Crystal structure of the class A extended-spectrum ß-lactamase CTX-M-96 in complex with relebactam at 1.03 Angstrom resolution.

The use of ß-lactam/ß-lactamase inhibitors constitutes an important strategy to counteract ß-lactamases in multidrug-resistant (MDR) Gram-negative bacteria. Recent reports have described ceftazidime-/avibactam-resistant isolates producing CTX-M variants with different amino acid substitutions (e.g., P167S, L169Q, and S130G). Relebactam (REL) combined with imipenem has proved very effective against Enterobacterales producing ESBLs, serine-carbapenemases, and AmpCs. Herein, we evaluated the inhibitory efficacy of REL against CTX-M-96, a CTX-M-15-type variant. The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL was covalently bound to the S70-Oγ atom upon cleavage of the C7-N6 bond. Compared with apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.81 Å), making the E166 and N170 side chains shift to create a proper hydrogen bonding network. Binding of REL also disturbs the hydrophobic patch formed by Y105, P107, and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 sidechain is also affected by the piperidine ring. Therefore, differences in the kinetic behavior of REL against class A ß-lactamases seem to rely, at least in part, on differences in the residues being involved in the association and stabilization of the inhibitor before hydrolysis. Our data provide the biochemical and structural basis for REL effectiveness against CTX-M-producing Gram-negative pathogens and essential details for further DBO design. Imipenem/REL remains an important choice for dealing with isolates co-producing CTX-M with other ß-lactamases.

A pH-dependent shift of redox cofactor specificity in a benzyl alcohol dehydrogenase of aromatoleum aromaticum EbN1.

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD + or NADP+ at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.

Structural characterization and hypoglycemic effect of polysaccharides of Polygonatum sibiricum.

Polygonatum sibiricum polysaccharide (PSP) was extracted and purified from raw material obtained from P. sibiricum. The structural features of PSP were investigated by Congo red, circular dichroism spectrum, high-performance gel permeation chromatography, scanning electron microscope, atomic force microscope, ultraviolet spectroscopy, and Fourier transform infrared spectroscopy analysis. In vitro simulations were conducted to investigate the kinetics of PSP enzyme inhibition. Moreover, a type II diabetes mouse model (T2DM) with streptozotocin-induced insulin resistance was established, and the indexes of lipid quadruple, insulin resistance index, oral glucose tolerance (OGTT), organ index, and pancreatic morphology of model mice were measured. The results showed that PSP mainly consists of monosaccharides, such as mannose, glucose, galactose, xylose, and arabinose. It also has a ß-glycosidic bond of a pyranose ring and an irregular reticulated aggregated structure with a triple helix. In vitro enzyme inhibition assays revealed that PSP acts as a reversible competitive inhibitor of α-glucosidase and α-amylase. Furthermore, PSP was found to reduce insulin resistance index, increase OGTT and serum insulin levels, decrease free fatty acid content to improve lipid metabolism, and lower glycated serum protein content to enhance glucose metabolism in T2DM mice, thereby leading to a reduction in blood glucose concentration. Additionally, PSP exhibited reparative effects on the damaged liver tissue cells and pancreatic tissue in T2DM mice. The experiment results provide a preliminary basis for the therapeutic mechanism of PSP about type II diabetes and a theoretical reference for application in food and pharmaceutical development.

Deciphering molecular mechanisms underlying the inhibition of ß-glucuronidase by xanthones from Centaurium spicatum.

Herein, we scrutinized the inhibitory potential of five xanthones and a flavonoid, sourced from Centaurium spicatum, against ß-glucuronidase activity. The results showed that gentisin and azaleatin emerged as the most potent inhibitors, with significantly lower IC50 values of 0.96 ± 0.10 and 0.57 ± 0.04 µM, respectively. The evaluation of enzyme kinetics unveiled that the isolated xanthones manifested inhibition of ß-glucuronidase through a mixed inhibition mode, whereas azaleatin exhibited a noncompetitive inhibition mechanism. The findings from molecular docking analysis unveiled that the compounds under investigation, particularly azaleatin, displayed comparatively diminished binding affinities towards ß-glucuronidase. Furthermore, the tested drugs were shown to occupy a common binding site as the employed reference drug. Our comprehensive Molecular Dynamics (MD) simulations analysis revealed consistent trajectories for the investigated drugs, wherein azaleatin and gentisin demonstrated notable stabilization of energy levels. Analysis of various MD parameters revealed that drugs with the lowest IC50 values maintained relatively stable interactions with ß-glucuronidase. These drugs were shown to exert notable alterations in their conformation or flexibility upon complexation with the target enzyme. Conversely, the flexibility and accessibility of ß-glucuronidase was reduced upon drug binding, particularly with azaleatin and gentisin, underscoring the stability of the drug-enzyme complexes. Analysis of Coul-SR and LJ-SR interaction energies unveiled consistent and stable interactions between certain isolated drugs and ß-glucuronidase. Azaleatin notably displayed the lowest average Coul-SR interaction energy, suggesting strong electrostatic interactions with the enzyme's active site and significant conformational variability during simulation. Remarkably, LJ-SR interaction energies across different xanthones complexes were more negative than their Coul-SR counterparts, emphasizing the predominant role of van der Waals interactions, encompassing attractive dispersion and repulsive forces, in stabilizing the drug-enzyme complexes rather than electrostatic interactions.

High-throughput kinetics in drug discovery.

The importance of a drug's kinetic profile and interplay of structure-kinetic activity with PK/PD has long been appreciated in drug discovery. However, technical challenges have often limited detailed kinetic characterization of compounds to the latter stages of projects. This review highlights the advances that have been made in recent years in techniques, instrumentation, and data analysis to increase the throughput of detailed kinetic and mechanistic characterization, enabling its application earlier in the drug discovery process.

The mechanistic insights into different aspects of promiscuity in metalloenzymes.

Enzymes are nature's ultimate machinery to catalyze complex reactions. Though enzymes are evolved to catalyze specific reactions, they also show significant promiscuity in reactions and substrate selection. Metalloenzymes contain a metal ion or metal cofactor in their active site, which is crucial in their catalytic activity. Depending on the metal and its coordination environment, the metal ion or cofactor may function as a Lewis acid or base and a redox center and thus can catalyze a plethora of natural reactions. In fact, the versatility in the oxidation state of the metal ions provides metalloenzymes with a high level of catalytic adaptability and promiscuity. In this chapter, we discuss different aspects of promiscuity in metalloenzymes by using several recent experimental and theoretical works as case studies. We start our discussion by introducing the concept of promiscuity and then we delve into the mechanistic insight into promiscuity at the molecular level.

Single-stranded DNA binding protein hitches a ride with the Escherichia coli YoaA-χ helicase.

The Escherichia coli XPD/Rad3-like helicase, YoaA, and DNA polymerase III subunit, χ, are involved in E. coli DNA damage tolerance and repair. YoaA and χ promote tolerance to the DNA chain-terminator, 3 -azidothymidine (AZT), and together form the functional helicase complex, YoaA-χ. How YoaA-χ contributes to DNA damage tolerance is not well understood. E. coli single-stranded DNA binding protein (SSB) accumulates at stalled replication forks, and the SSB-χ interaction is required to promote AZT tolerance via an unknown mechanism. YoaA-χ and SSB interactions were investigated in vitro to better understand this DNA damage tolerance mechanism, and we discovered YoaA-χ and SSB have a functional interaction. SSB confers a substrate-specific effect on the helicase activity of YoaA-χ, barely affecting YoaA-χ on an overhang DNA substrate but inhibiting YoaA-χ on forked DNA. A paralog helicase, DinG, unwinds SSB-bound DNA in a similar manner to YoaA-χ on the substrates tested. Through use of ensemble experiments, we believe SSB binds behind YoaA-χ relative to the DNA ds/ss junction and show via single-molecule assays that SSB translocates along ssDNA with YoaA-χ. This is, to our knowledge, the first demonstration of a mechanoenzyme pulling SSB along ssDNA.

Residues in the Fructose Binding Pocket Are Required for Ketohexokinase-A Activity.

Excessive fructose consumption is a primary contributor to the global surges in obesity, cancer, and metabolic syndrome. Fructolysis is not robustly regulated and is initiated by ketohexokinase (KHK). In this study, we determined the crystal structure of KHK-A, one of two human isozymes of KHK, in the apo-state at 1.85 Å resolution, and we investigated the roles of residues in the fructose-binding pocket by mutational analysis. Introducing alanine at D15, N42, or N45 inactivated KHK-A, whereas mutating R141 or K174 reduced activity and thermodynamic stability. Kinetic studies revealed that the R141A and K174A mutations reduced fructose affinity by 2- to 4-fold compared to WT KHK-A, without affecting ATP affinity. Molecular dynamics simulations provided mechanistic insights into the potential roles of the mutated residues in ligand coordination and the maintenance of an open state in one monomer and a closed state in the other. Protein-protein interactome analysis indicated distinct expression patterns and downregulation of partner proteins in different tumor tissues, warranting a re-evaluation of KHK's role in cancer development and progression. The connections between different cancer genes and the KHK signaling pathway suggest that KHK is a potential target for preventing cancer metastasis. This study enhances our understanding of KHK-A's structure and function and offers valuable insights into potential targets for developing treatments for obesity, cancer, and metabolic syndrome.

Educational activity of enzyme kinetics in an undergraduate biochemistry course: invertase enzyme as a model.

This article aims to simplify and facilitate the process of practical teaching of enzyme kinetics by utilizing minimal teaching laboratory requirements. Simultaneously, it ensures that students comprehend the enzyme kinetics experiment effectively. The focus is on teaching students how to estimate the maximum velocity (Vmax) and Michaelis constant (Km) of ß-fructofuranosidase enzyme (also known as invertase) isolated from dry yeast. The invertase enzyme catalyzes the hydrolysis of sucrose substrate into glucose and fructose, employing the Michaelis-Menten approach of evaluating invertase enzyme kinetics as well as Lineweaver-Burk linear graphic approach of evaluating the Michaelis-Menten enzyme kinetics. The practical experiment seeks to reinforce the concepts of initial velocity dependence on substrate concentration. The data presented in the work were generated from a genuine practical biochemistry course enrolled by second-year undergraduate students in the Department of Pharmacy and the Department of Medical Laboratory Science. While there were minor variations in the invertase enzyme kinetic parameters among students, they successfully carried out the experiment. The students accurately estimated the Vmax and Km of the invertase enzyme in the sucrose hydrolysis chemical reaction. Moreover, they demonstrated an understanding of the meanings of the kinetic parameters (Km and Vmax) and the utility of the Lineweaver-Burk plot.

Global kinetic mechanism describing single nucleotide incorporation for RNA polymerase I reveals fast UMP incorporation.

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3' end was fastest when the 3' terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

Cloning, expression, and purification of an α-carbonic anhydrase from Toxoplasma gondii to unveil its kinetic parameters and anion inhibition profile.

Toxoplasmosis, induced by the intracellular parasite Toxoplasma gondii, holds considerable implications for global health. While treatment options primarily focusing on folate pathway enzymes have notable limitations, current research endeavours concentrate on pinpointing specific metabolic pathways vital for parasite survival. Carbonic anhydrases (CAs, EC 4.2.1.1) have emerged as potential drug targets due to their role in fundamental reactions critical for various protozoan metabolic processes. Within T. gondii, the Carbonic Anhydrase-Related Protein (TgCA_RP) plays a pivotal role in rhoptry biogenesis. Notably, α-CA (TcCA) from another protozoan, Trypanosoma cruzi, exhibited considerable susceptibility to classical CA inhibitors (CAIs) such as anions, sulphonamides, thiols, and hydroxamates. Here, the recombinant DNA technology was employed to synthesise and clone the identified gene in the T. gondii genome, which encodes an α-CA protein (Tg_CA), with the purpose of heterologously overexpressing its corresponding protein. Tg_CA kinetic constants were determined, and its inhibition patterns explored with inorganic metal-complexing compounds, which are relevant for rational compound design. The significance of this study lies in the potential development of innovative therapeutic strategies that disrupt the vital metabolic pathways crucial for T. gondii survival and virulence. This research may lead to the development of targeted treatments, offering new approaches to manage toxoplasmosis.

Glyphosate-Induced Phosphonatase Operons in Soil Bacteria of the Genus Achromobacter.

Achromobacter insolitus and Achromobacter aegrifaciens, bacterial degraders of the herbicide glyphosate, were found to induce phosphonatase (phosphonoacetaldehyde hydrolase, EC 3.11.1.1) when grown on minimal media with glyphosate as the sole source of phosphorus. The phosphonatases of the strains were purified to an electrophoretically homogeneous state and characterized. The enzymes differed in their kinetic characteristics and some other parameters from the previously described phosphonatases. The phosphonatase of A. insolitus was first revealed to separate into two stable forms, which had similar kinetic characteristics but interacted differently with affinity and ion-exchange resins. The genomes of the investigated bacteria were sequenced. The phosphonatase genes were identified, and their context was determined: the bacteria were shown to have gene clusters, which, besides the phosphonatase operon, included genes for LysR-type transcription activator (substrate sensor) and putative iron-containing oxygenase PhnHD homologous to monooxygenases PhnY and TmpB of marine organophosphonate degraders. Genes of 2-aminoethylphosphonate aminotransferase (PhnW, EC 2.6.1.37) were absent in the achromobacterial phosphonatase operons; instead, we revealed the presence of genes encoding the putative flavin oxidase HpnW. In silico simulation showed 1-hydroxy-2-aminoethylphosphonate to be the most likely substrate of the new monooxygenase, and a number of glycine derivatives structurally similar to glyphosate to be substrates of flavin oxidase.

Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6.

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention.

Modeling the distribution of enzymes on lipid vesicles: A novel framework for surface-mediated reactions in coagulation.

Blood coagulation is a network of biochemical reactions wherein dozens of proteins act collectively to initiate a rapid clotting response. Coagulation reactions are lipid-surface dependent, and this dependence is thought to help localize coagulation to the site of injury and enhance the association between reactants. Current mathematical models of coagulation either do not consider lipid as a variable or do not agree with experiments where lipid concentrations were varied. Since there is no analytic rate law that depends on lipid, only apparent rate constants can be derived from enzyme kinetic experiments. We developed a new mathematical framework for modeling enzymes reactions in the presence of lipid vesicles. Here the concentrations are such that only a fraction of the vesicles harbor bound enzymes and the rest remain empty. We call the lipid vesicles with and without enzyme TF:VIIa+ and TF:VIIa- lipid, respectively. Since substrate binds to both TF:VIIa+ and TF:VIIa- lipid, our model shows that excess empty lipid acts as a strong sink for substrate. We used our framework to derive an analytic rate equation and performed constrained optimization to estimate a single, global set of intrinsic rates for the enzyme-substrate pair. Results agree with experiments and reveal a critical lipid concentration where the conversion rate of the substrate is maximized, a phenomenon known as the template effect. Next, we included product inhibition of the enzyme and derived the corresponding rate equations, which enables kinetic studies of more complex reactions. Our combined experimental and mathematical study provides a general framework for uncovering the mechanisms by which lipid mediated reactions impact coagulation processes.

The Human Paraoxonase 2: An Optimized Procedure for Refolding and Stabilization Facilitates Enzyme Analyses and a Proteomics Approach.

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.

Elucidating solvent effects on lipase-catalyzed peroxyacid synthesis through activity-based kinetics and molecular dynamics.

Peroxyacid synthesis is the first step in Prilezhaev epoxidation, which is an industrial method to form epoxides. Motivated by the development of a kinetic model as a tool for solvent selection, the effect of solvent type and acid chain length on the lipase-catalyzed peroxyacid synthesis was studied. A thermodynamic activity-based ping-pong kinetic expression was successfully applied to predict the effect of the reagent loadings in hexane. The activity-based reaction quotients provided a prediction of solvent-independent equilibrium constants. However, this strategy did not achieve satisfactory estimations of initial rates in solvents of higher polarity. The lack of compliance with some assumptions of this methodology could be confirmed through molecular dynamics calculations i.e. independent solvation energies and lack of solvent interaction with the active site. A novel approach is proposed combining the activity-based kinetic expression and the free binding energy of the solvent with the active site to predict kinetics upon solvent change. Di-isopropyl ether generated a strong interaction with the enzyme's active site, which was detrimental to kinetics. On the other hand, toluene or limonene gave moderate interaction with the active site rendering improved catalytic yield compared with less polar solvents, a finding sharpened when peroctanoic acid was produced.

Pharmacological and behavioural effects of tryptamines present in psilocybin-containing mushrooms.

BACKGROUND AND PURPOSE: Demand for new antidepressants has resulted in a re-evaluation of the therapeutic potential of psychedelic drugs. Several tryptamines found in psilocybin-containing "magic" mushrooms share chemical similarities with psilocybin. Early work suggests they may share biological targets. However, few studies have explored their pharmacological and behavioural effects. EXPERIMENTAL APPROACH: We compared baeocystin, norbaeocystin and aeruginascin with psilocybin to determine if they are metabolized by the same enzymes, similarly penetrate the blood-brain barrier, serve as ligands for similar receptors and modulate behaviour in rodents similarly. We also assessed the stability and optimal storage and handling conditions for each compound. KEY RESULTS: In vitro enzyme kinetics assays found that all compounds had nearly identical rates of dephosphorylation via alkaline phosphatase and metabolism by monoamine oxidase. Further, we found that only the dephosphorylated products of baeocystin and norbaeocystin crossed a blood-brain barrier mimetic to a similar degree as the dephosphorylated form of psilocybin, psilocin. The dephosphorylated form of norbaeocystin was found to activate the 5-HT2A receptor with similar efficacy to psilocin and norpsilocin in in vitro cell imaging assays. Behaviourally, only psilocybin induced head twitch responses in rats, a marker of 5-HT2A-mediated psychedelic effects and hallucinogenic potential. However, like psilocybin, norbaeocystin improved outcomes in the forced swim test. All compounds caused minimal changes to metrics of renal and hepatic health, suggesting innocuous safety profiles. CONCLUSIONS AND IMPLICATIONS: Collectively, this work suggests that other naturally occurring tryptamines, especially norbaeocystin, may share overlapping therapeutic potential with psilocybin, but without causing hallucinations.

Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.

Glutamate:glyoxylate aminotransferase (GGAT; EC 2.6.1.4) and serine:glyoxylate aminotransferase activities (SGAT; EC 2.6.1.45) are central photorespiratory reactions within plant peroxisomes. Both enzymatic reactions convert glyoxylate, a product of glycolate oxidase, to glycine, a substrate of the mitochondrial glycine decarboxylase complex. The GGAT reaction uses glutamate as an amino group donor and also produces α-ketoglutarate, which is recycled to glutamate in plastids by ferredoxin-dependent glutamate synthase. Using serine, a product of mitochondrial serine hydroxymethyltransferase, as an amino group donor, the SGAT reaction also produces hydroxypyruvate, a substrate of hydroxypyruvate reductase. The activities of these photorespiratory aminotransferases can be measured using indirect, coupled, spectrophotometric assays, detailed herein.

Spectrophotometric Assays for Measuring Hydroxypyruvate Reductase Activity.

Hydroxypyruvate reductase (HPR; EC 1.1.1.81) activity is integral to the photorespiratory pathway. Within photorespiration, HPR catalyzes the reduction of hydroxypyruvate, a product of the serine:glyoxylate aminotransferase reaction to glycerate, a substrate for glycerate kinase, using NADH as cofactor. Here we detail a spectrophotometric assay for measuring HPR activity in vitro by following the consumption of NADH at 340 nm.

Discovery of azaleatin as a potential allosteric inhibitor for dengue NS2B-NS3 protease using in vitro and in silico studies.

The rise in dengue cases in tropical and sub-tropical areas has become a significant health concern. At present, there is no definitive cure for dengue fever, which underscores the importance of identifying potent inhibitors. Dengue NS2B-NS3 protease is the prime drug target due to its vital function for replication. Quercetin, a flavone, has anti-dengue virus properties but is limited by low bioavailability. Previous studies have shown that methoxy substitution in flavones improves bioavailability and metabolic stability. Azaleatin is a derivative of quercetin with a methoxy substitution at the C5 position, however its ability to inhibit dengue is unknown. In this study, azaleatin was investigated for its inhibition against dengue NS2B-NS3 protease using in vitro and in silico techniques. The fluorescence assay was used to determine the IC50 value and inhibition kinetics. The molecular interaction between azaleatin and NS2B-NS3 was studied using CB-Dock2 and AutoDock Vina. The complex's stability was then analysed using GROMACS. Besides, the ADMETlab 2.0 was utilized to predict pharmacokinetic of the azaleatin. Results showed that azaleatin inhibits dengue NS2B-NS3 protease non-competitively with a Ki of 26.82 µg/ml and an IC50 of 38 µg/ml. Molecular docking indicated binding of the azaleatin to the allosteric pocket of NS2B-NS3 with a docking score of -8.2 kcal/mol. Azaleatin was found stable in the pocket along 100 ns, supporting its inhibitory mode. The compound has favourable pharmacokinetic profiles and conformed to Lipinski's Rule of Five. Taken together, azaleatin inhibits NS2B-NS3 protease in a non-competitive mode, suggesting its potential as safer anti-dengue compound.Communicated by Ramaswamy H. Sarma.