A Structural Model of the Endogenous Human BAF Complex Informs Disease Mechanisms.

Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.

Claudin-4 polymerizes after a small extracellular claudin-3-like substitution.

Tight junctions play a pivotal role in the functional integrity of the human body by forming barriers that compartmentalize tissues and protect the body from external threats. Essential components of tight junctions are the transmembrane claudin proteins, which can polymerize into tight junction strands and meshworks. This study delves into the structural determinants of claudin polymerization, using the close homology yet strong difference in polymerization capacity between claudin-3 and claudin-4. Through a combination of sequence alignment and structural modeling, critical residues in the second extracellular segment are pinpointed. Molecular dynamics simulations provide insights into the interactions of and the conformational changes induced by the identified extracellular segment 2 residues. Live-stimulated emission depletion imaging demonstrates that introduction of these residues from claudin-3 into claudin-4 significantly enhances polymerization in nonepithelial cells. In tight junction-deficient epithelial cells, mutated claudin-4 not only influences tight junction morphology but also partially restores barrier function. Understanding the structural basis of claudin polymerization is crucial, as it offers insights into the dynamic nature of tight junctions. This knowledge could be applied to targeted therapeutic interventions, offer insight to repair or prevent barrier defects associated with pathological conditions, or introduce temporary barrier openings during drug delivery.

Functional Characterization Supports Multiple Evolutionary Origins of Pheromone Receptors in Bark Beetles.

Chemical communication using pheromones is thought to have contributed to the diversification and speciation of insects. The species-specific pheromones are detected by specialized pheromone receptors (PRs). Whereas the evolution and function of PRs have been extensively studied in Lepidoptera, only a few PRs have been identified in beetles, which limits our understanding of their evolutionary histories and physiological functions. To shed light on these questions, we aimed to functionally characterize potential PRs in the spruce bark beetle Ips typographus ("Ityp") and explore their evolutionary origins and molecular interactions with ligands. Males of this species release an aggregation pheromone comprising 2-methyl-3-buten-2-ol and (4S)-cis-verbenol, which attracts both sexes to attacked trees. Using two systems for functional characterization, we show that the highly expressed odorant receptor (OR) ItypOR41 responds specifically to (4S)-cis-verbenol, with structurally similar compounds eliciting minor responses. We next targeted the closely related ItypOR40 and ItypOR45. Whereas ItypOR40 was unresponsive, ItypOR45 showed an overlapping response profile with ItypOR41, but a broader tuning. Our phylogenetic analysis shows that these ORs are present in a different OR clade as compared to all other known beetle PRs, suggesting multiple evolutionary origins of PRs in bark beetles. Next, using computational analyses and experimental validation, we reveal two amino acid residues (Gln179 and Trp310) that are important for ligand binding and pheromone specificity of ItypOR41 for (4S)-cis-verbenol, possibly via hydrogen bonding to Gln179. Collectively, our results shed new light on the origins, specificity, and ligand binding mechanisms of PRs in beetles.

Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.

Structural coverage of the human interactome.

Complex biological processes in cells are embedded in the interactome, representing the complete set of protein-protein interactions. Mapping and analyzing the protein structures are essential to fully comprehending these processes' molecular details. Therefore, knowing the structural coverage of the interactome is important to show the current limitations. Structural modeling of protein-protein interactions requires accurate protein structures. In this study, we mapped all experimental structures to the reference human proteome. Later, we found the enrichment in structural coverage when complementary methods such as homology modeling and deep learning (AlphaFold) were included. We then collected the interactions from the literature and databases to form the reference human interactome, resulting in 117 897 non-redundant interactions. When we analyzed the structural coverage of the interactome, we found that the number of experimentally determined protein complex structures is scarce, corresponding to 3.95% of all binary interactions. We also analyzed known and modeled structures to potentially construct the structural interactome with a docking method. Our analysis showed that 12.97% of the interactions from HuRI and 73.62% and 32.94% from the filtered versions of STRING and HIPPIE could potentially be modeled with high structural coverage or accuracy, respectively. Overall, this paper provides an overview of the current state of structural coverage of the human proteome and interactome.

Identification and classification of papain-like cysteine proteinases.

Papain-like cysteine peptidases form a big and highly diverse superfamily of proteins involved in many important biological functions, such as protein turnover, deubiquitination, tissue remodeling, blood clotting, virulence, defense, and cell wall remodeling. High sequence and structure diversity observed within these proteins hinders their comprehensive classification as well as the identification of new representatives. Moreover, in general protein databases, many families already classified as papain like lack details regarding their mechanism of action or biological function. Here, we use transitive remote homology searches and 3D modeling to newly classify 21 families to the papain-like cysteine peptidase superfamily. We attempt to predict their biological function and provide structural characterization of 89 protein clusters defined based on sequence similarity altogether spanning 106 papain-like families. Moreover, we systematically discuss observed diversity in sequences, structures, and catalytic sites. Eventually, we expand the list of human papain-related proteins by seven representatives, including dopamine receptor-interacting protein 1 as potential deubiquitinase, and centriole duplication regulating CEP76 as retaining catalytically active peptidase-like domain. The presented results not only provide structure-based rationales to already existing peptidase databases but also may inspire further experimental research focused on peptidase-related biological processes.

New CYP154C4 from Streptomyces cavourensis YBQ59 performs regio- and stereo- selective 3ß-hydroxlation of nootkatone.

Nootkatone, a sesquiterpenoid widely used in the food and cosmetics industries, exhibits diverse biological activities and pharmaceutical prospects. Modification of nootkatone to create new derivatives with desirable activities has attracted significant attention. For this purpose, cytochrome P450 monooxygenases (P450 or CYP) are attractive candidates due to their ability to perform regio- and stereoselective hydroxylation at allylic C-H bonds. In this study, CYP154C4 from Streptomyces cavourensis YBQ59 was cloned and expressed in Escherichia coli. By screening 64 candidate substrates, this P450 was found to catalyze the regio- and stereoselective hydroxylation of nootkatone, yielding a single product, 3ß-hydroxynootkatone. Using a whole-cell E. coli system expressing CYP154C4, supported by the heterologous redox partners YkuN from Bacillus subtilis and FdR from E. coli, 3ß-hydroxynootkatone was produced on a preparative scale. The structure of this compound was determined by 1H NMR, 13C NMR, NOESY, HMBC, and HSQC. The kinetics of product formation were analyzed using HPLC, and the Km and Kcat values were calculated. Furthermore, structural insights into the selective hydroxylation of nootkatone were elucidated by molecular docking. 3ß-Hydroxynootkatone, recently synthesized semi-synthetically from nootkatone, has been reported to exhibit a higher insecticidal activity than its parent compound. Additionally, the functionalization of nootkatone with N-acyl-2-aminothiazole at the C3 and C2 positions, yielding an α-glucosidase inhibitor, has also been previously described. Therefore, 3ß-hydroxynootkatone has great potential for further research and for synthesizing new derivatives with valuable biological activities for agricultural and medicinal applications.

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126.

To date, there are very limited reports on sequence analysis and structure-based molecular modeling of phosphatases produced by probiotic bacteria. Therefore, a novel protein tyrosine-like phosphatase was characterized from L. helveticus 2126 in this study. The purified bacterial phosphatase was subjected to mass spectrometric analysis, and the identity of constructed sequence was analyzed using peptide mass fingerprint. The 3-D structure of protein was elucidated using homology modeling, while its stability was assessed using Ramachandran plot, VERIFY 3D, and PROCHECK. The bacterium produced an extracellular phosphatase of zone diameter 15 ± 0.8 mm on screening medium within 24 h of incubation. This bacterial phosphatase was highly specific towards sodium phytate as it yielded the lowest Km value of 299.50 ± 4.95 µM compared to other phosphorylated substrates. The activity was effectively stimulated in the presence of zinc, magnesium, and manganese ions thereby showing its PTP-like behavior. The phosphatase showed a molecular mass of 43 kDa, and the corresponding M/Z ratio data yielded 46% query coverage to Bacillus subtilis (3QY7). This showed a 61.1% sequence similarity to Ligilactobacillus ruminis (WP_046923835.1). The final sequence construct based on these bacteria showed a conserved motif "HCHILPGIDD" in their active site. In addition, homology modeling showed a distorted Tim barrel structure with a trinuclear metal center. The final model after energy minimization showed 90.9% of the residues in the favorable region of Ramachandran's plot. This structural information can be used in genetic engineering for improving the overall stability and catalytic efficiency of probiotic bacterial phosphatases.

Design, synthesis of new 3H-imidazo[4,5-b]pyridine derivatives and evaluation of their inhibitory properties as mixed lineage kinase 3 inhibitors.

Mixed-lineage protein kinase 3 (MLK3) is implicated in several human cancers and neurodegenerative diseases. A series of 3H-imidazo[4,5-b]pyridine derivatives were designed, synthesized and evaluated as novel MLK3 inhibitors. A homology model of MLK3 was developed and all designed compounds were docked to assess their binding pattern and affinity toward the MLK3 active site. Based on this knowledge, we synthesized and experimentally evaluated the designed compounds. Majority of the compounds showed significant inhibition of MLK3 in the enzymatic assay. In particular, compounds 9a, 9e, 9j, 9 k, 12b and 12d exhibited IC50 values of 6, 6, 8, 11, 14 and 14 nM, respectively. Furthermore, compounds 9a, 9e, 9 k and 12b exhibited favorable physicochemical properties among these compounds.

Catalyzing a Cure: Discovery and development of LRRK2 inhibitors for the treatment of Parkinson's disease.

Parkinson's disease (PD) is an age-related second most common progressive neurodegenerative disorder that affects millions of people worldwide. Despite decades of research, no effective disease modifying therapeutics have reached clinics for treatment/management of PD. Leucine-rich repeat kinase 2 (LRRK2) which controls membrane trafficking and lysosomal function and its variant LRRK2-G2019S are involved in the development of both familial and sporadic PD. LRRK2, is therefore considered as a legitimate target for the development of therapeutics against PD. During the last decade, efforts have been made to develop effective, safe and selective LRRK2 inhibitors and also our understanding about LRRK2 has progressed. However, there is an urge to learn from the previously designed and reported LRRK2 inhibitors in order to effectively approach designing of new LRRK2 inhibitors. In this review, we have aimed to cover the pre-clinical studies undertaken to develop small molecule LRRK2 inhibitors by screening the patents and other available literature in the last decade. We have highlighted LRRK2 as targets in the progress of PD and subsequently covered detailed design, synthesis and development of diverse scaffolds as LRRK2 inhibitors. Moreover, LRRK2 inhibitors under clinical development has also been discussed. LRRK2 inhibitors seem to be potential targets for future therapeutic interventions in the treatment and management of PD and this review can act as a cynosure for guiding discovery, design, and development of selective and non-toxic LRRK2 inhibitors. Although, there might be challenges in developing effective LRRK2 inhibitors, the opportunity to successfully develop novel therapeutics targeting LRRK2 against PD has never been greater.

Sequence identification and in silico characterization of novel thermophilic lipases from Geobacillus species.

Microbial lipases are utilized in various biotechnological areas, including pharmaceuticals, food, biodiesel, and detergents. In this study, we cloned and sequenced Lip21 and Lip33 genes from Geobacillus sp. GS21 and Geobacillus sp. GS33, then we in silico and experimentally analyzed the encoded lipases. For this purpose, Lip21 and Lip33 were cloned, sequenced, and their amino acid sequences were investigated for determination of biophysicochemical characteristics, evolutionary relationships, and sequence similarities. 3D models were built and computationally affirmed by various bioinformatics tools, and enzyme-ligand interactions were investigated by docking analysis using six ligands. Biophysicochemical property of Lip21 and Lip33 was also determined experimentally and the results demonstrated that they had similar isoelectric point (pI) (6.21) and Tm (75.5°C) values as Tm was revealed by denatured protein analysis of the circular dichroism spectrum and pI was obtained by isoelectric focusing. Phylogeny analysis indicated that Lip21 and Lip33 were the closest to lipases from Geobacillus sp. SBS-4S and Geobacillus thermoleovorans, respectively. Alignment analysis demonstrated that S144-D348-H389 was catalytic triad residues in Lip21 and Lip33, and enzymes possessed a conserved Gly-X-Ser-X-Gly motif containing catalytic serine. 3D structure analysis indicated that Lip21 and Lip33 highly resembled each other and they were α/ß hydrolase-fold enzymes with large lid domains. BANΔIT analysis results showed that Lip21 and Lip33 had higher thermal stability, compared to other thermostable Geobacillus lipases. Docking results revealed that Lip21- and Lip33-docked complexes possessed common residues (H112, K115, Q162, E163, and S141) that interacted with the substrates, except paranitrophenyl (pNP)-C10 and pNP-C12, indicating that these residues might have a significant action on medium and short-chain fatty acid esters. Thus, Lip21 and Lip33 can be potential candidates for different industrial applications.

Homology modeling, virtual screening, molecular docking, and ADME approaches to identify a potent agent targeting NK2R protein.

Neurokinin/tachykinin receptors are classified as the G-protein coupled receptor superfamily. The neurokinin 2 receptor (NK2R) is widely expressed in different tissues. NK2R is associated with a range of biological events, such as inflammation, smooth muscle contraction, intestinal motor functions, and asthma. Despite these diverse activities, no approved drugs targeting NK2R have been developed yet. Our study focuses on finding potential inhibitors for NK2R using virtual screening, molecular docking, and ADME (absorption, distribution, metabolism, and excretion) approaches. We used a homology modeling approach and AlphaFold DB to obtain the three-dimensional structure of mouse and human NK2R proteins, respectively. The homology model of NK2R was predicted using MODELLER v10.3 and further refined and validated using the 3Drefine tool and RAMPAGE server, respectively. Molecular docking was performed using a library of 910 structurally similar molecules to four NK1R antagonists: aprepitant, casopitant, fosaprepitant, and rolapitant. Molecular docking revealed six small molecules that displayed high Chemscore fitness scores, and binding energies with desirable ligand-NK2R interactions. The evaluation of the in silico ADME profile, solubility, and permeability of the ligand molecules has revealed that the small molecules are potentially nontoxic and have the chance of exhibiting biological activity after oral administration. Further experimental studies (in vitro and in vivo assays) are required to evaluate the effectiveness of these inhibitors as therapeutic targets.

Functional characterization and structural prediction of hypothetical proteins in monkeypox virus and identification of potential inhibitors.

The excessive activation of the monkeypox virus (MPXV-Congo_8-156) is linked to various skin and respiratory disorders such as rashes, fluid-filled blisters, swollen lymph nodes and encephalitis (inflammation of the brain), highlighting MPXV-Congo_8-156 as a promising target for drug intervention. Despite the effectiveness of Cidofovir, in inhibiting MPXV activity, its limited ability to penetrate the skin and its strong side effects restrict its application. To address this challenge, we screened 500 compounds capable of penetrating the skin and gastrointestinal tract to identify potent MPXV inhibitors. Various characterization schemes and structural models of MPXV-Congo_8-156 were explored with bioinformatics tools like PROTPARAM, SOPMA, SWISS-MODEL and PROCHECK. Using molecular docking in PyRx, we evaluated the binding affinities of these compounds with MPXV-Congo_8-156 and identified the top five candidates ranging from - 9.2 to - 8.8 kcal/mol. ADMET analysis indicated that all five compounds were safer alternatives, showing no AMES toxicity or carcinogenicity in toxicological assessments. Molecular dynamics (MD) simulations, conducted for 100 ns each, confirmed the docking interactions of the top five compounds alongside the control (Cidofovir), validating their potential as MPXV inhibitors. The compounds with PubChem CID numbers 4061636, 4422538, 3583576, 4856107 and 4800629 demonstrated strong support in terms of root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA) value, hydrogen bond analysis, and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis. Thus, our investigation identified these five compounds as promising inhibitors of MPXV, offering potential therapeutic avenues. However, further in vivo studies are necessary to validate our findings.

Structural Perspective of NR4A Nuclear Receptor Family and Their Potential Endogenous Ligands.

There are 48 nuclear receptors in the human genome, and many members of this superfamily have been implicated in human diseases. The NR4A nuclear receptor family consisting of three members, NR4A1, NR4A2, and NR4A3 (formerly annotated as Nur77, Nurr1, and NOR1, respectively), are still orphan receptors but exert pathological effects on immune-related and neurological diseases. We previously reported that prostaglandin A1 (PGA1) and prostaglandin A2 (PGA2) are potent activators of NR4A3, which bind directly to the ligand-binding domain (LBD) of the receptor. Recently, the co-crystallographic structures of NR4A2-LBD bound to PGA1 and PGA2 were reported, followed by reports of the neuroprotective effects of these possible endogenous ligands in mouse models of Parkinson's disease. Based on these structures, we modeled the binding structures of the other two members (NR4A1 and NR4A3) with these potential endogenous ligands using a template-based modeling method, and reviewed the similarity and diversity of ligand-binding mechanisms in the nuclear receptor family.

Molecular insights on Eltrombopag: potential mitogen stimulants, angiogenesis, and therapeutic radioprotectant through TPO-R activation.

The purpose of this study is to investigate the molecular interactions and potential therapeutic uses of Eltrombopag (EPAG), a small molecule that activates the cMPL receptor. EPAG has been found to be effective in increasing platelet levels and alleviating thrombocytopenia. We utilized computational techniques to predict and confirm the complex formed by the ligand (EPAG) and the Thrombopoietin receptor (TPO-R) cMPL, elucidating the role of RAS, JAK-2, STAT-3, and other essential elements for downstream signaling. Molecular dynamics (MD) simulations were employed to evaluate the stability of the ligand across specific proteins, showing favorable characteristics. For the first time, we examined the presence of TPO-R in human umbilical cord mesenchymal stem cells (hUCMSC) and human gingival mesenchymal stem cells (hGMSC) proliferation. Furthermore, treatment with EPAG demonstrated angiogenesis and vasculature formation of endothelial lineage derived from both MSCs. It also indicated the activation of critical factors such as RUNX-1, GFI-1b, VEGF-A, MYB, GOF-1, and FLI-1. Additional experiments confirmed that EPAG could be an ideal molecule for protecting against UVB radiation damage, as gene expression (JAK-2, ERK-2, MCL-1, NFkB, and STAT-3) and protein CD90/cMPL analysis showed TPO-R activation in both hUCMSC and hGMSC. Overall, EPAG exhibits significant potential in treating radiation damage and mitigating the side effects of radiotherapy, warranting further clinical exploration.

What is the context?● Chemotherapy, radiation treatment, or immunological disorders can cause a decrease in platelet count (thrombocytopenia) or decrease all blood cell types (pancytopenia) in the bone marrow. This can make it challenging to choose the appropriate cancer treatment plan.● Eltrombopag (EPAG) is an oral non-peptide thrombopoietin (TPO) mimetic that activates the cMPL receptor in the body. This activation leads to cell differentiation and proliferation, stimulating platelet production and reducing thrombocytopenia. The cMPL receptor is present in liver cells, megakaryocytes, and hematopoietic cells. However, its effects on stem cell proliferation and differentiation are not entirely understood.What is the new?● This study delves into the molecular interactions and therapeutic applications of EPAG, a small molecule that activates cMPL (TPO-R).● The study offers a comprehensive analysis of the ligand-receptor complex formation, including an examination of downstream signaling elements. Furthermore, molecular dynamics simulations demonstrate the stability of the ligand when interacting with targeted proteins.● The research investigates the presence of TPO-R on stem cell-derived endothelial cells, shedding insight into the ability of EPAG TPO-mimetic to promote angiogenesis and vasculature formation.● The study revealed that EPAG has the potential to protect against UVB-induced radiation damage and stimulate stem cell growth.What is the implications?The study emphasizes the potential of EPAG as a promising option for addressing radiation injury and minimizing the adverse effects of radiotherapy. It could revolutionize treatments not only for thrombocytopenia but also for enhancing the growth of stem cells. Furthermore, the research deepens our understanding of EPAG's molecular mechanisms, providing valuable insights for developing future drugs and therapeutic approaches for cell therapy to treat radiation damage.

Falcaria vulgaris extract: A mixture of quorum sensing inhibitors for controlling Pectobacterium carotovorum subsp. carotovorum.

A promising strategy to control bacterial diseases involves using Quorum Sensing Inhibitor (QSI) compounds. This study aimed to evaluate the potential of Falcaria vulgaris plant extract to combat the phytopathogenic Pectobacterium carotovorum subsp. carotovorum (Pcc) via its QSI activity. Using biosensors and Minimum Inhibitory Concentration (MIC) assays, the QSI and antimicrobial aspects of the extract were assessed. Furthermore, the effect of the extract on the reduction of tuber maceration in potatoes was examined. Subsequently, homology modeling based on LasR was conducted to analyze interactions between ligand 3-oxo-C8-AHL, and ExpR2 protein. Docking studies were performed on all extract compounds identified via Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The extract effectively reduced maceration at sub-MIC concentrations across various pathogenic strains. Furthermore, Cyclopentadecanone, 2-hydroxy, showed more negative docking energy than the native ligand. Z,E-2,13-Octadecadien-1-ol showed energy equivalence to the native ligand. Additionally, this plant included certain compounds or their analogs that had previously been discovered as QSI compounds. These compounds included oleic acid, n-Hexadecanoic acid, cytidine, and linoleic acid, and they had energies that were comparable to that of the native ligand. In conclusion, the remarkable QSI property showed by this plant is likely attributed to a combination of compounds possessing this characteristic.

Rational design of ASCT2 inhibitors using an integrated experimental-computational approach.

ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting specific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final structures combined with molecular dynamics simulations reveal multiple pharmacologically relevant conformations in the ASCT2 binding site as well as a previously unknown mechanism of stereospecific inhibition. Furthermore, this integrated analysis guided the design of a series of unique ASCT2 inhibitors. Our results provide a framework for future development of cancer therapeutics targeting nutrient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery.

Deciphering the 4',7-Dihydroxy-3'-Methylflavone from Boerhavia, Agonist/Antagonist Interactions Against 5-HT2 Receptors using Homology Modeling, Molecular Docking, and Dynamic Simulation Studies.

Seizures, depression, and anxiety are neurological disorders that affected innumerable people worldwide. Recent research has revealed that targeting 5-hydroxytryptamine 2 (5-HT2) receptors can help suppress these conditions. An in-depth literature study has identified that phytocompounds from the Boerhavia genus could reduce seizures. Therefore, homology models of 5-HT2 receptors were generated and validated using techniques such as the alignment of amino acid sequences and the Ramachandran plot. Later, a comparison of modeled structures was made with a non-redundant set of PDB structures. The pharmacokinetics, drug-likeness, blood-brain barrier (BBB) permeability, and Lipinski's rule of five shed light on 22 phytocompounds, which are the potential candidates for molecular docking among 127 Boerhavia's bioactive. Notably, molecular docking analysis revealed 4',7-dihydroxy-3'-methylflavone as the most potent lead compound, which has a strong binding affinity to all modeled receptors. Additionally, with a remarkably high docking score of -9.1 kcal/mol, 4',7-dihydroxy-3'-methylflavone showed promising interactions, particularly with 5-HT2A receptor, as seen from the RMSD, SASA, Rg, and number of hydrogen bonds during 100 ns molecular dynamic (MD) simulation. Principal component analysis (PCA) and Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) further confirmed that 4',7-dihydroxy-3'-methylflavone is the best novel phytocompound in Boerhavia genus for 5-HT2 receptor as agonist/antagonist activity against seizures.

Identification of linear B cell epitopes on the leukotoxin protein of Fusobacterium necrophorum.

OBJECTIVE: Fusobacterium necrophorum can casuse Lemierre's syndrome in humans and a range of illnesses, including foot rot and liver abscesses, in animals. The main virulence factor released by F. necrophorum is leukotoxin, which has been shown to have a strong correlation with the severity of the disease. Leukotoxin is commonly employed as the key antigen in the formulation of subunit vaccines. Therefore, identification of the B-cell epitope of F. necrophorum leukotoxin is necessary. METHODS: In this research, we utilized lymphocyte hybridoma technology to develop a monoclonal antibody (mAb), 3D7, targeting the F. necrophorum leukotoxin protein. Identification of B-cell epitopes recognized by 3D7 mAb was achieved through Western blot, ELISA and dot blots using leukotoxin-truncated recombinant proteins and peptides, and through SWISS-MODEL homology modeling and PyMOL visualization. RESULTS: The 3D7 mAb was identified as belonging to the IgG1 subclass with a κ-chain light chain. It demonstrated reactivity with the natural leukotoxin. The results showed that the 3D7 mAb recognizes a B-cell epitope of the F. necrophorum leukotoxin protein, I2168SSFGVGV2175 (EP-3D7). Sequence comparison analysis showed that EP-3D7 was highly conserved in F. necrophorum strains, but less conserved in other bacteria, indicating the specificity of EP-3D7. EP-3D7 is present on the surface of leukotoxin proteins in a ß-folded manner. CONCLUSIONS: In summary, these results establish EP-3D7 as a conserved antigenic epitope of F. necrophorum leukotoxin. It could be valuable in the development of vaccines and diagnostic reagents for F. necrophorum epitopes.

Homology Modeling, Molecular Dynamics Simulation, and Prediction of Bovine TLR2 Heterodimerization.

Toll-like receptor 2 (TLR2) is a major membrane-bound receptor with ligand and species specificity that activates the host immune response. Heterodimerization of TLR2 with TLR1 (TLR2/1) or TLR6 (TLR2/6), triggered by ligand binding, is essential to initiating the signaling pathway. Bovine TLR2 (bTLR2) heterodimerization has not been defined yet compared with human and mouse TLR2s (hTLR2 and mTLR2). The aim of the present study was to model bovine TLRs (TLRs 1, 2 and 6) and create the heterodimeric forms of the bovine TLR2 using molecular dynamics (MD) simulations. We compared the intermolecular interactions in bTLR2/1-PAM3 and bTLR2/6-PAM2 with the hTLR2 and mTLR2 complexes through docking simulations and subsequent MD analyses. The present computational findings showed that bTLR2 dimerization could have a biological function and activate the immune response, similar to hTLR2 and mTLR2. Agonists and antagonists that are designed for hTLR2 and mTLR2 can target bTLR2. However, the experimental approaches to comparing the functional immune response of TLR2 across species were missing in the present study. This computational study provides a structural analysis of the bTLR2 interaction with bTLR1 and bTLR6 in the presence of an agonist/antagonist and reveals the three-dimensional structure of bTLR2 dimerization. The present findings could guide future experimental studies targeting bTLR2 with different ligands and lipopeptides.