ABSTRACT
Pro-inflammation, which is developed due to the increased production of cytokines, mainly interleukin-6 (IL-6), during the working of immune system pathways, becomes a major concern these days for many researchers. So, it is desired to design, screen, and synthesize new molecules with multi-parametric features showing their efficacy for Toll-like receptors (TLRs) and inhibiting the disease-causing receptor sites like viral infections, cancers, etc. along with controlling inflammation, fever, and other side effects during such pathways. Further, looking at the literature, curcumin a multi-targeted agent is showing its efficiency toward various receptor sites involved in many diseases as mentioned above. This fascinated us to build up new molecules which behave like curcumin with minimum side effects. In silico studies, involving ADMET studies, toxicological data, and docking analyses, of newly synthesized compounds (3-5) along with tautomers of curcumin i.e., (1-2), and some reported compounds like 9 and 10 have been studied in detail. Great emphasis has been made on analyzing binding energies, protein-ligand structural interactions, stabilization of newly synthesized molecules against various selected receptor sites using such computational tools. Compound 3 is the most efficient multifunctional agent, which has shown its potential toward most of the receptor sites in docking analysis. It has also responded well in Molecular dynamics (MD) simulation toward 5ZLN, 4RJ3, 4YO9, 4YOJ, and 1I1R sites. Finally, studies were extended to understand in vitro anti-inflammatory activity for particularly compound 3 in comparison to diclofenac and curcumin, which signifies the efficiency of compound 3.
Subject(s)
Antineoplastic Agents , Curcumin , Humans , Curcumin/pharmacology , Curcumin/chemistry , Molecular Docking Simulation , Antineoplastic Agents/pharmacology , Pyrimidines , Immune System , Inflammation , PurinesABSTRACT
Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.
Subject(s)
Enzyme Stability , Escherichia coli , Pyrococcus abyssi , Recombinant Fusion Proteins , Temperature , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Kinetics , Pyrococcus abyssi/genetics , Pyrococcus abyssi/enzymology , Small Ubiquitin-Related Modifier Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , Genetic Vectors/metabolism , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , SUMO-1 Protein/chemistry , Cloning, Molecular , SolubilityABSTRACT
Hennekam Lymphangiectasia-Lymphedema Syndrome 3 (HKLLS3) is a rare genetical disorder caused by mutations in a few genes including ADAMTS3. It is characterized by lymphatic dysplasia, intestinal lymphangiectasia, severe lymphedema and distinctive facial appearance. Up till now, no extensive studies have been conducted to elucidate the mechanism of the disease caused by various mutations. As a preliminary investigation of HKLLS3, we sorted out the most deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs) that might affect the structure and function of ADAMTS3 protein by using a variety of in silico tools. A total of 919 nsSNPs in the ADAMTS3 gene were identified. 50 nsSNPs were predicted to be deleterious by multiple computational tools. 5 nsSNPs (G298R, C567Y, A370T, C567R and G374S) were found to be the most dangerous and can be associated with the disease as predicted by different bioinformatics tools. Modelling of the protein shows it can be divided into segments 1, 2 and 3, which are connected by short loops. Segment 3 mainly consists of loops without substantial secondary structures. With prediction tools and molecular dynamics simulation, some SNPs were found to significantly destabilize the protein structure and disrupt the secondary structures, especially in segment 2. The deleterious effects of mutations in segment 1 are possibly not from destabilization but from other factors such as the change in phosphorylation as suggested by post-translational modification (PTM) studies. This is the first-ever study of ADAMTS3 gene polymorphism, and the predicted nsSNPs in ADAMST3, some of which have not been reported yet in patients, will serve for diagnostic purposes and further therapeutic implications in Hennekam syndrome, contributing to better diagnosis and treatment.
Subject(s)
Lymphedema , Polymorphism, Single Nucleotide , Humans , Molecular Dynamics Simulation , Lymphedema/genetics , Protein Stability , Computational BiologyABSTRACT
OBJECTIVE: Periodontitis is a chronic oral disease prevalent worldwide, and natural products are recommended as adjunctive therapy due to their minor side effects. Curcumin, a widely used ancient compound, has been reported to possess therapeutic effects in periodontitis. However, the exact mechanism underlying its activity remains unclear. In this context, the present study aimed to conduct computational simulations to uncover the potential mechanism of action of Curcumin in the treatment of periodontitis. MATERIALS AND METHODS: Single-cell analysis was conducted using a dataset (i.e., GSE164241) curated from the Gene Expression Omnibus (GEO) database through an R package "Seurat package." Bulk RNA sequencing data were curated from GSE10334 and GSE16134 and processed by R package "Limma." Then, the marker genes in the single-cell transcriptome and differentially expressed genes (DEGs) in the bulk transcriptome were integrated. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were also carried out to reveal their functionalities. Key targets were mined from their protein-protein interaction (PPI) network topologically. Afterward, molecular docking was performed. The top-ranked pose was subjected to molecular dynamics simulations to investigate the stability of the docking result. RESULTS: FOS, CXCL1, CXCL8, and IL1B, were filtered after a series of selected processes. The results of molecular modeling suggested that except for IL1B, the Vena Scores of the rest exceeded -5 kcal/mol. Furthermore, the molecular dynamic simulation indicated that the binding of the CXCL8-Curcumin complex was stable over the entire 100 ns simulation. CONCLUSION: The present study unlocked the binding modes of CXCL1, FOS, and CXCL8 with the Curcumin molecule, which were relatively stable, especially for CXCL8, hindering its promising potential to serve as the critical targets of Curcumin in periodontitis treatment.
Subject(s)
Curcumin , Periodontitis , Humans , Gene Expression Profiling/methods , Curcumin/pharmacology , Curcumin/therapeutic use , Molecular Docking Simulation , Periodontitis/drug therapy , Periodontitis/genetics , Protein Interaction Maps/genetics , Computational Biology/methodsABSTRACT
KDM5A over-expression mediates cancer cell proliferation and promotes resistance toward chemotherapy through epigenetic modifications. As its complete mechanism of action is still unknown, there is no KDM5A specific drug available at clinical level. In the current study, lead compounds for KDM5A were determined through pharmacophore modeling and high-throughput virtual screening from Asinex libraries containing 0.5 million compounds. These virtual hits were further evaluated and filtered for ADMET properties. Finally, 726 compounds were used for docking analysis against KDM5A. On the basis of docking score, 10 top-ranked compounds were selected and further evaluated for non-central nervous system (CNS) and CNS drug-like properties. Among these compounds, N-{[(7-Methyl-4-oxo-1,2,3,4-tetrahydrocyclopenta [c] chromen-9-yl) oxy]acetyl}-l-phenylalanine (G-score: -11.363 kcal/mol) was estimated to exhibit non-CNS properties while 2-(3,4-Dimethoxy-phenyl)-7-methoxy-chromen-4-one (G-score: -7.977 kcal/mol) was evaluated as CNS compound. Docked complexes of both compounds were finally selected for molecular dynamic simulation to examine the stability. This study concluded that both these compounds can serve as lead compounds in the quest of finding therapeutic agents against KDM5A associated cancers.
Subject(s)
Antineoplastic Agents/chemistry , Phenylalanine/chemistry , Retinoblastoma-Binding Protein 2/metabolism , Antineoplastic Agents/pharmacology , Binding Sites , Computer-Aided Design , Drug Screening Assays, Antitumor , High-Throughput Screening Assays , Ligands , Molecular Docking Simulation , Phenylalanine/pharmacology , Protein Binding , Structure-Activity Relationship , ThermodynamicsABSTRACT
(1) Background: SARS-CoV-2 Omicron BA.1 is the most common variation found in most countries and is responsible for 99% of cases in the United States. To overcome this challenge, there is an urgent need to discover effective inhibitors to prevent the emerging BA.1 variant. Natural products, particularly flavonoids, have had widespread success in reducing COVID-19 prevalence. (2) Methods: In the ongoing study, fifteen compounds were annotated from Echium angustifolium and peach (Prunus persica), which were computationally analyzed using various in silico techniques. Molecular docking calculations were performed for the identified phytochemicals to investigate their efficacy. Molecular dynamics (MD) simulations over 200 ns followed by molecular mechanics Poisson-Boltzmann surface area calculations (MM/PBSA) were performed to estimate the binding energy. Bioactivity was also calculated for the best components in terms of drug likeness and drug score. (3) Results: The data obtained from the molecular docking study demonstrated that five compounds exhibited remarkable potency, with docking scores greater than -9.0 kcal/mol. Among them, compounds 1, 2 and 4 showed higher stability within the active site of Omicron BA.1, with ΔGbinding values of -49.02, -48.07, and -67.47 KJ/mol, respectively. These findings imply that the discovered phytoconstituents are promising in the search for anti-Omicron BA.1 drugs and should be investigated in future in vitro and in vivo research.
ABSTRACT
Pectinase enzymes from different plants have many possible biotechnological applications in various industries. Considering industrial significance of pectinolytic enzymes, the polygalacturonase (PG) gene from grapes was cloned into Escherichia coli DH5α using pTZ57R/T vector. Homologous sequences established a close link to Vitis vinifera. Conserve domain analysis predicted PLN02218 domain belongs to the cl31843 superfamily, showing its function as polygalacturonase. After confirmation by PCR and restriction analysis, the PG gene was expressed in E. coli BL21 and induced by IPTG. Expression level was assessed by 12% SDS-PAGE, which showed a 47 kDa protein. High expression level in the soluble fraction indicated that the protein is intracellular or transmembrane. Maximum expression was obtained with 1 mM IPTG and 6 hour induction time, and autoinduction with lactose increased production of the recombinant enzyme. Zymogram analysis revealed that the induced protein was an active enzyme. Expressed PG showed pectinolytic effect on the physiochemical properties of lemon juice. Natural biopolymers are greatly needed because synthetic fibers can negatively affect health. Pectin hydrolysis of banana pseudostem by the PG enzyme produced better quality fiber. Morphological studies by scanning electron microscopy revealed pectin degradation within the fiber cell architecture, showing the effectiveness of PG treatment on banana pseudostem.
Subject(s)
Polygalacturonase/metabolism , Vitis/enzymology , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Genes, Plant , Microscopy, Electron, Scanning , Polygalacturonase/genetics , Polymerase Chain Reaction , Vitis/geneticsABSTRACT
The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The Km value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding Vmax value was 25.00 ± 0.67 µmol min-1 mg-1 in the presence of 2 mM CaCl2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.
Subject(s)
Pyrococcus abyssi , Starch , alpha-Amylases , alpha-Amylases/metabolism , alpha-Amylases/chemistry , alpha-Amylases/genetics , Enzyme Stability , Hydrogen-Ion Concentration , Models, Molecular , Pyrococcus abyssi/enzymology , Starch/metabolism , Starch/chemistry , TemperatureABSTRACT
One of the most common viral infections worldwide is the Human Papilloma Virus (HPV) which has been linked to cancer and other diseases in many countries. Monosaccharide esters are significant in the field of carbohydrate chemistry because they are efficient in the synthesis of pharmacologically active compounds. Therefore, the present study aimed to perform thermodynamic, molecular docking and molecular dynamics study of a series of previously designed monosaccharaides, methyl ß-d-galactopyranoside (MGP, 1) esters (2-10) with along with their physicochemical and pharmacokinetic properties. We have optimized the MGP esters employing the DFT study at the B3LYP/6-311 + G (d,p) level of theory. The subsequent analysis also investigated the electronic energies, enthalpies, entropies, polarizability, and natural bond orbital (NBO) of these modified esters. Then, MGP esters were docked into CTX-M-15 extended-spectrum beta-lactamase from Escherichia coli (PDB: 4HBT) and E2 DNA-binding domain from human papillomavirus type 31 (PDB: 1A7G), and the results revealed that most of the esters can efficiently bind to the target. Desmond was used to doing molecular dynamics simulations at 200 ns in addition to molecular docking to look at the binding conformational stability of the protein-ligand complex. Based on RMSD and RMSF, it was determined that the stability of the protein-ligand combination was maintained during the whole 200 ns simulations for all compounds. Finally, a pharmacokinetic study suggests that modified esters of MGP exhibited better pharmacokinetic characteristics and were less hazardous than the parent drug. This work demonstrated that potential MGP esters can efficiently bind to 4HBT and 1A7G proteins and opened avenues for the development of newer antimicrobial agents that can target dangerous pathogens.Communicated by Ramaswamy H. Sarma.
Subject(s)
Anti-Infective Agents , Galactose , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Ligands , Escherichia coli , Esters , Antiviral Agents/pharmacologyABSTRACT
Cancer is the uncontrolled spread of abnormal cells that results in abnormal tissue growth in the affected organ. One of the most important organs is exposed to the growth of colon cancer cells, which start in the large intestine (colon) or the rectum. Several therapeutic protocols were used to treat different kinds of cancer. Recently, several studies have targeted tubulin and microtubules due to their remarkable prefoliation. Also, recent research shows that quinoline compounds have significant efficacy against human colorectal cancer. So, the present work investigated the potential of thirty quinoline compounds as tubulin inhibitors using computational methods. A 3D-QSAR approach using two contours (CoMFA and CoMSIA), molecular docking simulation to determine the binding type of the complexes (ligand-receptor), molecular dynamics simulation and identifying pharmacokinetic characteristics were used to design molecules. For all compounds designed (T1-5), molecular docking was used to compare the stability by type of binding. The ADMET has been utilized for molecules with good stability in molecular docking (T1-3); these compounds have good medicinal characteristics. Furthermore, a molecular dynamics simulation (MD) at 100 ns was performed to confirm the stability of the T1-3 compounds; the molecules (T1-3) remained the most stable throughout the simulation. The compounds T1, T2 and T3 are the best-designed drugs for colorectal carcinoma treatments.Communicated by Ramaswamy H. Sarma.
Subject(s)
Colorectal Neoplasms , Quinolines , Humans , Molecular Docking Simulation , Quantitative Structure-Activity Relationship , Molecular Dynamics Simulation , Quinolines/pharmacology , Quinolines/chemistry , Colorectal Neoplasms/drug therapyABSTRACT
Hepatocellular carcinoma (HCC) incidence varies widely around the world and is impacted by factors such as the prevalence of chronic hepatitis B and C infections, alcohol intake, and access to healthcare. The proteins (BRAF_human, VGFR3_human, EGFR_human and UFO_human) play a vital role in hepatocellular carcinoma prognosis, which involves cell proliferation, cell growth, transmission of extracellular signals to the cell nucleus and consequently regulating many other cellular processes. Fostamatinib has been studied for its possible use in the treatment of hepatocellular cancer because it is a more convenient therapy choice for patients and has minor side effects on the human body. However, resveratrol phytochemical has been investigated for its potential use in the prevention and treatment of a wide range of disorders, including cancer, cardiovascular disease, diabetes, and neurological problems due to its frequently antioxidant, anti-inflammatory, and immune-modulating characteristics, which can aid in the prevention of chronic illnesses. This study developed de novo-based fragment-optimized resveratrol (FOR), enhancing therapeutic potential and lowering toxicity. The docking study was performed with four target proteins, and the findings reveal that the vascular endothelial growth factor receptor 3 protein possessed the highest binding energy values of -7.6 kcal/mol with FOR. Additionally, it completely fulfills the criteria of drug-likeliness rules. Thus, FOR proves to be an efficient drug candidate for future in-vivo studies against hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular , Drug Design , Liver Neoplasms , Molecular Docking Simulation , Resveratrol , Resveratrol/pharmacology , Resveratrol/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Humans , Liver Neoplasms/drug therapy , Computer SimulationABSTRACT
New vanillin derivatives, namely, ethyl (4-formyl-2-methoxyphenoxy)acetate (2a) and 2-(4-formyl-2-methoxyphenoxy)-N-phenylacetamide (2b), respectively, were synthesized and characterized by NMR (1H and 13C), IR, mass spectra and confirmed by single-crystal X-ray analysis. Hirshfeld surface (HS) analysis was performed to probe intra- and intermolecular interactions and surface reactivity. 2D fingerprint plots (FP) were used to study the nature and percentage contribution of intermolecular interactions leading to the formation of the crystal unit. Density functional theory (DFT) simulations were used to obtain the electronic structure and reactivity of the new molecules. Natural population analysis (NPA) and frontier molecular orbital (FMO) calculations reveal significant charge transfer and a reduced HOMO-LUMO gap up to 4.34 eV for 2b. Bader's quantum theory of atoms in molecules (QTAIM) study is utilized to understand the surface topological and bonding nature of 2a and 2b. The performed molecular electrostatic potential (MESP) and density of states (DOS) study further suggest sites likely to be attractive to incoming reagents. At the same time, hyperpolarizability (ßo) is used to characterize the nonlinear optical properties, and TD-DFT study shows the excitation energy and absorption behavior. In silico studies were performed, including docking, binding free energies (MMBGSA) and molecular dynamics simulations. Compounds 2a and 2b were docked with RdRp of SARS-Cov-2, and the MMBGSA for 2a and 2b were -30.70 and -28.47 kcal/mol, respectively, while MD simulation showed the stability of protein-ligand complexes.Communicated by Ramaswamy H. Sarma.
ABSTRACT
Tuberculosis (TB), caused by Mycobacterium tuberculosis, ranks among the top causes of global human mortality, as reported by the World Health Organization's 2022 TB report. The prevalence of M. tuberculosis strains that are multiple and extensive-drug resistant represents a significant barrier to TB eradication. Fortunately, having many completely sequenced M. tuberculosis genomes available has made it possible to investigate the species pangenome, conduct a pan-phylogenetic investigation, and find potential new drug targets. The 442 complete genome dataset was used to estimate the pangenome of M. tuberculosis. This study involved phylogenomic classification and in-depth analyses. Sequential filters were applied to the conserved core genome containing 2754 proteins. These filters assessed non-human homology, virulence, essentiality, physiochemical properties, and pathway analysis. Through these intensive filtering approaches, promising broad-spectrum therapeutic targets were identified. These targets were docked with FDA-approved compounds readily available on the ZINC database. Selected highly ranked ligands with inhibitory potential include dihydroergotamine and abiraterone acetate. The effectiveness of the ligands has been supported by molecular dynamics simulation of the ligand-protein complexes, instilling optimism that the identified lead compounds may serve as a robust basis for the development of safe and efficient drugs for TB treatment, subject to further lead optimization and subsequent experimental validation.
Subject(s)
Antitubercular Agents , Drug Design , Mycobacterium tuberculosis , Proteomics , Tuberculosis , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Antitubercular Agents/pharmacology , Humans , Tuberculosis/drug therapy , Tuberculosis/microbiology , Proteomics/methods , Genome, Bacterial , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Phylogeny , Molecular Docking Simulation , Molecular Dynamics Simulation , Genomics/methodsABSTRACT
Acute myeloid leukemia, a serious condition affecting stem cells, drives uncontrollable myeloblast proliferation, leading to accumulation. Extensive research seeks rapid, effective chemotherapeutics. A potential option is a BRD4 inhibitor, known for suppressing cell proliferation. Sulfonamide derivatives probed essential structural elements for potent BRD4 inhibitors. To achieve this goal, we employed 3D-QSAR molecular modeling techniques, including CoMFA, CoMSIA, and HQSAR models, along with molecular docking and molecular dynamics simulations. The validation of the 2D/3D QSAR models, both internally and externally, underscores their robustness and reliability. The contour plots derived from CoMFA, CoMSIA, and HQSAR analyses played a pivotal role in shaping the design of effective BRD4 inhibitors. Importantly, our findings highlight the advantageous impact of incorporating bulkier substituents on the pyridinone ring and hydrophobic/electrostatic substituents on the methoxy-substituted phenyl ring, enhancing interactions with the BRD4 target. The interaction mode of the new compounds with the BRD4 receptor (PDB ID: 4BJX) was investigated using molecular docking simulations, revealing favorable binding energies, supported by the formation of hydrogen and hydrophobic bonds with key protein residues. Moreover, these novel inhibitors exhibited good oral bioavailability and demonstrated non-toxic properties based on ADMET analysis. Furthermore, the newly designed compounds along with the most active one from series 58, underwent a molecular dynamics simulation to analyze their behavior. The simulation provided additional evidence to support the molecular docking results, confirming the sustained stability of the analyzed molecules over the trajectory. This outcome could serve as a valuable reference for designing and developing novel and effective BRD4 inhibitors.Communicated by Ramaswamy H. Sarma.
ABSTRACT
The current study focuses on molecular cloning, expression and structural characterization of growth hormone-receptor (GHR) and its extracellular domain as growth hormone binding protein (GHBP) from the liver of Nili-Ravi buffalo (Bubalus bubalis; Bb). RNA was isolated, genes were amplified by reverse transcriptase-polymerase chain reaction and sequence was characterized. The BbGHR sequence showed three amino acid variations in the extracellular domain when compared with Indian BbGHR. For the production of full length BbGHR and BbGHBP in Escherichia coli (E. coli) BL21 (RIPL) Codon Plus, expression plasmids were constructed under the control of T7lac promoter and isopropyl ß-D thiogalactopyranoside was used as an inducer. BbGHR and BbGHBP were expressed as inclusion bodies at ~ 40% and > 30% of the total E. coli proteins, respectively. The BbGHBP was solubilized and refolded by dilution method using cysteine-cystine redox potential. The recombinant BbGHBP was purified and biological activity was checked on HeLa cell lines showing increase cell proliferation in the presence of ovine GH (oGH), hence justifying the increase in the half-life of GH in the presence of BbGHBP. For the molecular interactions of oGH-BbGHBP multiple docking programs were employed to explore the subsequent interactions which showed high binding affinity and presence of large number of hydrogen bonds. Molecular Dynamics studies performed to examine the stability of proteins and exhibited stable structures along with favorable molecular interactions. This study has described the sequence characterization of BbGHR in Nili-Ravi buffaloes and hence provided the basis for the assessment of GH-GHR binding in other Bovidae species.
Subject(s)
Buffaloes , Receptors, Somatotropin , Humans , Sheep/genetics , Animals , Buffaloes/genetics , Buffaloes/metabolism , Receptors, Somatotropin/genetics , Receptors, Somatotropin/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , HeLa Cells , Cloning, Molecular , Growth Hormone/genetics , Growth Hormone/metabolismABSTRACT
NEK7 is a NIMA related-protein kinase that plays a crucial role in spindle assembly and cell division. Dysregulation of NEK7 protein leads to development and progression of different types of malignancies including colon and breast cancers. Therefore, NEK7 could be considered as an attractive target for anti-cancer drug discovery. However, few efforts have been made for the development of selective inhibitors of NIMA-related kinase but still no FDA approved drug is known to selectively inhibit the NEK7 protein. Dacomitinib and Neratinib are two Enamide derivatives that were approved for treatment against non-small cell lung cancer and breast cancer respectively. Drug repurposing is a time and cost-efficient method for re-evaluating the activities of previously authorized medications. Thus, the present research involves the repurposing of two FDA-approved medications via comprehensive in silico approach including Density functional theory (DFTs) studies which were conducted to determine the electronic properties of the Dacomitinib and Neratinib. Afterward, binding orientation of selected drugs inside NEK7 activation loop was evaluated through molecular docking approach. Selected drugs exhibited potential molecular interactions engaging important amino acid residues of active site. The docking score of Dacomitinib and Neratinib was -30.77 and -26.78 kJ/mol, respectively. The top ranked pose obtained from molecular docking was subjected to Molecular Dynamics (MD) Simulations for investigating the stability of protein-ligand complex. The RMSD pattern revealed the stability of protein-ligand complex throughout simulated trajectory. In conclusion, both drugs displayed inhibitory efficacy against NEK7 protein and provide a prospective therapy option for malignant malignancies linked with NEK7.
ABSTRACT
Targeted delivery of therapeutic anticancer chimeric molecules enhances the efficacy of drug by improving cellular uptake and circulation time. Engineering the molecules to facilitate the specific interaction between chimeric protein and its receptor is critical to elucidate biological mechanism as well as accuracy in modeling of complexes. A theoretically designed novel protein-protein interfaces can serve as a bottom-up method for comprehensive understanding of interacting protein residues. This study was aimed for in silico analyses of a chimeric fusion protein against breast cancer. The amino acid sequences of the interleukin 24 (IL-24) and LK-6 peptide were used to design the chimeric fusion protein via a rigid linker. The secondary and tertiary structures along with physicochemical properties by ProtParam and solubility were predicted using online software. The validation and quality of the fusion protein was confirmed by Rampage and ERRAT2. The newly designed fusion construct has a total length of 179 amino acids. The top-ranked structure from alpha fold2 showed 18.1 KD molecular weight by ProtParam, quality factor of 94.152 by ERRAT, and a valid structure by a Ramachandran plot with 88.5% residues in the favored region. Finally, the docking and simulation studies were performed using HADDOCK and Desmond module of Schrodinger. The quality, validity, interaction analysis, and stability of the fusion protein depict a functional molecule. The fusion gene IL24-LK6 after cloning and expression in a suitable prokaryotic cell might be a useful candidate for developing a novel anticancer therapy.
ABSTRACT
Azurin is a natural protein produced by Pseudomonas aeruginosa that exhibits potential anti-tumor, anti-HIV, and anti-parasitic properties. The current study aimed to investigate the potential of azurin protein against breast cancer using both in silico and in vitro analyses. The amino acid sequence of Azurin was used to predict its secondary and tertiary structures, along with its physicochemical properties, using online software. The resulting structure was validated and confirmed using Ramachandran plots and ERRAT2. The mature azurin protein comprises 128 amino acids, and the top-ranked structure obtained from I-TASSER was shown to have a molecular weight of 14 kDa and a quality factor of 100% by ERRAT2, with 87.4% of residues in the favored region of the Ramachandran plot. Docking and simulation studies of azurin protein were conducted using HDOCK and Desmond servers, respectively. The resulting analysis revealed that Azurin docked against p53 and EphB2 receptors demonstrated maximum binding affinity, indicating its potential to cause apoptosis. The recombinant azurin gene was successfully cloned and expressed in a BL21 (DE3) strain using a pET20b expression vector under the control of the pelB ladder, followed by IPTG induction. The azurin protein was purified to high levels using affinity chromatography, yielding 70 mg/L. In vitro cytotoxicity assay was performed using MCF-7 cells, revealing the significant cytotoxicity of the azurin protein to be 105 µg/mL. These findings highlight the potential of azurin protein as an anticancer drug candidate.
ABSTRACT
Zika virus (ZIKV) pandemic and its implication in congenital malformations and severe neurological disorders had created serious threats to global health. ZIKV is a mosquito-borne flavivirus which spread rapidly and infect a large number of people in a shorter time-span. Due to the lack of effective therapeutics, this had become paramount urgency to discover effective drug molecules to encounter the viral infection. Various anti-ZIKV drug discovery efforts during the past several years had been unsuccessful to develop an effective cure. The NS2B-NS3 protein was reported as an attractive therapeutic target for inhibiting viral proliferation, due to its central role in viral replication and maturation of non-structural viral proteins. Therefore, the current in silico drug exploration aimed to identify the novel inhibitors of Zika NS2B-NS3 protease by implementing an e-pharmacophore-based high-throughput virtual screening. A 3D e-pharmacophore model was generated based on the five-featured (ADPRR) pharmacophore hypothesis. Subsequently, the predicted model is further subjected to the high-throughput virtual screening to reveal top hit molecules from the various small molecule databases. Initial hits were examined in terms of binding free energies and ADME properties to identify the candidate hit exhibiting a favourable pharmacokinetic profile. Eventually, molecular dynamic (MD) simulations studies were conducted to evaluate the binding stability of the hit molecule inside the receptor cavity. The findings of the in silico analysis manifested affirmative evidence for three hit molecules with -64.28, -55.15 and -50.16 kcal/mol binding free energies, as potent inhibitors of Zika NS2B-NS3 protease. Hence, these molecules holds the promising potential to serve as a prospective candidates to design effective drugs against ZIKV and related viral infections.
ABSTRACT
The recent outbreak of the Ebola virus (EBOV) has marked it as one of the most severe health threats globally. Among various anti-EBOV inhibitors studied, galidesivir (BCX4430) has shown remarkable efficacy. This study aims to identify novel potential anti-EBOV drugs among galidesivir analogs, focusing on the Zaire ebolavirus (Z-EBOV), which exhibits a mortality rate of 90%. We subjected 200 candidate compounds to molecular docking calculations, followed by an evaluation of the bioactivity of the top 25 compounds using the OSIRIS Property Explorer. Initial 50 ns molecular dynamics (MD) simulations were then performed. According to our findings, only six compounds exhibited positive drug scores. We further performed molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations of binding energy over 50 ns, selecting the two top-performing compounds for extended 150 ns MD simulations. CID 117698807 and CID 117712809 showed higher binding stability compared to galidesivir, with ΔGbinding values of -36.7 and -53.4 kcal/mol, respectively. Both compounds demonstrated high stability within the Z-EBOV-V24 active site over the 150 ns MD simulations. Hence, our study proposes CID 117698807 and CID 117712809 as potential anti-Z-EBOV-V24 drug candidates, warranting further investigation.Communicated by Ramaswamy H. Sarma.