed fed‐batch strategy, resulting in FPase activity of 11 FPU mL–1 at 220 h using 90 g L–1 BHD controlled fed into the bioreactor. An enzymatic hydrolysis procedure using the generated CEC was also optimized using a central composite rotational design (CCRD). The optimized enzyme hydrolysis conditions achieved a reducing sugar concentration of 80.9 g L–1 in 48 h using 170 g L–1 of BHD as the substrate at a ratio of 15 FPU of enzyme substrate per g of BHD. A preliminary economic assessment demonstrated that, for a first‐ and second‐generation (1G + 2G) ethanol biorefinery, the cost contribution of enzymes would be about US$0.2/L of biofuel. In conclusion, an efficient and highly productive procedure was developed successfully for the production of a CEC. It was particularly effective for the enzymatic hydrolysis of pretreated sugarcane bagasse.

A Potentially Versatile Enzyme Sensor Platform: Enzyme-Loaded, Tagged, Porous Polymeric Nanocapsules.

Enzymes are essential to life and indispensable in a wide range of industries (food, pharmaceutical, medical, biosensing, etc.); however, a significant shortcoming of these fragile biological catalysts is their poor stability. To address this challenge, a variety of immobilization methods have been described to enhance the enzyme's stability. These immobilization methods generally are specific to an individual enzyme or optimal for a particular application. The aim of this study is to explore the utility of porous, indicator moiety-tagged, polymeric nanocapsules (NCs) for the encapsulation of enzymes and measurement of the enzyme's substrate. As a model enzyme, glucose oxidase (GOx) is used. The GOx enzyme-loaded, fluorophore-tagged NCs were synthesized by using self-assembled surfactant vesicle templates. To show that the biological activity of GOx is preserved during entrapment, the rate of the GOx enzyme catalyzed reaction was measured. To evaluate the protective features of the porous NCs, the encapsulated GOx enzyme activity was followed in the presence of hydrolytic enzymes. During the encapsulation of GOx and the purification of the GOx-loaded NCs, the GOx activity decayed less than 10%, and up to 30% of the encapsulated GOx activity could be retained for 3-5 days in the presence of hydrolytic enzymes. In support of the potentially unique advantages of the enzyme-loaded NCs, as a proof-of-concept example, the fluorophore-tagged, GOx-loaded NCs were used for the determination of glucose in the concentration range between 18 and 162 mg/dL and for imaging the distribution of glucose concentration in imaging experiments.

Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells

Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner. By integrating enzymatic PTM-mechanisms, and genetically encoding residue-selective photo-crosslinker into PTM-enzymes, our strategy expands the substrate profiles of both bacterial and mammalian lysine acylation enzymes, including bacterial lysine acylases PatZ, YiaC, LplA, TmcA, and YjaB, as well as mammalian acyltransferases GCN5 and Tip60, leading to discovery of distinct yet functionally important substrates and acylation sites. The concept of direct capturing substrates of PTM-enzymes via residue crosslinking may extend to the other types of amino acid residues beyond lysine, which has the potential to facilitate the investigation of diverse types of PTMs and substrate-enzyme interactive proteomics.

Pseudomonas effector AvrB is a glycosyltransferase that rhamnosylates plant guardee protein RIN4.

The plant pathogen Pseudomonas syringae encodes a type III secretion system avirulence effector protein, AvrB, that induces a form of programmed cell death called the hypersensitive response in plants as a defense mechanism against systemic infection. Despite the well-documented catalytic activities observed in other Fido (Fic, Doc, and AvrB) proteins, the enzymatic activity and target substrates of AvrB have remained elusive. Here, we show that AvrB is an unprecedented glycosyltransferase that transfers rhamnose from UDP-rhamnose to a threonine residue of the Arabidopsis guardee protein RIN4. We report structures of various enzymatic states of the AvrB-catalyzed rhamnosylation reaction of RIN4, which reveal the structural and mechanistic basis for rhamnosylation by a Fido protein. Collectively, our results uncover an unexpected reaction performed by a prototypical member of the Fido superfamily while providing important insights into the plant hypersensitive response pathway and foreshadowing more diverse chemistry used by Fido proteins and their substrates.

Synthesis and evaluation of nanosystem containing chondroitinase ABCI based on hydroxyapatite

The bacterial enzyme chondroitinase ABCI (chABCI), which has been isolated from Proteus Vulgaris, is crucial in the treatment of spinal cord injuries. However, due to its short lifespan, the maintenance and clinical application of this enzyme are very constrained. In this study, the immobilization of this enzyme on hydroxyapatite has been carried out and assessed with the aim of enhancing the characteristics and efficiency of chABCI. Hydroxyapatite particles (HAPs) are a potential candidate for drug-delivery carriers because of their excellent biocompatibility, shape controllability, and high adsorption. The use of the nanometer scale allows efficient access to the enzyme's substrate. It demonstrates important biological application capabilities in this way. Field emission gun-scanning electron microscopy (FEG-SEM), X-ray diffraction (XRD), infrared spectroscopy (FT-IR), in vitro release study, and cytotoxicity test were used to characterize the drug nanosystem's properties. According to the findings, electrostatic bindings was formed between charged groups of the enzyme and hydroxyapatite nanoparticles. The results also demonstrated that immobilized chABCI on hydroxyapatite has beneficial properties, such as more manageable drug release, minimal toxicity and side effects, and a high potential to enhance the efficacy of drug delivery and decrease the need for repeated injections.

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi.

Glycosyltransferases (GTs) are pivotal enzymes in the biosynthesis of various biological molecules. This study focuses on the scale-up, expression, and purification of a plant flavonol-specific 3-O glucosyltransferase (Cp3GT), a key enzyme from Citrus paradisi, for structural analysis and modeling. The challenges associated with recombinant protein production in Pichia pastoris, such as proteolytic degradation, were addressed through the optimization of culture conditions and purification processes. The purification strategy employed affinity, anion exchange, and size exclusion chromatography, leading to greater than 95% homogeneity for Cp3GT. In silico modeling, using D-I-TASSER and COFACTOR integrated with the AlphaFold2 pipeline, provided insights into the structural dynamics of Cp3GT and its ligand binding sites, offering predictions for enzyme-substrate interactions. These models were compared to experimentally derived structures, enhancing understanding of the enzyme's functional mechanisms. The findings present a comprehensive approach to produce a highly purified Cp3GT which is suitable for crystallographic studies and to shed light on the structural basis of flavonol specificity in plant GTs. The significant implications of these results for synthetic biology and enzyme engineering in pharmaceutical applications are also considered.

Long-Term Outcomes of Disease Modifying Therapies in Gaucher Disease.

To study clinical response to treatment with enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) in a cohort of Gaucher disease. Retrospective data of 8 patients of Gaucher disease was compiled. The treatment included all three currently available enzyme replacement therapies as well as substrate reduction therapy with Eliglustat. The relevant blood investigations were done in follow-up visits. The assessment of the effects of long-term treatment over varying periods up to 13 y was done with various issues related to the course of therapy documented. Improvement in hematological parameters was seen in all patients. Reduction of spleen size occurred in 7 of 8 patients (87.5%). One patient had 2 successful pregnancies while on therapy. A distinct patient with type 3 Gaucher disease developed complication in the form of Gaucheroma within the spleen. Awareness about the disease and the efficacy of the therapies amongst pediatricians will help in early diagnosis and better outcomes. The available therapies have changed the outcome of the patients and improved the quality of life in patients with Gaucher disease. The data of Indian patients is important at this juncture when under Rare Disease Policy, government funding has become available for ERT for Gaucher disease patients in India.

Global identification of palmitoylating enzyme substrates in cardiomyocytes provides mechanistic insights into why upregulation of these enzymes aggravates myocyte injury in response to ischemia/reperfusion

Global identification of palmitoylating enzyme substrates in cardiomyocytes provides mechanistic insights into why upregulation of these enzymes aggravates myocyte injury in response to ischemia/reperfusion

The Mutational Road not Taken: Using Ancestral Sequence Resurrection to Evaluate the Evolution of Plant Enzyme Substrate Preferences.

We investigated the flowering plant salicylic acid methyl transferase (SAMT) enzyme lineage to understand the evolution of substrate preference change. Previous studies indicated that a single amino acid replacement to the SAMT active site (H150M) was sufficient to change ancestral enzyme substrate preference from benzoic acid to the structurally similar substrate, salicylic acid (SA). Yet, subsequent studies have shown that the H150M function-changing replacement did not likely occur during the historical episode of enzymatic divergence studied. Therefore, we reinvestigated the origin of SA methylation preference here and additionally assessed the extent to which epistasis may act to limit mutational paths. We found that the SAMT lineage of enzymes acquired preference to methylate SA from an ancestor that preferred to methylate benzoic acid as previously reported. In contrast, we found that a different amino acid replacement, Y267Q, was sufficient to change substrate preference with others providing small positive-magnitude epistatic improvements. We show that the kinetic basis for the ancestral enzymatic change in substate preference by Y267Q appears to be due to both a reduced specificity constant, kcat/KM, for benzoic acid and an improvement in KM for SA. Therefore, this lineage of enzymes appears to have had multiple mutational paths available to achieve the same evolutionary divergence. While the reasons remain unclear for why one path was taken, and the other was not, the mutational distance between ancestral and descendant codons may be a factor.

Designing Drug Delivery Vehicles based on N-(2-Hydroxypropyl) Methacrylamide.

The development of polymeric-based drug delivery has seen faster growth in the past two decades. In polymers, copolymers as drug carriers are increasing to decrease the drug compounds' side effects and dosage-related toxicity. The study's primary objective is to utilize computational resources to design drug molecules and perform in silco physicochemical property analysis. In our study, we designed new copolymers based on N-(2-Hydroxypropyl) methacrylamide (HPMA) as backbone along with polyethylene glycol (PEG) and lauryl methacrylate (LMA). Different functional groups were selected for attaching to the side chain of the copolymers through a random trial and error approach. In order to predict the pharmacokinetic properties (absorption, distribution, metabolism, excretion, and toxicity), the designed copolymer molecules were evaluated utilizing ADME and PkCSM pharmacokinetics servers. Molecular interaction between the designed copolymer molecules and human serum albumin (HSA) was performed using AutoDock Vina and PatchDock server. The designed molecules are shown to be soluble in water and have high gastrointestinal absorption. Only one molecule is predicted to pass through the blood-brain barrier. Two designed molecules have been shown to have carcinogenic properties. Lethal dose 50 (LD50), cytochrome P450, and permeability glycoprotein Enzyme's substrate formation were also analyzed for toxicity and metabolism. Our study will provide insight for designing new drug compounds or carriers and analyzing their physicochemical properties to help further optimize compounds for clinical studies.

Investigation of natural compounds as methyltransferase inhibitors against dengue virus: an in silico approach

The global challenge posed by Dengue virus (DENV) infection persists, exacerbated by the absence of specific antiviral therapies. The viral methyltransferase (MTase) enzyme, crucial for viral RNA methylation and immune system evasion, has emerged as a promising drug target for combating Dengue fever. In this study, a comprehensive exploration of natural compounds derived from the COCONUT database was conducted, selecting 224 compounds based on their structural similarity to the native substrate of the MTase enzyme, S-adenosyl-L-methionine (SAM). Employing virtual screening techniques, four natural compounds (CNP0307160, CNP0082902, CNP0449158, and CNP0296775) with acceptable docking scores were selected for further re-docking after geometry optimization by the DFT method. Re-docking analyses unveiled significant interactions, including hydrogen bonds and hydrophobic interactions, between these selected ligands and the MTase protein. To gain deeper insights into the dynamic stability of these complexes, we conducted molecular dynamics simulations which showed lower RMSD values for CNP0307160, CNP0082902, and CNP0296775 when compared to the reference molecule. Furthermore, we assessed the structural and dynamic stability of the protein-ligand complexes through free binding energy calculations and Principal Component Analysis (PCA) of the simulation trajectories. In these analyses, the CNP0296775 compound exhibited promising results compared to the other three compounds. The cumulative findings of these investigations underscore the potential of CNP0296775 as a strong inhibitor of DENV MTase, thus offering a promising starting point for its further experimental validation and optimization. Communicated by Ramaswamy H. Sarma