Protein Binding Affinity Research Articles

Extreme Tolerance of Nanoparticle-Protein Corona to Ultra-High Abundance Proteins Enhances the Depth of Serum Proteomics.

The serum nanoparticle-protein corona (NPC) provides specific disease information, thus opening a new avenue for high-throughput in-depth proteomics to facilitate biomarker discovery. Yet, little is known about the interactions between NPs and proteins, and its role in enhanced depth of serum proteomics. Herein, a series of protein spike-in experiments are conducted to systematically investigate protein depletion and enrichment during NPC formation. Proteomic depth is serum concentration-dependent, and NPC exhibits powerful tolerance to ultra-high abundant proteins. In addition, protein-protein interactions (PPI), especially those involving albumin, play a pivotal role in promoting proteomic depth. Furthermore, a triple-protein assay is established to interrogate the relationship between protein binding affinity and concentration. NPC formation is a product of balancing binding affinity, concentration, and PPI. Overall, this study elucidates how NPs achieve protein depletion and enrichment for enhanced serum proteomic depth to gain a better understanding of NPC as an essential tool of proteome profiling.

Optimized peptide inhibitor Aqs1C targets LasR to disrupt quorum sensing and biofilm formation in Pseudomonas aeruginosa: Insights from MD simulations and in vitro studies.

Pseudomonas aeruginosa (PA) is a critical pathogen, and its antibiotic resistance is largely driven by the quorum-sensing regulator LasR. Herein, we report the design, synthesis, and characterization of Aqs1C, a mutated peptide derivative of Aqs1, optimized to inhibit LasR and its quorum-sensing pathway. By introducing a targeted mutation, Aqs1C exhibited enhanced stability and binding affinity for LasR protein compared to its predecessor, Aqs1B. Using molecular dynamics simulations (MDS), the Aqs1C-LasR complex demonstrated a marked increase in structural stability, reflected in reduced root mean square deviation (RMSD) values and lower binding free energy. Electrostatic complementarity analysis showed stronger and more favorable interactions between Aqs1C and LasR. Further, GaMD experiments were able to reproduce the binding state between Aqs1C and LasR, indicating the binding mechanism between them. These molecular insights correlated with functional in vitro assays. Aqs1C effectively inhibited quorum-sensing-associated virulence factors in PA, involving biofilm formation (77.6 % inhibition), pyocyanin production (75.7 % inhibition), protease secretion (61.1 % inhibition), and rhamnolipid production (74.1 % inhibition), at a 100 μg/mL concentration, in a comparable or superior pattern to azithromycin (AZM). Molecular modelling, MDS, and GaMD insights and in vitro assays established Aqs1C as a promising candidate for therapeutic development to mitigate PA infections through targeted quorum-sensing disruption.

Coumarin derivatives containing the 1,3,4 oxadiazole/thiadiazole moiety discovered as potential anti-tobacco mosaic virus agents.

In this paper, a series of oxadiazole/thidiazole containing coumarin derivative derivatives were designed, synthesized and characterized using NMR and HRMS. The evaluation of antiviral activity revealed that some of the synthesized compounds exhibited good in vivo antiviral efficacy against tobacco mosaic virus (TMV). Notably, compounds H6 and Y5 demonstrated exceptional therapeutic and protective effects against TMV, with EC50 values of 180.7, 190.3 and 215.8, 218.6μg/mL, respectively, surpassing the efficacy of NingNanmycin, which exhibited EC50 values of 284.1 and 247.1μg/mL. The preliminary mechanistic studies indicated that H6 and Y5 had ahigh binding affinity for the tobacco mosaic virus capsid protein (TMV-CP), potentially obstructing the self-assembly and replication processes of TMV particles. Furthermore, the chlorophyll content and superoxide dismutase (SOD) activity in tobacco leaves increased, while the malondialdehyde (MDA) content decreased. H6 has the potential to be developed as a novel antiviral.

Isorauhimbine and Vinburnine as Novel 5-HT2A Receptor Antagonists From Rauwolfia serpentina for the Treatment of Insomnia: An In Silico Investigation

Objective: This study aimed to identify and delineate the antagonistic effects of phytochemicals present in Rauwolfia serpentina (Indian snakehead) on the serotonin 2A receptor (5HT2AR) for insomnia treatment. Methods: Molecular docking and molecular dynamics simulations were performed to analyze the binding affinity and stability of protein–ligand complexes. The dynamic behavior of the complexes was studied via normal mode analysis performed via the iMODS server. The blood–brain barrier permeability of the phytochemicals was hence analyzed via the Brain or IntestinaL EstimateD permeation method (BOILED-Egg). The bioactivity of phytochemicals as “g-protein coupled receptor (GPCR) ligands” was analyzed via the MolInspiration server. Lipinski’s rule of five was used as a benchmark to assess the drug likeness of the phytochemicals. The SWISS ADME server was used to analyze the physiochemical properties of the selected phytocompounds. Furthermore, the preclinical efficacy and safety of the compounds were assessed via absorption, distribution, metabolism, excretion, and toxicity (ADME/T) analysis performed via the Deep-PK and ProTox 3.0 servers. Results: Cumulative results from various computational parameters yielded isorauhimbine and vinburnine as hit compounds because they are 5HT2AR antagonists. These phytochemicals exhibited promising docking scores, and their root mean square deviation, radius of gyration, and solvent accessible surface area confirmed their stability with the target protein. The ADME/T profile and drug-likeness values of these two compounds validated their safety, efficacy, and potential as drug candidates. Conclusion: We conclude that isorauhimbine and vinburnine stand out as potential phytochemicals from R. serpentina that could potentially serve as potential antagonistic drug candidates against 5HT2AR for insomnia treatment. However, these findings need to be validated through wet lab experiments to fully elucidate their therapeutic potential against insomnia.

Phytochemical analysis, therapeutic and molecular docking studies for the compounds of wild type and Senkambu variants of curry leaves targeting HER2 Kinase domain a potential gastric cancer receptor

Gastric cancer is the fifth most notable health concern globally. In recent years, molecular docking, a computational technique, has emerged as tool in drug discovery. The present investigation aimed to identify the major bioactive compounds in the wild-type curry leaves found in the Shevaroy Hills and the local Senkambu variant from Karamadai. Virtual screening of 40 ligands from Curry leaves of wild type and Senkambu type was identified through GC-MS profiling. These compounds were targeted against HER2 Kinase domain which is a potential receptor for Gastric cancer. Information regarding the binding site residues for the receptor was predicted using CASTp server. Molecular docking was performed for HER2 kinase domain with the predicted compounds through GC-MS profiling. The top 3 hits reported with least binding affinity for the target protein were considered for further interaction analysis using Biovia Discovery studio visualizer. Upon analyzing the interacted compounds, the Piperine from Wild type curry leaves was found to have good interaction with HER2 Kinase domain by forming two hydrogen bonds and binding score of -8.3 k cal/mol. The current study might guide the designing of analogues of piperine in the evolution of effective broad spectrum drug development in cancer therapy.

In-silico identification of potential peptide inhibitors to disrupt NLRP3 inflammasome complex formation by blocking NLRP3-ASC pyrin-pyrin interactions

The NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome is a well-known and frequently cited regulator of caspase-1 activation. It plays a significant role in several pathophysiological processes and is a major regulator of the innate immune response. A growing amount of scientific evidences for its aberrant activation in various chronic inflammatory diseases attracts a growing interest in the development of new NLRP3 inhibitors. One of the successful strategies used to identify new inhibitors is peptide inhibitors. Compared to small molecule inhibitors, peptide inhibitors show greater selectivity and less toxicity. In this study, we used an in-silico mutagenesis approach to design new peptide inhibitors from reported peptide inhibitor of NLRP3. The sequence of the peptide inhibitor against NLRP3 was searched from the literature and modeled using the online server PEP-FOLD3. The in-silico alanine scanning mutagenesis of the reference peptide revealed that residues, Y23, R28, E6, I26, R20, L19, Q33, K11, L14, and K13 have positive affinity values and are therefore better candidates for substitution to increase binding affinity. By replacing these residues, the affinity of the newly designed peptide inhibitors for the NLRP3 PYD protein was significantly increased. Further, molecular dynamics simulations and binding energy calculations validated the stability and higher binding affinities of the newly designed peptide inhibitors compared to the wild-type peptide inhibitor. Our research revealed that all the suggested peptide inhibitors have higher binding affinities for the NLRP3 protein as compared to the native wild-type peptide inhibitor and could block NLRP3-ASC pyrin-pyrin interaction.

Assessment of inhibitory potentiality of natural compounds against worrisome rice blast fungus

Rice blast, a severe fungal disease, is a substantial threat to global food security, particularly in rice-oriented areas. The Magnaporthe oryzae fungus is increasingly resistant and fast developing in nature. However, chemical fungicides are not only detrimental to the environment but eventually also lose their efficiency. To Tackle this issue, we used an in silico based strategy to identify plant metabolites as bio-fungicides to combat rice blast. Therefore, we screened a total of 56 antifungal natural compounds for the ability to inhibit fungal development through the targeted inhibition of essential proteins in the rice blast pathogen. Molecular docking analysis identified curcumin, myricetin, sterigmatocystin, and versicolorin B as promising candidates with superior binding affinities compared to conventional fungicides like strobilurin, azoxystrobin, and tricyclazole. Notably, myricetin showed the docking score for the SD protein of −233.20, whereas versicolorin B demonstrated the highest binding affinity for the SD protein of −234.23. Among the control fungicides, azoxystrobin displayed the lowest docking score of −177.53. The docked complexes were found to be stable based on molecular dynamics simulations results; the binding free energies of SD-Versicolorin B (−156.018 ± 24.881 kJ/mol) and SD-Myricetin (−137.526 ± 19.977 kJ/mol) complexes were also found to be favorable. Taking everything considered, these naturally occurring substances showed strong fungicidal effects against rice blast causative agent while remaining non-toxic, providing encouraging substitutes for traditional fungicides. In conclusion, these non-toxic natural compounds exhibited strong fungicidal action against rice blast, suggesting promising alternatives to conventional fungicides.

Sodium, Potassium-Adenosine Triphosphatase as a Potential Target of the Anti-Tuberculosis Agents, Clofazimine and Bedaquiline.

Multidrug-resistant tuberculosis (MDR-TB) patients are treated with a standardised, short World Health Organization (WHO) regimen which includes clofazimine (CFZ) and bedaquiline (BDQ) antibiotics. These two antibiotics lead to the development of QT prolongation in patients, inhibiting potassium (K+) uptake by targeting the voltage-gated K+ (Kv)11.1 (hERG) channel of the cardiomyocytes (CMs). However, the involvement of these antibiotics to regulate other K+ transporters of the CMs, as potential mechanisms of QT prolongation, has not been explored. This study determined the effects of CFZ and BDQ on sodium, potassium-adenosine triphosphatase (Na+,K+-ATPase) activity of CMs using rat cardiomyocytes (RCMs). These cells were treated with varying concentrations of CFZ and BDQ individually and in combination (1.25-5 mg/L). Thereafter, Na+,K+-ATPase activity was determined, followed by intracellular adenosine triphosphate (ATP) quantification and cellular viability determination. Furthermore, molecular docking of antibiotics with Na+,K+-ATPase was determined. Both antibiotics demonstrated dose-response inhibition of Na+,K+-ATPase activity of the RCMs. The greatest inhibition was demonstrated by combinations of CFZ and BDQ, followed by BDQ alone and, lastly, CFZ. Neither antibiotic, either individually or in combination, demonstrated cytotoxicity. Molecular docking revealed an interaction of both antibiotics with Na+,K+-ATPase, with BDQ showing higher protein-binding affinity than CFZ. The inhibitory effects of CFZ and BDQ, individually and in combination, on the activity of Na+,K+-ATPase pump of the RCMs highlight the existence of additional mechanisms of QT prolongation by these antibiotics.

Silver Nanoparticles Reduce ACE2 Expression via Changing Mitochondrial Function in Human Fibroblast-Like Lung Cell and Periodontal Ligament Fibroblast Cells

Silver nanoparticles (AgNPs) have demonstrated antibacterial properties and are widely recognized as one of the most prominent types of nanoparticles. Recent studies have highlighted their effectiveness against coronaviruses. However, the detailed molecular mechanisms underlying the action of AgNPs on viruses and their impacts on the human body remain to be fully elucidated. Thus, we attempt to delineate the preventive effects of AgNPs against SARS-CoV-2 infection. Our findings indicate that treatment with AgNPs reduces ACE2 expression, a key receptor for SARS-CoV-2 particularly in normal oral and lung cells. Additionally, we observed a decrease in the binding affinity of the spike protein to the cell after the AgNP treatment. Through western blot analysis, we identified the involvement of the AKT and/or mTOR signaling pathway in this process. Because AKT and mTOR signaling affects mitochondrial function, we investigated the effects of the AgNP treatment on mitochondria. As a result, we observed the localization of AgNPs within mitochondria. Further, an increase in mitochondrial Fe2+ and reactive oxygen species levels was observed, ultimately resulting in mitochondrial dysfunction. Our results show the efficacy of the AgNP treatment in preventing coronavirus infections.

Assessment of Punicalagin activity Against Influenza a Virus Proteins via In-silico Approaches

Background: To investigate the anti-influenza mechanism of punicalagin and its inhibitory effects on influenza virus proteins, we conducted molecular docking studies targeting 11 viral proteins. Additionally, molecular dynamics simulations were performed for the protein with the lowest free energy and the minimum concentration required for binding in a simulated environment. Methods: The molecular structure and data of punicalagin were obtained from the PubChem database and converted into a PDB file. FASTA sequences of viral proteins were retrieved from UniProt, and their PDB structures were predicted using the I-TASSER server. Molecular docking was performed using AutoDock 4.2 software, while molecular dynamics simulations were conducted with Gromacs 2022 software. Results: The PB1-F2 protein exhibited the best inhibitory performance, with a binding energy of -5.76 kcal/mol and the lowest inhibition constant (Ki). Docking of punicalagin to the PB1-F2 protein led to a significant decrease in the average total energy (TE), radius of gyration (Rg), and root mean square deviation (RMSD), as well as alterations in the secondary structure of the protein (P < 0.001). The most prominent secondary structural change was a reduction in coil structures and an increase in turn structures. Conclusions: Punicalagin displayed a strong binding affinity for the PB1-F2 protein compared to other influenza viral proteins. Considering the role of PB1-F2 in exacerbating inflammation caused by influenza, punicalagin may mitigate inflammation in patients by modulating the activity of this protein.

The interaction of isoquinoline alkaloid crebanine with immunoglobulin G and cytotoxic effects toward MCF-7 breast cancer cell line.

In this study, the interaction of crebanine, an isoquinoline alkaloid, with immunoglobulin G (IgG) was evaluated. Subsequently, the anticancer effects of crebanine in MCF-7 breast cancer cells were assessed. The results demonstrate that static quenching plays a key role in the fluorescence quenching of the IgG by crebanine, and some embedded hydrophobic patches of the IgG are exposed upon interaction with crebanine, while the characteristic β-sheet conformation of the IgG was almost preserved. Theoretical studies also show that several hydrophilic and hydrophobic residues play a crucial role in the formation of hydrogen bonds between crebanine and IgG, along with the stability of the complex. Cellular studies indicate that crebanine induces selective anticancer effects in MCF-7 cells (IC50: 36.76 μM) compared to human embryonic kidney cells (HEK-293, IC50: 723.77 μM) through the inhibition of colony formation, induction of oxidative stress and lipid peroxidation, upregulation of the Bax/Bcl-2 ratio, and cytochrome c release, which are indicative of the intrinsic apoptosis pathway. In conclusion, this study provides valuable information regarding the protein binding affinity and anticancer activity of crebanine, which are essential factors for determining the pharmacological activity of small molecules as drug candidates.

In vitro anticancer effects in hepatocellular carcinoma (HCC) and protein interaction study of xanthoangelol.

Xanthoangelol (C25H28O4), a natural flavonoid derived from chalcones, has shown potential pharmacological activities. However, its primary interaction mechanism with proteins and cells is not well understood. In the present study, we focus on the anticancer effects of xanthoangelol against hepatocellular carcinoma (HCC) as well as its binding affinity with a plasma drug carrier protein, α2-macroglobulin. The anticancer effects of xanthoangelol on human HCC cell line HepG2 cells were assayed using MTT, LDH, qPCR, and caspase activity assays. Efficient binding of the xanthoangelol with α2-macroglobulin was established by experimental and molecular docking studies. It was found that xanthoangelol significantly mitigates cell viability through upregulating intrinsic (Bax/Bcl-2, caspase-9) and extrinsic (caspase-8) apoptotic pathways. Moreover, it was detected that xanthoangelol induces ER stress through the upregulation of CHOP in HepG2 cells. Fluorescence spectra show that xanthoangelol strongly interacts with α2-macroglobulin mediated by a static quenching mechanism and Trp1237 and Tyr1323 residues were exposed to the solvent with the addition of xanthoangelol. Meanwhile, both experimental and theoretical studies display that hydrophilic forces play a key role in the formation of xanthoangelol-α2-macroglobulin complex, leading to a slight conformational change in α2-macroglobulin. In conclusion, our findings suggest that xanthoangelol, which has a high binding affinity for a plasma carrier protein, may inhibit the viability of HCC by inducing apoptosis and ER stress.

Microbial Proteomics Approach for Synthetic Vaccine Development form Bacillus Weihenstephanensis

Bacillus weihenstephanensis causes systemic infections leading to be an obligatory step in infection. Peptide fragments of antigen protein can be used to select nonamers for use in rational vaccine design and to increase the understanding of roles of the immune system in infectious diseases. Analysis shows MHC class II binding peptides of antigen protein from Bacillus weihenstephanensis are important determinant for protection of host form bacteral infection. In this assay, we used PSSM and SVM algorithms for antigen design and predicted the binding affinity of bacterial protein having 210 amino acids, which shows 202 nonamers. Binding ability prediction of antigen peptides to major histocompatibility complex (MHC) class I & II molecules is important in vaccine development from Bacillus weihenstephanensis.

The lack of trade-off between conformational stability and binding affinity in a nanobody with therapeutic potential for a misfolding disease

To improve protein pharmaceuticals, we need to balance protein stability and binding affinity with in vivo efficiency. We have recently developed a nanobody (NB-AGT-2) against the alanine:glyoxylate aminotransferase with high stability (Tm ~ 85 °C) that may be useful to treat a misfolding disease called primary hyperoxaluria type 1. In this work, we characterize the relationships between protein stability and binding affinity in NB-AGT-2 by generating single and double cavity-creating mutants in its hydrophobic core. These mutations decrease thermal stability by 10–20 °C, reflecting changes in thermodynamic stability of up to 8 kcal·mol−1, hardly affecting their binding affinity for its target. Statistical mechanics analysis support long-range propagation of stability effects due to mutations. Our results thus show that NB stability can be largely challenged without an effect on its binding.

Incorporating Water Molecules into Highly Accurate Binding Affinity Prediction for Proteins and Ligands.

In the binding process between proteins and ligand molecules, water molecules play a pivotal role by forming hydrogen bonds that enable proteins and ligand molecules to bind more strongly. However, current methodologies for predicting binding affinity overlook the importance of water molecules. Therefore, we developed a model called GraphWater-Net, specifically designed for predicting protein-ligand binding affinity, by incorporating water molecules. GraphWater-Net employs topological structures to represent protein atoms, ligand atoms and water molecules, and their interactions. Leveraging the Graphormer network, the model extracts interaction features between nodes within the topology, alongside the interaction features of edges and nodes. Subsequently, it generates embeddings with attention weights, inputs them into a Softmax function for regression prediction, and ultimately outputs the predicted binding affinity value. Experimental results on the Comparative Assessment of Scoring Functions (CASF) 2016 test set show that the introduction of water molecules into the complex significantly improves the prediction performance of the proposed model for protein and ligand binding affinity. Specifically, the Pearson correlation coefficient (Rp) exceeds that of current state-of-the-art methods by a margin of 0.022 to 0.129. By integrating water molecules, GraphWater-Net has the potential to facilitate the rational design of protein-ligand interactions and aid in drug discovery.

Phytogenic synthesis and antimicrobial activity of ZnO nano bow ties (ZnO NBTs): An experimental and computational study

Phytogenic synthesis is a sustainable and eco-friendly approach for producing nanoscale particles, using biological entities such as plants and their byproducts. In this study, Allium sativum extract was selected as a capping and reducing agent due to the presence of phytochemicals such as allicin, diallyl disulfide (DADS), vinyl dithiins, ajoene (E- and Z-ajoene), diallyl trisulfide (DATS), and thiol (sulfhydryl) groups. The resulting ZnO Nano Bow Ties (ZnO NBTs) were characterized using FE-SEM, XRD, EDX, DLS, zeta potential, FTIR, and UV-Vis spectroscopy to evaluate the size, morphology, and crystallinity. The obtained XRD, SEM, and DLS results suggested an average longitudinal length of ∼372 nm with a maximum lateral width of ∼64 nm and a Bow Tie shape. Gas Chromatography-Mass Spectroscopy (GC-MS) analysis was employed to elucidate the prominent phytochemical constituents of the Allium sativum extract. Preliminary antibacterial assays reveal significant inhibition zones and growth inhibition effects against gram-negative bacteria of both Klebsiella pneumoniae and Escherichia coli, suggesting the promising antimicrobial potential of these ZnO NBTs. Monte Carlo simulations revealed that the cone-shaped ZnO NBTs bind strongly to the active sites of the target proteins with binding affinities of −36.20 and −32.14 kcal/mol for Klebsiella pneumoniae and Escherichia coli respectively, which correlates with their activities. The ZnO NBTs complexes formed stronger hydrophobic interactions and hydrogen bonds with amino acid residues of Escherichia coli than with Klebsiella pneumoniae. This integrated experimental and computational study underscores the potential of the use of ZnO NBTs as a sustainable and effective strategy to combat bacterial pathogens. The findings of this study indicate that efficient morphology (shape) is a major contributor to the protein binding affinities of ZnO NBTs, with promising implications for the design of antibacterial drugs in nanomedicine.

Exploring potential therapeutic candidates against COVID-19: a molecular docking study

The COVID-19 pandemic, caused by SARS-CoV-2, has made it urgently necessary to develop effective therapeutic options, and in recent years, computational biological tool assisted to achieve this goal. We conducted MD simulations using six SARS-CoV-2 proteins and 31 ligands to identify possible inhibitors as well as evaluate the binding efficiency of them. Post simulation study showed that 6VSB-Dactinomycin complex reveals the most stable binding, protein flexibility, and robust structural integrity, making it a promising model for therapeutic studies. Our study assessed the therapeutic potential of antiviral candidates against covid-19 virus using computational and experimental method, including the flexibility and stability of twelve docked protein–ligand complexes using normal mode analysis (NMA) and molecular dynamics (MD) simulations. After MD simulation, Dactinomycin and Ivermectin showed significant deformability and high binding affinities for Spike protein and RNA-dependent RNA polymerase, respectively. Remarkably, Dactinomycin showed greater binding to the Helicase protein, but Hesperidin and Epigallocatechin gallate (EGCG) exhibited encouraging interactions with the Nucleocapsid protein and Main protease. Analysis of docking experiments and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) showed the toxicity profiles and binding affinity of various drugs with key viral proteins. Toxicity of major drugs exhibited low to moderate although Dactinomycin, Ivermectin and vitamin-D exhibited higher degree of toxicity. Carcinogenicity observed in Quercetin, Baricitinib, Luteolin, Berberine, and Favipiravir while hepatotoxicity was not found in most case. Although EGCG and Hesperidin exhibit potential, more studies are required to evaluate effectiveness against approved drugs such as Remdesivir. This integrated approach shows that an integration of computational predictions with experimental data can help to support the development of antiviral drugs by adding novel perspectives in the safety profiles and efficiency of potential drugs.

The transmission of mutation effects in a multiprotein machine: A comprehensive metadynamics study of the cardiac thin filament.

The binding of Ca2+ ions within the troponin core of the cardiac thin filament (CTF) regulates normal contraction and relaxation. Mutations within the troponin complexes are known to alter normal functions and result in the eventual development of cardiomyopathy. However, despite the importance of the problem, detailed microscopic knowledge of the mechanism of pathogenic effect of point mutations and their effects on the conformational free energy surface of CTF remains elusive. Mutations are known to transmit their effects hundreds of angstroms along this protein complex and between different component proteins. To explore the impact of point mutations on the conformational free energy barrier between the closed and blocked state of CTF, and to understand the transmission of mutation, we have carried out metadynamics simulations for the wild-type (WT) and two mutants (cardiac troponin T Arg92Trp (R92W) and Arg92Leu (R92L)). Specifically, we have investigated the conformational modification of the tropomyosin (Tm) and the troponin (Tn) complex during the closed-to-blocked state transition for both the WT and two hypertrophic cardiomyopathy causing mutations. Our calculations demonstrated that mutations within the cardiac troponin T (cTnT) protein alter conformational properties of the Tm and the other proteins of the Tn complex as well as the Ca2+ binding affinity of the cTnC protein through the indirect mediation of cardiac troponin I (cTnI). Importantly, the data revealed a significant influence of the mutations on the conformational transition free energy barriers for both the Tm and cTnC proteins. Furthermore, we found both mutations independently alter the free energy barrier of transitions of cTnT. Such alteration in the free energy upon mutation of one protein in a complex, allosterically affects the others through structural and dynamical changes, leading to a pathogenic effect on the function of the thin filament.

Potential Inhibitor of DENV-2 Virus Protease (NS2B-NS3): An In-Silico Studies of Anti-Viral Plants

Dengue virus (DENV) is a mosquito-borne pathogen that affects millions of people worldwide. The DENV-2 protease is a vital enzyme responsible for viral replication and is a promising target for antiviral therapy. The objective of the study is to identify potential inhibitors of DENV-2 protease using In-Silico approaches with phytocompounds from ten antiviral plants. Initially, 133 phytoconstituents were collected with anti-dengue properties from previously reported studies which were virtually screened using SWISS ADME for ADME properties. The DENV-2 protease structure (2FOM) was obtained from the Protein Data Bank and molecular docking was performed using AutoDock Vina. The best-scoring compounds were evaluated and top five potential inhibitors with high binding affinity and stability were selected. The top-scoring compounds were Ligand-91 (Terchebin, -8.1 kcal/mol), Ligand-13 (7-desacetyl-7-benzoylgedunin, -7.8 kcal/mol), Ligand-100 (Triterpenoid, -7.8 kcal/mol), Ligand-12 (7-desacetyl-7-benzoylazadiradione, -7.7 kcal/mol), Ligand-20 (Azadirolic acid, -7.7 kcal/mol), Ref.1 (Doxycycline, -6.6 kcal/mol), Ref.2(Monosdenvir, -7.5 kcal/mol), and Ref.3 (Zanamivir, -5.6 kcal/mol). The result of the study shows that 7-desacetyl-7-benzoylazadiradione and 7-desacetyl-7-benzoylgeduninas compounds with high binding affinity for the target protein. These compounds are found in Azadirachta indica making it a promising candidate for further experimental validation and development of antiviral agents against DENV-2. Keywords: Molecular docking, Anti-dengue, Anti-viral, ADME analysis

Specific binding between Arabidopsis thaliana phytochrome-interacting factor 3 (AtPIF3) bHLH and G-box originated prior to embryophyte emergence

The basic helix-loop-helix (bHLH) domain via critical amino acid residues on basic region binding to E-box (5′-CANNTG-3′) is known in embryophyte. However, the dictated E-box types selection by bHLH dimers and the significant impact of these critical amino acid residues along embryophyte evolution remain unclear. The Arabidopsis thaliana PIF3-bHLH (AtPIF3-bHLH) recombinant protein and a series of AtPIF3-bHLH mutants were synthesized and analyzed. The reduced DNA binding ability and affinity of AtPIF3-bHLH point-mutation proteins, observed via fluorescence-based electrophoretic mobility shift assay (fEMSA) and isothermal titration calorimetry (ITC), suggest the critical role of these DNA-recognition sites in maintaining the AtPIF3-bHLH–DNA interaction. The purifying selection signals and the DNA-recognition-site conservation at the species level suggest the invariant roles of these sites throughout embryophyte evolution. The G-box outcompeted other types of E-box for binding in our competitive fEMSAs. The dynamic hydrogen bond formed between AtPIF3-bHLH and the G-box core indicates flexible identification of the core region. These features highlight a fast fixation of the bHLH-G-box recognition mechanism through embryophyte evolution and serve as a blueprint for studying DNA recognition determinants of other TF families.