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Quorum sensing enables cell-cell communication in bacteria and regulates biofilm formation. Biofilm production promotes pathogenicity of Escherichia coli and causes infections. However, antibiotic resistance limits conventional treatment efficacy against biofilm infections. Quorum quenching offers an alternative by disrupting quorum sensing signals. Allicin, extracted from garlic, possesses antimicrobial and anti-quorum sensing properties. This study employed molecular docking and dynamics simulations to investigate allicin's interaction with the E. coli quorum sensing system, specifically targeting the cytoplasmic SidA receptor protein. SidA binds to N-acyl-homoserine lactone ligands and regulates quorum sensing in E. coli . The crystal structure of SidA was obtained from the PDB. Molecular docking revealed that allicin competitively binds to the ligand-binding pocket of SidA. Simulations analyzed the effects of allicin binding on SidA stability and affinity for N-acyl-homoserine lactones over 200 ns. Parameters like RMSD, RMSF, and hydrogen bonding indicated SidA was more stable when bound to allicin compared to unbound. Binding free energies suggested allicin reduced SidA's affinity for native ligands. Therefore, allicin binding to SidA alters its conformation and inhibits interaction with N-acyl-homoserine lactones, disrupting quorum sensing signaling and biofilm production in E. coli .Communicated by Ramaswamy H. Sarma.

5-Fluorouracil (5FU) is a chemotherapy drug used to treat various cancers, such as colorectal, prostate, skin, pancreas, and stomach, as an ointment or solution. However, its consumption has several side effects. Therefore, a new derivative of fluorouracil containing 5-Amino-1H-tetrazole was designed and synthesized through multi-step synthesis to reduce urea excretion and toxicity. The effectiveness of the synthesized drug on the Adenocarcinoma gastric cell line (AGS) gastric cancer cell line was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, which showed that the new 5-fluorouracil (5FU) analog, with an IC 50 of 15.67 µg/mL, is more effective in inhibiting the proliferation of AGS cells after 24 h compared to both synthesized and reported 5FU. In addition, In-silico studies showed that the new 5FU derivative based on amino tetrazole, with a binding energy of -7.2 kcal/mol, exhibits greater anti-cancer activity against the BCL2 enzyme than 5FU, with a binding energy of - 4.8 kcal/mol. It is predicted that the new 5FU derivative will be effective in treating gastric and colorectal cancers. The new derivative of the 5-fluorouracil drug was characterized and identified using FTIR and NMR spectroscopy.Communicated by Ramaswamy H. Sarma.

Staphylococcus aureus is a prevalent Gram-positive bacteria leading cause of a wide range of human pathologies. Moreover, antibiotic résistance of pathogenesis bacteria is one of the worldwide health problems. In Gram-positive bacteria, the enzyme of SrtA, is responsible for the anchoring of surface-exposed proteins to the cell wall peptidoglycan. Because of its critical role in Gram-positive bacterial pathogenesis, SrtA is an attractive target for anti-virulence during drug development. To date, some SrtA inhibitors have been discovered most of them being derived from flavonoid compounds, like Myricetin. In order to provide potential hit molecules against SrtA for clinical use, we obtained a total of 293 compounds by performing in silico shape-based screening of compound libraries against Myristin as a reference structure. Employing molecular docking and scoring functions, the top 3 compounds Apigenin, Efloxate, and Compound 8261032 were screened by comparing their docking scores with Myricetin. Furthermore, MD simulations and MM-PBSA binding energy calculation studies revealed that only Compound 8261032 strongly binds to the catalytic core of the SrtA enzyme than Myricetin, and stable behavior was consistently observed in the docking complex. Compound 8261032 showed a good number of hydrogen bonds with SrtA and higher MM-PBSA binding energy when compared to all three molecules. Also, it makes strength interactions with Arg139 and His62, which are critical for SrtA biological activity. This study showed that the development of this inhibitor could be a fundamental strategy against resistant bacteria, but further studies in vitro are needed to confirm this claim.Communicated by Ramaswamy H. Sarma.

Carbonic anhydrase owing to its potential as an industrial biocatalyst for carbon dioxide sequestration from flue gas has attracted considerable attention in solving global warming problems. A large body of research has been conducted to increase the thermal stability of carbonic anhydrase from different sources against the harsh operational conditions of CO 2 capture systems. In contrast to cost-intensive protein engineering methods, solvation with aqueous-organic binary mixtures offers a convenient and economical alternative strategy for retention of protein structure and stability. This study aimed to examine the stabilizing effect of methyl diethanolamine (MDEA) as a component of an aqueous-organic solvent mixture on human carbonic anhydrase II (HCA II) at extreme temperatures. Computational and also spectroscopic examinations were employed for tracking conformational changes and stability evaluation of HCA II in 50:50 (vol %) water: MDEA binary mixture at high temperature. Molecular dynamic (MD) simulation studies predicted the high thermal stability of HCA II in the presence of MDEA. UV absorbance spectra confirmed the thermo-stabilizing effect of the binary solvent mixture on HCA II. While the enzymatic activity of HCA II at 25 °C in the presence of 10, 25, and 50 (vol%) of MDEA was substantially increased, no obvious effect on retention of HCA II activity in the water-MDEA binary solvent mixture at 85 °C was seen. It is shown that the solvation of HCA II in the presence of MDEA could result in the prevention of aggregate formation in high temperatures but not functional stability.Communicated by Ramaswamy H. Sarma.

Molecular docking techniques are routinely employed for predicting ligand binding conformations and affinities in the in silico phase of the drug design and development process. In this study, a reliable semiempirical quantum mechanics (SQM) method, PM7, was employed for geometry optimization of top-ranked poses obtained from two widely used docking programs, AutoDock4 and AutoDock Vina. The PDBbind core set (version 2016), which contains high-quality crystal protein - ligand complexes with their corresponding experimental binding affinities, was used as an initial dataset in this research. It was shown that docking pose optimization improves the accuracy of pose predictions and is very useful for the refinement of docked complexes via removing clashes between ligands and proteins. It was also demonstrated that AutoDock Vina achieves a higher sampling power than AutoDock4 in generating accurate ligand poses (RMSD ≤ 2.0 Å), while AutoDock4 exhibits a better ranking power than AutoDock Vina. Finally, a new protocol based on a combination of the results obtained from the two docking programs was proposed for structure-based virtual screening studies, which benefits from the robust sampling abilities of AutoDock Vina and the reliable ranking performance of AutoDock4.Communicated by Ramaswamy H. Sarma.

Toll-like receptor 8 (TLR8), as an endosomal transmembrane receptor, plays a crucial role in the innate immune response to neoplasia and viruses. Previous studies have shown that TLR8 agonists e.g. Motolimod can be used to treat patients with last-stage cancer. In this study, in order to find new suitable ligands for TLR8, 16 PBD codes related to TLR8 complexes were collected to design the pharmacophore models using the Pharmit server. Then the PubChem, and ZINC databases were screened by them. Subsequently, the ADME-Tox features of the compounds were detected using FAF-Drugs4 and the selected compounds were docked to TLR8 (PDB: 3w3j). Molecular dynamics simulation was used to compare compounds with the best docking scores, with Motolimod in complex with TLR8. Finally, two compounds were identified, PubChem: 124126919 (A) and PubChem: 18559540 (B), each with advantages over Motolimod. As the RMSD results showed that compound A has very good flexibility, in terms of energy calculated using the MM-GBSA method, complex B and TLR8 showed the lowest energy level compared to the rest of the complexes. These observations suggest that these two compounds could be used as TLR8 agonists with the desired pharmacological features in future experimental studies.Communicated by Ramaswamy H. Sarma.

Hyperpigmentation is a disorder caused by increased melanin deposition and changes in skin pigmentation. Inhibition of tyrosinase activity contributes to the control of food browning and skin pigmentation diseases. The effects of arachidonic acid (AA) on tyrosinase activity were examined using different spectroscopy methods including UV-VIS spectrophotometry, fluorescence spectroscopy, circular dichroism (CD) differential scanning calorimetry, and molecular dynamics (MD) simulations. Based on the kinetic results, arachidonic acid showed mixed-type of inhibition with Ki = 4.7 µM. Fluorescence and CD studies showed changes of secondary and tertiary structures of enzyme and a reduction of α-helix* amino acids after its incubation with different concentrations of AA, which is also confirmed by DSSP analysis. In addition, differential scanning calorimetry (DSC) studies showed a decrease in thermodynamic stability of enzyme from Tm = 338.65k for sole enzyme after incubation with AA in comparison with complex enzyme with Tm= 334.26k, ΔH =7.52 kJ/mol, and ΔS = 0.15 kJ/mol k. Based on the theoretical methods, it was found that the interaction between enzyme and AA follows an electrostatic manner with ΔG = -8.314 kJ/mol and ΔH = -12.9 kJ/mol. The MD results showed the lowest flexibility in the complex amino acids and minimal fluctuations in AA interaction with tyrosinase in Residue 240 to 260 and 66 to 80. Thus, AA inhibitory and structural and thermodynamic instability of tyrosinase supported advantages of this fatty acid for prevention of medical hyperpigmentation. Therefore, it is a good candidate for cosmetic applications. Communicated by Ramaswamy H. Sarma.

Anti-VEGF therapies are common for the treatment of cancer. Carboxypeptidase G (CPG-2) enzyme is a zinc-dependent metalloenzyme that metabolizes non-toxic synthetic 'benzoic mustard prodrugs' to cytotoxic moieties in tumor cells. In this study, we designed a dual-activity agent by combining a designed anti-VEGF- and CPG-2 enzyme to convert methotrexate (MTX). VEGF-A was docked against a set of scaffolds, and suitable inverse rotamers were made. Rosetta design was used for the interface design. The top 1200 binders were chosen by flow cytometry and displayed in yeast. The activity of CPG-2 enzyme was analyzed at different temperature conditions and in the presence of the substrate, MTX. Optimal binders were selected and protein was eluted using immobilized metal affinity chromatography and size-exclusion chromatography. Both, native PAGE and on-yeast flow cytometry confirmed the binding of the binder to VEGF-A. The activity of truncated enzymes was slightly lower than that of full-length enzymes linked to VEGF-A. The method should be generally useful as a dual-activity agent for targeting VEGF-A and combination therapy with the enzyme CPG-2 for metabolizing non-toxic prodrugs to cytotoxic moieties.Communicated by Ramaswamy H. Sarma.

The inhibitory effects of ferulic and chlorogenic acids on tyrosinase activity were investigated through multi-spectroscopic and molecular docking techniques. Ferulic and chlorogenic acids, flavonoid compounds, demonstrated inhibitory monophenolase activities of tyrosinase. The inhibitor effects against monophenolase activity were in a reversible and competitive manner with ki value equal to 6.8 and 7.5 µM respectively. The affinity between tyrosinase and L-DOPA decreased when fatty acids were added to the solution. The multi-spectroscopic techniques like UV-vis, fluorescence, and isothermal calorimetry are employed to investigate changes. Intrinsic fluorescence quenching and conformational changes of tyrosinase by hydrophobic interaction were confirmed. Tyrosinase had two and three binding sites for ferulic and chlorogenic acids with a binding constant in the order of magnitude of -6.8 and -7.2 kcal/mol. In addition, the secondary structural changes with Circular dichroism (CD) analysis, secondary structure (DSSP), radius of gyration (Rg) and analysis of hydrogen bonds (H-bonds) confirmed. Ferulic acid effect can be observed obviously and also content of α-helix decreased. Thermodynamic parameters indicated that the interaction between enzyme and ferulic and chlorogenic acids followed a spontaneous reaction dynamic manner with ΔG = -14.78 kJ/mol and ΔG = -14.61 kJ/mol (298k). The findings highlighted the potential applications of ferulic acid and chlorogenic acids in food and drug industries as potent inhibitors of tyrosinase.Communicated by Ramaswamy H. Sarma.

The inhibition of protein-protein interactions (PPIs) by small molecules is an exciting drug discovery strategy. Here, we aimed to develop a pipeline to identify candidate small molecules to inhibit PPIs. Therefore, KPI, a Knowledge-based Protein-Protein Interaction Inhibition pipeline, was introduced to improve the discovery of PPI inhibitors. Then, phytochemicals from a collection of known Middle Eastern antiviral herbs were screened to identify potential inhibitors of key PPIs involved in COVID-19. Here, the following investigations were sequenced: 1) Finding the binding partner and the interface of the proteins in PPIs, 2) Performing the blind ligand-protein inhibition (LPI) simulations, 3) Performing the local LPI simulations, 4) Simulating the interactions of the proteins and their binding partner in the presence and absence of the ligands, and 5) Performing the molecular dynamics simulations. The pharmacophore groups involved in the LPI were also characterized. Aloin, Genistein, Neoglucobrassicin, and Rutin are our new pipeline candidates for inhibiting PPIs involved in COVID-19. We also propose KPI for drug repositioning studies.Communicated by Ramaswamy H. Sarma.

Amyloid fibrillar aggregates play a critical role in many neurodegenerative disorders. Conversion of globular proteins into fibrils is associated with global conformational rearrangement and involves the transformation of α-helices to β-sheets. In the present work, the accelerated molecular dynamics technique was applied to study the unfolding of hen egg-white lysozyme at elevated temperatures, and the transformation of the native structure to a disordered one was analyzed. The influence of the disulfide bonds on the conformational dynamics and the energy landscape of denaturation process was considered. Our results show that formation of the metastable β-enriched conformers of individual protein molecules may precede the aggregation process. These β-rich intermediates can play a role of bricks making up fibrils.Communicated by Ramaswamy H. Sarma.

In the present work, we studied the structure-activity relationship and kinetics of thermal inactivation of α-glucosidase A (AglA) in a 50 mM potassium phosphate buffer at pH 6.8 using p -nitrophenyl α-d-glucopyranoside ( p NPG) as the synthetic substrate following absorbance at 410 nm by UV-Vis spectrophotometer. The interface structure and residual activity plot were analyzed via biochemical measurements by means of conformational lock theory, as well. The thermal inactivation curves were plotted in temperature interval from 30 to 50 °C. Based on experimental and structural data we suggested intermediates during inactivation before the loss of enzyme activity. Arrhenius plot for thermal inactivation rate constant showed biphasic appearance related to before and after 45°C temperature. The contact areas between two subunits were ruptured and unlocked stepwise during dimer dissociation. Cleavage of these areas induced the dissociation of the subunits along with destruction of the active centers and subsequently the loss of activity. It seems that the contact areas interact with active centers by conformational changes involving secondary structural elements.

The widespread antigenic changes lead to the emergence of a new type of coronavirus (CoV) called as severe acute respiratory syndrome (SARS)-CoV-2 that is immunologically different from the previous circulating species. Angiotensin-converting enzyme-2 (ACE-2) is one of the most important receptors on the cell membrane of the host cells (HCs) which its interaction with spike protein (SP) with a furin-cleavage site results in the SARS-CoV-2 invasion. Hence, in this review, we presented an overview on the interaction of ACE-2 and furin with SP. As several kinds of CoVs, from various genera, have at their S1/S2 binding site a preserved site, we further surveyed the role of furin cleavage site (FCS) on the life cycle of the CoV. Furthermore, we discussed that the small molecular inhibitors can limit the interaction of ACE-2 and furin with SP and can be used as potential therapeutic platforms to combat the spreading CoV epidemic. Finally, some ongoing challenges and future prospects for the development of potential drugs to promote targeting specific activities of the CoV were reviewed. In conclusion, this review may pave the way for providing useful information about different compounds involved in improving the effectiveness of CoV vaccine or drugs with minimum toxicity against human health.Communicated by Ramaswamy H. Sarma.

Based on the accumulative evidences during recent decades, miRNAs have been found overexpressed in several human cancer types and also in Down syndrome patients, contributing to the neuropathology of Down syndrome. From this point of view, investigations on the structure and dynamic mechanisms related to the Argonaute 2 miRNAs binding in which silencing of the mRNA occurs, have inspired many clinical researchers to target this complex to inhibit the silencing process. In the current research, we have virtually screened the OTAVA_CNS_library to introduce new inhibitor compounds for the Ago2/miRNA complex. Ten hit compounds were obtained, with just one of them nominated as the best compound. Following the interaction analysis, by utilizing molecular dynamics (MD) simulations, effects of two mutations (Thr526 to isoleucine and Gln545 to alanine) on the dynamic properties of Ago2 in the complex with the best inhibitor compound were investigated. RMSD, RMSF and h-bond number beside other analyses, highlighted the importance of the Thr526 and Gln545 mutations for the stability and flexibility of the (Ago2)/ligand complex.[Formula: see text]Communicated by Ramaswamy H. Sarma.

Glucokinase (GK) plays a key role in the regulation of hepatic glucose metabolism. An unusual mechanism of positive cooperativity of monomeric GK containing only a single binding site for glucose is very interesting and still unclear. The activation process of GK is associated with a large-scale conformational change from the inactive to the active state. Here, conventional and targeted molecular dynamics simulations were used to study the conformational dynamics of GK in the stable configurations and in the transition from active to inactive state. Three phases of the structural reorganization of GK were detected. The first step is a transformation of GK from the active state to the intermediate structure, where the cleft between the domains is open, but alpha helix 13 is still inside the small domain. From this point, there are two alternative paths. One path leads to the inactive state through the release of helix 13 from the inside of small domain to the outside. Other path goes back to the active state. Simulation results reveal the critical role of helix 13 in the transformation of GK from the open state to inactive one and the influence of the loop 2 on the protein transformation between the open and the closed active states. Principal component analysis and covariance matrix analysis were carried out to analyze the dynamics of protein. Importance of hydrogen bonds in the stability of the closed conformation is shown. Overall, our simulations provide new information about the dynamics of GK and its structural transformation.Communicated by Ramaswamy H. Sarma.

The interaction ability of bovine serum albumin (BSA) with 2,6-divanillylidenecyclohexanone (DVH) as a stable curcumin derivative was investigated using fluorescence and circular dichroism (CD) spectroscopy techniques under simulative physiological conditions (pH = 7.2). Following the obtained results of binding studies, bovine serum albumin nanoparticles (BSANPs) were synthesized and characterized using Fourier transform infrared spectroscopy (FT-IR), filed emission scanning electron microscopy (FE-SEM), atomic force microscope (AFM) and dynamic light scattering (DLS). The stable BSANPs showed a spherical shape with a diameter of 149.14 ± 46.69 nm and the formulation of BSA had no change during the fabrication process. DVH was loaded on BSANPs (DVH@BSANPs) and the release studies showed sustained release of DVH from BSANPs. The validation of DVH@BSANPs system confirmed that the Fickian release mechanism of DVH followed on Korsmeyer-Pepas model. The in vitro studies on HFFF2 and MDA-MB-231 were investigated using MTT assay, DAPI and annexinV/PI staining that showed biocompatible BSANPs reduced the cytotoxicity of DVH in normal cell lines significantly, and antitumor activity of DVH was increased when it was loaded onto BSANPs without necrosis. These results suggest that DVH@BSANPs are a novel biocompatible sustained release system for effective therapeutic approach.Communicated by Ramaswamy H. Sarma.

The aim of this study was to investigate the spermine effect on the thermal denaturation, conformation and activity of elastase at three temperatures of 303, 313 and 323 K in the Tris buffer, at pH 8.5, using UV-vis spectrophotometry, spectrofluorometry and circular dichroism as well as molecular docking and molecular simulation. The increased absorption of elastase in the presence of spermine suggested a change in the environment of tryptophan. It was found that under the influence of spermine, the emission intensity of elastase extremely was reduced, and the use of the Stern-Volmer equation showed that some static quenching had occurred. The thermodynamic parameters values (enthalpy and entropy) and the molecular docking technique also revealed that van der Waals forces or hydrogen bonding interactions played an important role in the binding process. The spermine-elastase complex formation led to increasing the value of the catalytic constant ( k ). So it could be considered as an activator. Slight changes were observed in the second structure of elastase (1.06% increase for the α-helix and 0.048% decrease the β-sheet) and the thermal stability effect. Molecular docking results also demonstrated that spermine could bind to porcine pancreatic elastase, and van der Waals forces or hydrogen bonding interactions played the major role in the binding process. Overall, our results showed that spermine could induce structural alterations in elastase, acting as a partial stabilizer and an activator for the enzyme.Communicated by Ramaswamy H. Sarma.cat ). So it could be considered as an activator. Slight changes were observed in the second structure of elastase (1.06% increase for the α-helix and 0.048% decrease the β-sheet) and the thermal stability effect. Molecular docking results also demonstrated that spermine could bind to porcine pancreatic elastase, and van der Waals forces or hydrogen bonding interactions played the major role in the binding process. Overall, our results showed that spermine could induce structural alterations in elastase, acting as a partial stabilizer and an activator for the enzyme.Communicated by Ramaswamy H. Sarma.

Carboxypeptidase A (EC.3.4.17.1) is a zinc-containing proteolytic enzyme that removes the C-terminal amino acid from a peptide chain with the free carboxylate-terminal. In this study, the effect of spermine interaction on the structure and thermal stability of Carboxypeptidase A was investigated by ultraviolet - visible spectroscopy, fluorescence spectroscopy, circular dichroism, Kinetic measurement, molecular docking and simulation studies have also been followed at the pH of 7.5. The transition temperature of Carboxypeptidase A, as a criterion of protein thermal stability, in the presence of spermine was enhanced by increasing the concentration of spermine. The results of fluorescence intensity changes, at two temperatures of 308 and 318 K, also suggested that spermine had a great ability to quench the fluorescence of Carboxypeptidase A through the static quenching procedure. The thermodynamic parameters changes, including standard Gibbs free-energy, entropy and enthalpy, showed that the binding of spermine to Carboxypeptidase A was spontaneous and the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A-spermine complex. The changes in the content of the α-helix and the β-sheet of the Carboxypeptidase A with binding to spermine were shown by the CD spectra method. Further, kinetic studies revealed that by increasing concentration of spermine, the activity of Carboxypeptidase A was enhanced. Also, the docking study revealed that the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A-spermine complex. As a result, spermine could be considered as an activator and a stabilizer for Carboxypeptidase A.Communicated by Ramaswamy H. Sarma.

To perform biological evaluations of newly-designed Pt(II) and Pd(II) complexes, the present study was conducted with targeted protein human serum albumin (HSA) and HCT116 cell line as model of human colorectal carcinoma. The binding of Pt(II) and Pd(II) complexes to HSA was analyzed using fluorescence spectroscopy and molecular docking. The thermal stability and alterations in the secondary structure of HSA in the presence of Pt(II) and Pd(II) complexes were investigated using the thermal denaturation method and circular dichroism (CD) spectroscopy. The cytotoxicity of the Pt(II) and Pd(II) complexes was studied against the HCT116 cell line using MTT assay. The binding analysis revealed that the fluorescence findings were well in agreement with docking results such that there is only one binding site for each complex on HSA. Binding constants of 8.7 × 10 3 M -1 , 2.65 × 10 3 M -1 , 0.3 × 10 3 M -1 , and 4.4 × 10 3 M -1 were determined for Pd(II) and Pt(II) complexes (I-IV) at temperature of 25 °C, respectively. Also, binding constants of 1.9 × 10 3 M -1 , 15.17 × 10 3 M -1 , 1.9 × 10 3 M -1 , and 13.1 × 10 3 M -1 were determined for Pd(II) and Pt(II) complexes (I-IV) at temperature of 37 °C, respectively. The results of CD and thermal denaturation showed that the molecular structure of HSA affected by interaction with Pt(II) and Pd(II) complexes is stable. Cytotoxicity studies represented the growth suppression effect of the Pt(II) and Pd(II) complexes toward the human colorectal carcinoma cell line. Therefore, the results suggest that the new designed Pt(II) and Pd(II) complexes are well promising candidates for use in cancer treatment, particularly for human colorectal cancer. Communicated by Ramaswamy H. Sarma.

In this study, a nanoemulsion containing mebudipine [composed of ethyl oleate (oil phase), Tween 80 (T80), Span 80 (S80) (surfactants), polyethylene glycol 400, ethanol (cosurfactants), and deionized water] was prepared with the aim of improving its bioavailability for an effective antihypertensive therapy. Particle size of the formulation was measured by dynamic light scattering. Then, artificial neural networks were used in identifying factors that influence the particle size of the nanoemulsion. Three variables, namely, amount of surfactant system (T80 + S80), amount of polyethylene glycol, and amount of ethanol as cosurfactants, were considered as input values and the particle size was used as output. The developed model showed that all the three inputs had some degrees of effect on particles size: increasing the value of each input decreased the size. Furthermore, amount of surfactant was found to be the dominant factor in controlling the final particle size of nanoemulsion. Communicated by Ramaswamy H. Sarma.

The interaction of ZnO nanoparticles with biological molecules such as proteins is one of the most important and challenging problems in molecular biology. Molecular dynamics (MD) simulations are useful technique for understating the mechanism of various interactions of proteins and nanoparticles. In the present work, the interaction mechanism of insulin with ZnO nanoparticles was studied. Simulation methods including MD and replica exchange molecular dynamics (REMD) and their conditions were surveyed. According to the results obtained by REMD simulation, it was found that insulin interacts with ZnO nanoparticle surface via its polar and charged amino acids. Unfolding insulin at ZnO nanoparticle surface, the terminal parts of its chains play the main role. Due to the linkage between chain of insulin and chain of disulfide bonds, opposite directional movements of N terminal part of chain A (toward nanoparticle surface) and N termini of chain B (toward solution) make insulin unfolding. In unfolding of insulin at this condition, its helix structures convert to random coils at terminal parts chains.

This study was designed to investigate the interaction of three oxovanadium (IV) Schiff base complexes with bovine serum albumin (BSA) by means of various spectroscopic and electrochemical methods along with molecular docking study and molecular dynamics simulations. Binding constants were estimated by fluorescence and UV-Vis spectroscopy. The results indicated a good affinity of the complexes for BSA in which furyl derivative had more activity. Molecular docking study showed that these complexes have the similar binding modes and located within subdomain IB in site III of BSA. The supporting of molecular docking and molecular dynamics results by experimental data, confirms the validity of the interactions data obtained by these methods. Biological activity against cancer cell showed that furyl derivative has higher activity than other complexes. Pharmaceutical analysis also showed that, these complexes potentially can be used as anticancer agents.

Successful clinical experience of using cisplatin and its derivatives in cancer therapy has encouraged scientists to synthesize new metal complexes with the aim of interacting with special targets such as proteins In this regard, biological effects of [Pt(FIP)(Phen)](NO 3 ) 2 compound which contains a novel phen-imidazole ligand, FIP, was investigated on bovine liver catalase (BLC) structure and function. Various spectroscopic methods such as UV-visible, fluorescence, and circular dichroism (CD) were applied at two temperatures 25 and 37°C for kinetics and structural studies. As a consequence, the enzymatic activity decreased slightly with increasing the platinum compound's concentration up to 30 μM and then remained constant at near 80% after this concentration. On the other hand, the fluorescence quenching measurements revealed that despite slight changes in activity, catalase experiences notable alterations in three-dimensional environment around the chromophores of the enzyme structure with increasing platinum complex concentration. Moreover, quenching data showed that BLC has two binding sites for Pt complex and hydrogen bonding interactions play a major role in the binding process. Furthermore, CD spectroscopy data showed that Pt(II) complex induces significant decrease in α-helix content of the secondary structure of BLC, but notable increase in random coil proportion accompanying a slight decrease in β-sheet content. All in all, hydrogen bonding interactions which are mainly involved in the binding process of the novel phen-imidazole compound to BLC significantly alter the protein structure but slightly change its function. This might be a promising outcome for chemotherapists and medicinal chemists to investigate in vivo properties of this novel metal complex with significant binding tendency to a macromolecule in the low concentrations without decreasing its intrinsic function.

Due to widespread exposure of human being to various sources of static magnetic fields (SMF), their effect on the spatial and temporal status of structure, arrangement, and polymerization of tubulin was studied at the molecular level. The intrinsic fluorescence intensity of tubulin was increased by SMF, indicating the repositioning of tryptophan and tyrosine residues. Circular Dichroism spectroscopy revealed variations in the ratios of alpha helix, beta, and random coil structures of tubulin as a result of exposure to SMF at 100, 200, and 300 mT. Transmission Electron microscopy of microtubules showed breaches and curvatures whose risk of occurrence increased as a function of field strength. Dynamic light scattering revealed an increase in the surface potential of tubulin aggregates exposed to SMF. The rate and extent of polymerization increased by 9.8 and 33.8%, at 100 and 300 mT, respectively, but decreased by 36.16% at 200 mT. The conductivity of polymerized tubulin increased in the presence of 100 and 300 mT SMF but remained the same as the control at 200 mT. The analysis of flexible amino acids along the sequence of tubulin revealed higher SMF susceptibility in the helical electron conduction pathway set through histidines rather than the vertical electron conduction pathway formed by tryptophan residues. The results reveal structural and functional effects of SMF on tubulin assemblies and microtubules that can be considered as a potential means to address the safety issues and for manipulation of bioelectrical characteristics of cytosol, intracellular trafficking and thus, the living status of cells, remotely.

Herein, the interaction of iron nanoparticle (Fe-NP) with cytochrome c (Cyt c) was investigated, and a range of techniques such as dynamic light scattering (DLS), zeta potential measurements, static and synchronous fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and ultraviolet-visible (UV-vis) spectroscopy were used to analyze the interaction between Cyt c and Fe-NP. DLS and zeta potential measurements showed that the values of hydrodynamic radius and charge distribution of Fe-NP are 83.95 ± 3.7 nm and 4.5 ± .8 mV, respectively. The fluorescence spectroscopy results demonstrated that the binding of Fe-NP with Cyt c is mediated by hydrogen bonds and van der Waals interactions. Also Fe-NP induced conformational changes in Cyt c and reduced the melting temperature value of Cyt c from 79.18 to 71.33°C. CD experiments of interaction between Fe-NP and Cyt c revealed that the secondary structure of Cyt c with the dominant α-helix structures remained unchanged whereas the tertiary structure and heme position of Cyt c are subjected to remarkable changes. Absorption spectroscopy at 695 nm revealed that Fe-NP considerably disrupt the Fe…S(Met80) bond. In addition, the UV-vis experiment showed the peroxidase-like activity of Cyt c upon interaction with Fe-NP. Hence, the data indicate the Fe-NP results in unfolding of Cyt c and subsequent peroxidase-like activity of denatured species. It was concluded that a comprehensive study of the interaction of Fe-NP with biological system is a crucial step for their potential application as intracellular delivery carriers and medicinal agents.

A vast research has been conducted to find suitable and safe carriers for vital and pH-sensitive drugs including antibiotics. This article reports the use of easily accessible and abundant purified beta-lactoglobulin (β-LG) protein as the potential carrier of widely used Kanamycin (Kana) and Ciprofloxacin (Cip) antibiotics. Spectroscopic techniques (Fluorescence, UV-vis, Circular Dichroism) combined with molecular docking were used to determine the binding mechanism of these drugs. Fluorescence studies showed moderate binding affinity with the calculated binding constants K Cip  = 60.1 (±0.2) × 10 3  M -1 and K kana  = 2.5 (±0.6) × 10 3  M -1 with the order of Cip > Kana. Results of UV-vis were consistent with fluorescence measurements and demonstrated a stronger complexation for Cip rather than Kana. The secondary structure of β-LG was preserved upon interaction with Kana; however, a reduction in β-sheet content from 39.1 to 31.9% was convoyed with an increase in α-helix from 12.8 to 20.5% due to complexation of Cip. Molecular docking studies demonstrated that preferred binding sites of these drugs are not the same and several amino acids are involved in stabilizing the interaction. Based on the achieved results, Kana and Cip can spontaneously bind to β-LG and this protein may serve as their transport vehicle.

Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris-HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine-α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.

In this study, a novel method to probe molecular interactions and binding of human hemoglobin (Hb) with nanodiamond (ND) was introduced based on the surface tension measurement. This method complements conventional techniques, which are basically done by zeta potential and dynamic light scattering (DLS) measurements, near and far circular dichroism (CD) spectroscopy, intrinsic and extrinsic fluorescence spectroscopy. Addition of ND to Hb solution increased the surface tension value of Hb-ND complex relative to those of Hb and ND molecules. The zeta potential values reveled that Hb and ND provide identical charge distribution at pH 7.5. DLS measurements demonstrated that Hb, ND, and ND-Hb complex have hydrodynamic radiuses of 98.37 ± 4.57, 122.07 ± 7.88 nm and 62.27 ± 3.70 at pH of 7.5 respectively. Far and near UV-CD results indicated the loss of α-helix structure and conformational changes of Hb, respectively. Intrinsic fluorescence data demonstrated that the fluorescence quenching of Hb by ND was the result of the static quenching. The hydrophobic interaction plays a pivotal role in the interaction of ND with Hb. Fluorescence intensity changes over time revealed conformational change of Hb continues after the mixing of the components (Hb-ND) till 15 min, which is indicative of the denaturation of the Hb relative to the protein control. Extrinsic fluorescence data showed a considerable enhancement of the ANS fluorescence intensity of Hb-ND system relative to the Hb till 60 nM of ND, likely persuaded by greater exposure of nonpolar residues of Hb hydrophobic pocket. The remarkable decrease in T m value of Hb in Hb-ND complex exhibits interaction of Hb with ND conducts to conformational changes of Hb. This study offers consequential discrimination into the interaction of ND with proteins, which may be of significance for further appeal of these nanoparticles in biotechnology prosecution.

Acetoacetate (AA) is an important ketone body, which produces reactive oxygen species (ROS). Advanced glycation end products (AGEs) are defined as final products of glycation process whose production is influenced by the levels of ROS. The accumulation of AGEs in the body contributes to pathogenesis of many diseases including complications of diabetes, and Alzheimer's and Parkinson's disease. Here, we evaluated the impact of AA on production of AGEs upon incubation of human serum albumin (HSA) with glucose. The effect of AA on the AGEs formation of HSA was studied under physiological conditions after incubation with glucose for 35 days. The physical techniques including circular dichroism (CD) and fluorescence spectroscopy were used to assess the impact of AA on formation and structural changes of glycated HSA (GHSA). Our results indicated that the secondary and tertiary structural changes of GHSA were increased in the presence of AA. The fluorescence intensity measurements of AGEs also showed an increase in AGEs formation. Acetoacetate has an activator effect in formation of AGEs through ROS production. The presence of AA may result in enhanced glycation in the presence of glucose and severity of complications associated with accumulation of AGEs.

The interaction between human hemoglobin (Hb) and oxali-palladium was studied using different spectroscopic methods of UV-vis, fluorescence, circular dichroism (CD), and chemiluminescence at two temperatures of 25 and 37°C. The experimental results showed that both dynamic and static quenching is occurred simultaneously when oxali-palladium quenches the fluorescence of Hb. According to the fluorescence quenching method, the binding site number, apparent binding constant, and corresponding thermodynamic parameters were measured at two temperatures. The values of ΔH°, ΔS°, and ΔG° indicate that process of the formation of oxali-palladium-Hb complex is a spontaneous interaction procedure in which electrostatic interaction plays a major role. In addition, UV-vis and CD results showed that the addition of oxali-palladium changes the conformation of Hb. To evaluate the functional changes of Hb via destruction of the heme structure, fluorescence studies were performed. The results demonstrated that two fluorescent heme degradation products are found during the interaction of oxali-palladium with Hb. Also, the amount of hydrogen peroxide produced in the solution of Hb due to the interaction of oxali-palladium with Hb using chemiluminescence method indicated heme degradation in the protein is occurred. Structural and functional changes induced in Hb via heme degradation are considered as side effects of this synthesized anticancer drug.

The binding of thiamine (vitamin B1) on lysozyme has been examined at various ionic strengths of phosphate buffer (pH 6.9), various pH values, and various protein concentrations at 25°C using thiamine selective membrane electrode. This method is faster and more precise than equilibrium dialysis technique which can obtain sufficient and accurate data for binding analysis. The values of Hill equation parameters were estimated for each set using binding capacity concept and used for calculation of intrinsic binding affinity. The results represent two binding sets for thiamine on lysozyme at various experimental conditions.

Recently, the great interests in manufacturing and application of metal oxide nanoparticles in commercial and industrial products have led to focus on the potential impact of these particles on biomacromolecules. In the present study, the interaction of copper oxide (CuO) nanoparticles with bovine serum albumin (BSA) was studied by spectroscopic techniques. The zeta potential value for BSA and CuO nanoparticles with average diameter of around 50 nm at concentration of 10 μM in the deionized (DI) water were -5.8 and -22.5 mV, respectively. Circular dichroism studies did not show any changes in the content of secondary structure of the protein after CuO nanoparticles interaction. Fluorescence data revealed that the fluorescence quenching of BSA by CuO nanoparticles was the result of the formed complex of CuO nanoparticles - BSA. Binding constants and other thermodynamic parameters were determined at three different temperatures. The hydrogen bond interactions are the predominant intermolecular forces to stabilize the CuO nanoparticle - BSA complex. This study provides important insight into the interaction of CuO nanoparticles with proteins, which may be of importance for further application of these nanoparticles in biomedical applications.

Imidazole analogs are the agents that attract both bioinorganic chemist and drug designer. Numerous methods have been proposed for synthesis of imidazole derivatives. In this study, a series of heterocyclic system with p-conjugated system such as 2-aryl-imidazo[4,5-f][1,10]phenanthroline analogs were synthesized. Then, three new palladium(II) complexes containing 2-(Furan-2-yl)-1H-Imidazo[4,5-f][1,10]Phenanthroline (FIP) and 2-(thiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (TIP) ligands were synthesized. The structures of the compounds, [Pd(Phen)(TIP)](NO3)2, [Pd(Phen)(FIP)](NO3)2, and [Pd(FIP)2]Cl were determined by spectroscopic methods and elemental analysis. Biological activity of the complexes synthesized was assessed against chronic myelogenous leukemia cell line, K562. Also, the interactions of human serum albumin with complexes were investigated using isothermal titration in the Tris buffer, pH 7.4. According to the results obtained, it was found that there is a set of six binding sites for these complexes on HSA with positive cooperativity in the binding process. Docking technique was also applied to confirm the experimental results. The results showed that smaller complexes have higher interaction affinity.

Effects of sodium (octyl, dodecyl, hexadecyl) sulfate and their cationic analogous on the structure of adenosine deaminase (ADA) were investigated by fluorescence and circular dichroism spectroscopy as well as molecular dynamics simulation and docking calculation. Root-mean-square derivations, radius of gyration, solvent accessible surface area, and radial distribution function were obtained. The results showed that anionic and cationic surfactants reduce protein stability. Cationic surfactants have more effect on the ADA structure in comparison with anionic surfactants. More concentration and longer surfactants are parallel to higher denaturation. Furthermore, aggregation in the presence of anionic surfactants is more than cationic surfactants. Docking data showed that longer surfactants have more interaction energy and smaller ones bound to the active site.

In recent years, using heavy metal compounds such as platinum as anticancer agent is one of the common ways in chemical therapy. In this study, a new anticancer compound of glycine derivatives of Pt(II) complex (amyl-glycine1, 10-phenanthroline Platinum nitrate) was designed, and the biological effects of this novel compound on the alterations in the function and structure of human hemoglobin (Hb) at different temperatures of 25 and 37°C were assessed by applying various spectroscopic (fluorescence and circular dichroism (CD)) and theoretical methods. Fluorescence data indicated the strong ability of Pt(II) complex to quench the intrinsic fluorescence of Hb. The binding constant, number of binding sites, and thermodynamic parameters at two temperatures were calculated, and the results indicated the major possibility of occurring van der Waals force or hydrogen bond interactions in the Pt(II) complex-Hb interaction. For evaluating the alteration of secondary structure of Hb upon interaction with various concentrations of complex, far-UV CD spectra were used and it was observed that in high dose of complex, significant changes were occurred which is indicative of some side effects in overdosing of this complex. On the other hand, the molecular docking results illustrate that are well in agreement in obtaining data with spectroscopy. Above results suggested that using Pt(II) complex as an anticancer agent, model drug in high-dose usage might cause some disordering in structure and function of Hb as well as improve understanding of the side effects of newly designed metal anticancer drugs undergoing.

Herein we have engineered a micellar Cu protoporphyrin catalyst that mediates carbon bond activation using peroxide as an electron source. Cu protoporphyrin is a biomimetic model of active site of chloroperoxidase enzyme, which catalyzes the carbon bond halogenation in the presence of a suitable amount of H 2 O 2 . The encapsulation of Cu(II) Protoporphyrin IX/L-Cysteine inside of cetyltrimethylammonium bromide micelle increases the rate of chlorination at pH 3. The cited catalyst resists high concentrations of hydrogen peroxide, which is previously reported as a suicide inactivator component of hemo-enzymes. Isothermal Titration Calorimetry (ITC) and Dynamic Light Scattering (DLS) data have revealed the formation of a micellar complex by encapsulation of six Cu(II) proporphyrins within each micelle. Moreover, electrochemical investigations indicate that L-Cysteine increases the intensity of electron transferred due to the formation of self-assembled monolayer on Au electrode. Our results paved a road toward the design of a more robust mimetic catalysis based on Protoporphyrin IX derivatives.

The simple aggregation of insulin under various chemical and physical stresses is still an important challenge for both pharmaceutical production and clinical formulation. In the storage form, this protein is subjected to various chemical modifications which alter its physicochemical and aggregation properties. Aspirin (acetylsalicylic acid) which is the most widely used medicine worldwide has been indicated to acetylate a large number of proteins both in vitro and in vivo. In this study, as insulin treated with aspirin at 37°C, a significant level of acetylation was observed by flourescamine and o-phthalaldehyde assay. Also, different spectroscopic techniques, gel electrophoresis, and microscopic assessment were applied to compare the structural variation and aggregation/fibrillation propensity among acetylated and non-acetylated insulin samples. The results of spectroscopic assessments elucidate that acetylation induces insulin unfolding which is accompanied with the exposure of protein hydrophobic patches, a transition from alpha-helix to beta-sheet and increased propensity of the protein for aggregation. The kinetic studies propose that acetylation increases aggregation rate of insulin under both thermal and chemical stresses. Also, gel electrophoresis and dynamic light scattering experiments suggest that acetylation induces insulin oligomerization. Additionally, the results of Thioflavin T fluorescence study, Congo red absorption assessment, and microscopic analysis suggest that acetylation with aspirin enhances the process of insulin fibrillation. Overall, the increased susceptibility of acetylated insulin for aggregation may reflect the fact that this type of modification has significant structural destabilizing effect which finally makes the protein more vulnerable for pathogenic aggregation/fibrillation.

Some amino acid derivatives, such as R-glycine, have been synthesized together with their full spectroscopic characterization. The sodium salts of these bidentate amino acid ligands have been interacted with [M(bpy)(H2O)2](NO3)2 giving the corresponding some new complexes with formula [M(bpy)(R-gly)]NO3 (where M is Pt(II) or Pd(II), bpy is 2,2'-bipyridine and R-gly is butyl-, hexyl- and octyl-glycine). Due to less solubility of octyl derivatives, the biological activities of butyl and hexyl derivatives have been tested against chronic myelogenous leukemia cell line, K562. The interaction of these complexes with highly polymerized calf thymus DNA has been extensively studied by means of electronic absorption, fluorescence and other measurements. The experimental results suggest that these complexes positive cooperatively bind to DNA presumably via groove binding. Molecular dynamic results show that the DNA structure is largely maintained its native structure in hexylglycine derivative-water mixtures and at lower temperatures. The simulation data indicates that the more destabilizing effect of butylglycine is induced by preferential accumulation of these molecules around the DNA and due to their more negative free energy of binding via groove binding.

A novel peroxidase-like artificial enzyme, named "caseoperoxidase", was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and k(cat) performance toward the native horseradish peroxidase demonstrated by the steady state kinetics using UV-vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein) was selected as an appropriate apo-protein for the heme active site because of its innate flexibility and exalted hydrophobicity. This selection was confirmed by homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation and provided a suitable protective role for the heme active-site. Additional experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.

Low-mobility group nonhistone chromatin protein, LMG160, is a nuclear matrix ribonucleoprotein particle (RNP) which has a RNA molecule with approximately 300 bases. In this study, structural stability of the intact LMG160 (I-LMG160) was investigated at different ionic strength and in the absence of its RNA moiety (T-LMG160) employing spectroscopic and thermodynamic techniques. The UV absorption spectra showed hypochromicity and red shift under increasing ionic strength for both forms of LMG160 but in different extents. The fluorescence emission intensity was decreased as ionic strength was increased and the Stern-Volmer quenching constant (Ksv) for T-LMG160 was 3.7 times less than for I-LMG160. In the absence of sodium chloride, I-LMG160 exhibited a very stable structure against the temperature change compared to T-LMG160. The thermodynamic parameters showed that the positive values of ΔHm and ΔSm increased by increasing ionic strength in both forms of LMG160. Removal of the RNA moiety altered secondary structure: as T-LMG160 showed more helical content than I-LMG160. From the results, it is concluded that I-LMG160 is more sensitive to alteration of environment and the RNA has an important role in this RNP conformation. Also, interaction of both I- and T-LMG160 with sodium chloride is entropy driven and is usually accompanied by surface hydrophobicity.

Sodium benzoate (SB), a powerful inhibitor of microbial growth, is one of the most commonly used food preservative. Here, we determined the effects of SB on human serum albumin (HSA) structure in the presence or absence of glucose after 35 days of incubation under physiological conditions. The biochemical, biophysical, and molecular approaches including free amine content assay (TNBSA assay), fluorescence, and circular dichroism spectroscopy (CD), differential scanning calorimetry (DSC), and molecular docking and LIGPLOT studies were utilized for structural studies. The TNBSA results indicated that SB has the ability to bind Lys residues in HSA through covalent bonds. The docking and LIGPLOT studies also determined another specific site via hydrophobic interactions. The CD results showed more structural helicity for HSA incubated with SB, while HSA incubated with glucose had the least, and HSA incubated with glucose + SB had medium helicity. Fluorescence spectrophotometry results demonstrated partial unfolding of HSA incubated with SB in the presence or absence of glucose, while maximum partial unfolding was observed in HSA incubated with glucose. These results were confirmed by DSC and its deconvoluted thermograms. The DSC results also showed significant changes in HSA energetic structural domains due to HSA incubation with SB in the presence or absence of glucose. Together, our studies showed the formation of three different intermediates and indicate that biomolecular investigation are effective in providing new insight into safety determinations especially in health-related conditions including diabetes.

Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H2O2 into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25 °C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (Tm) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes.

Alterations in the synthesis of melanin contribute to a number of diseases; therefore, the design of new tyrosinase inhibitors is very important. Mushroom tyrosinase (MT) is a metalloenzyme, which plays an important role in melanin biosynthesis. In this study, the inhibitory effect of a novel designed compound, i.e. 2-((1Z)-(2-(2,4-dinitrophenyl)hydrazin-1-ylidene)methyl)phenol, as a specific ligand which can bind to the copper ion of MT, has been assessed. The ligand was found to competitively inhibit both the cresolase and catecholase activities of MT, with small inhibition constants of 2.8 and 2.6 μM, respectively. Intrinsic fluorescence studies were performed to gain more information on the binding constants. Docking results indicated that the ligand binds to copper ions in the active site of MT via the OH group of the ligand. The ligand makes four hydrogen bonds with aspartic acid and one hydrogen bond with the histidine residue in the active site. Molecular dynamics results show that ligand binds to the MT via both electrostatic and hydrophobic interactions with its different parts.