Protein Binding Sites Research Articles

DeepProSite: structure-aware protein binding site prediction using ESMFold and pretrained language model.

Identifying the functional sites of a protein, such as the binding sites of proteins, peptides, or other biological components, is crucial for understanding related biological processes and drug design. However, existing sequence-based methods have limited predictive accuracy, as they only consider sequence-adjacent contextual features and lack structural information. In this study, DeepProSite is presented as a new framework for identifying protein binding site that utilizes protein structure and sequence information. DeepProSite first generates protein structures from ESMFold and sequence representations from pretrained language models. It then uses Graph Transformer and formulates binding site predictions as graph node classifications. In predicting protein-protein/peptide binding sites, DeepProSite outperforms state-of-the-art sequence- and structure-based methods on most metrics. Moreover, DeepProSite maintains its performance when predicting unbound structures, in contrast to competing structure-based prediction methods. DeepProSite is also extended to the prediction of binding sites for nucleic acids and other ligands, verifying its generalization capability. Finally, an online server for predicting multiple types of residue is established as the implementation of the proposed DeepProSite. The datasets and source codes can be accessed at https://github.com/WeiLab-Biology/DeepProSite. The proposed DeepProSite can be accessed at https://inner.wei-group.net/DeepProSite/.

PoSSuM v.3: A Major Expansion of the PoSSuM Database for Finding Similar Binding Sites of Proteins.

Information on structures of protein-ligand complexes, including comparisons of known and putative protein-ligand-binding pockets, is valuable for protein annotation and drug discovery and development. To facilitate biomedical and pharmaceutical research, we developed PoSSuM (https://possum.cbrc.pj.aist.go.jp/PoSSuM/), a database for identifying similar binding pockets in proteins. The current PoSSuM database includes 191 million similar pairs among almost 10 million identified pockets. PoSSuM drug search (PoSSuMds) is a resource for investigating ligand and receptor diversity among a set of pockets that can bind to an approved drug compound. The enhanced PoSSuMds covers pockets associated with both approved drugs and drug candidates in clinical trials from the latest release of ChEMBL. Additionally, we developed two new databases: PoSSuMAg for investigating antibody-antigen interactions and PoSSuMAF to simplify exploring putative pockets in AlphaFold human protein models.

Density Functional Theory-Guided Photo-Triggered Anticancer Activity of Curcumin-Based Zinc(II) Complexes.

Photodynamic therapy (PDT) has evolved as a new therapeutic modality for cancer treatment with fewer side effects and drug resistance. Curcumin exhibits PDT activity, but its low bioavailability restricts its clinical application. Here, the bioavailability of curcumin was increased by its complex formation with the Zn(II) center. For a structure-activity relationship study, Zn(II)-based complexes (1-3) comprising N^N-based ligands (2,2'-bipyridine in 1 and 2 or 1,10-phenanthroline in 3) and O^O-based ligands (acetylacetone in 1, monoanionic curcumin in 2 and 3) were synthesized and thoroughly characterized. The X-ray structure of the control complex, 1, indicated a square pyramidal shape of the molecules. Photophysical and TD-DFT studies indicated the potential of 2 and 3 as good visible light type-II photosensitizers for PDT. Guided by the TD-DFT studies, the low-energy visible light-triggered singlet oxygen (1O2) generation efficacy of 2 and 3 was explored in solution and in cancer cells. As predicted by the TD-DFT calculations, these complexes produced 1O2 efficiently in the cytosol of MCF-7 cancer cells and ultimately displayed excellent apoptotic anticancer activity in the presence of light. Moreover, the molecular docking investigation showed that complexes 2 and 3 have very good binding affinities with caspase-9 and p-53 proteins and could activate them for cellular apoptosis. Further molecular dynamics simulations confirmed the stability of 3 in the caspase-9 protein binding site.

Induced Circular Dichroism Analysis of Thermally Induced Conformational Changes on Protein Binding Sites Under a Crowding Environment.

Protein-ligand interactions in crowded cellular environments play a crucial role in biological functions. The crowded environment can perturb the overall protein structure and local conformation, thereby influencing the binding pathway of protein-ligand reactions within the cellular milieu. Therefore, a detailed understanding of the local conformation is crucial for elucidating the intricacies of protein-ligand interactions in crowded cellular environments. In this study, we investigated the feasibility of induced circular dichroism (ICD) using 8-anilinonaphthalene-1-sulfonic acid (ANS) for local conformational analysis at the binding site in a crowding environment. Bovine serum albumin (BSA) concentration-dependent measurements were performed to assess the feasibility of ANS-ICD for analyzing protein interior binding sites. The results showed distinct changes in the ANS-ICD spectra of BSA solutions, indicating their potential for analyzing the internal conformation of proteins. Moreover, temperature-dependent measurements were performed in dilute and crowding environments, revealing distinct denaturation pathways of BSA binding sites. Principal component analysis of ANS-ICD spectral changes revealed lower temperature pre-denaturation in the crowded solution than that in the diluted solution, suggesting destabilization of binding sites owing to self-crowding repulsive interactions. The established ANS-ICD method can provide valuable conformational insights into protein-ligand interactions in crowded cellular environments.

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq.

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false positive rates. Here, we present the R/Bioconductor package DEWSeq that makes use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.

Molecular Recognition of Moringa oleifera Active Compounds for Stunted Growth Prevention Using Network Pharmacology and Molecular Modeling Approach.

In this study, network pharmacology was used to analyze the active compounds of Moringa oleifera as food supplements for stunted growth prevention. Thirty-eight important proteins were discovered that may be strongly related to stunting. Those proteins were uploaded to several online tool platforms in order to determine the shared genes' pathways. Six pathways were identified that may be correlated with human growth. Furthermore, ligands for molecular docking analysis were retrieved from the top 5 active substances discovered through experimental investigation. In the meantime, the first-degree rank based on the protein-protein interaction (PPI) topological analysis was utilized to choose albumin protein (ALB) as a receptor. Our docking results showed that every ligand binds to the receptors, indicating that they can bind to the binding site of the ALB protein to form a complex formation. Further, MD simulation was used to verify the stability of the ligand in complex with the protein in the TIP3P water model. Based on the validation parameters, our results suggested that all models achieved a stable phase along the simulation. Additionally, the MM-GBSA method was used to calculate the binding energies of all models. Ligands 2 and 4 have strong binding to the binding pocket of ALB, followed by ligands 3, 5, and 2, suggesting that those ligands could be promising food supplements that can be utilized for the prevention of stunted growth in children.

Low-Entropy Hydration Shells at the Spike RBD's Binding Site May Reveal the Contagiousness of SARS-CoV-2 Variants.

The infectivity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is primarily determined by the binding affinity between the receptor-binding domain (RBD) of the spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. Here, through screening off pseudo hydrophilic groups on protein surfaces, the distribution of low-entropy regions on hydration shells of the ACE2 receptor and the RBDs of multiple SARS-CoV-2 variants was demonstrated. Shape matching between the low-entropy hydration shells of multiple SARS-CoV-2 variants and the ACE2 receptor has been identified as a mechanism that drives hydrophobic attraction between the RBDs and the ACE2 receptor, which estimates the binding affinity. Low-entropy regions of the hydration shells, which play important roles in determining the binding of other viruses and their receptors, are demonstrated. The RBD-ACE2 binding is thus found to be guided by hydrophobic collapse between the shape-matched low-entropy regions of the hydration shells of the proteins. A measure of the low-entropy status of the hydration shells can be estimated by calculating genuine hydrophilic groups within the binding sites. An important indicator of the contagiousness of SARS-CoV-2 variants is the low-entropy level of its hydration shells at the spike protein binding site.

‘Synthesis, antiviral activity, molecular docking, and molecular dynamics studies of ethoxy phthalimide pyrazole derivatives against Cytomegalovirus and Varicella-Zoster virus: potential consequences and strategies for developing new antiviral treatments’

Substituted ethoxy phthalimide pyrazole derivatives (6a–e) have been produced using a one-pot synthesis technique. Spectral analysis was used to establish the molecular structure of the synthesized compounds, and they were examined in silico and in vitro for their ability to bind to and inhibit replication of the AD-169 strain, the Davis strain of CMV, the OKA strain and the 07/1 strain of Varicella-Zoster virus (VZV). Molecular Docking was used to estimate the binding mechanism and energy of compounds 4, 6a–e to their respective target proteins, thymidine kinase (TK), Varicella-Zoster protease (VZP) of VZV and tegument protein pp71 (TPpp71) of Cytomegalovirus (CMV). The MIC50 and EC50 were utilized to evaluate the antiviral and cytotoxic activities of test compounds in human embryonic lung (HEL) cells against the two reference medicines, Ganciclovir and Acyclovir. The chemicals studied showed a high affinity for binding sites and near binding sites of target proteins by generating H-bonds, carbon-hydrogen bonds, π-anion, π-sulfur, π-sigma, alkyl and π-alkyl interactions. All of the test compounds (6a–e) had higher binding energy than the standard medications. The ADME/T data suggests that these potential inhibitors are less toxic. Drug-protein complexes are structurally compact and demonstrate minimal conformational change in molecular dynamics (MDs) simulations, indicating stability and stiffness. MM-PBSA and post-simulation analysis can predict lead compound active cavity binding stability. By inhibiting multitargeted proteins, these synthetic compounds may improve antiviral therapy. Our research suggests that these unique synthesized chemicals may be useful and accessible adjuvant antiviral therapy for Varicella Zoster and CMV. Highlights Two components synthesis of substituted ethoxy phthalimide pyrazole derivatives (6a–e). Tested compounds (6a–e) have antiviral and cytotoxicity activity against CMV and Varicella-Zoster virus (VZV) in HEL cells. Compounds bind to TK, Varicella-Zoster protease (VZP) of VZV, and modeled TPpp71 of Cytomegalovirus (CMV). In comparison to reference drugs, compounds have strong binding free energy and interactions with VZV and CMV protein complexes. The RMSD, RMSF, Rg, residual correlative motion (RCM), No. of hydrogen bonds, protein secondary structure content, per-residue protein secondary structure and MM/PBSA energy calculated for the selected compound with thymidine kinase (TK), VZP of VZV, and modeled tegument protein pp71 (TPpp71) of CMV through MD simulation studies for 50 ns. In comparison to the two reference drugs, ligands/compounds were found to meet the Lipinski rule of five and to have strong biological activity. Communicated by Ramaswamy H. Sarma

Broad-spectrum antagonistic potential of Bacillus spp. volatiles against Rhizoctonia solani and Xanthomonas oryzae pv. oryzae.

Rhizoctonia solani and Xanthomonas oryzae pv. oryzae (Xoo) are the two major diseases affecting the quality and quantity of rice production. In the current study, volatile organic compounds (VOCs) of Bacillus spp. were used as green biocontrol agents for plant diseases. In in vitro experiments, Bacillus spp. FZB42, NMTD17, and LLTC93-VOCs displayed strong antimicrobial volatile activity with inhibition rates of 76, 66, and 78% for R. solani and 78, 81, and 76% for Xoo, respectively, compared to control. The synthetic VOCs, namely Pentadecane (PDC), Benzaldehyde (BDH), 1,2-Benz isothiazol-3(2H)-one (1,2-BIT), and mixture (MIX) of VOCs showed high volatile activity with inhibition rates of 86, 86, 89, and 92% against R. solani and 81, 81, 82, and 86%, respectively, against Xoo as compared to control. In addition, the scanning and transmission electron microscopes (SEM and TEM) analyses were performed to examine the effect of Bacillus and synthetic VOC treatments on R. solani and Xoo morphology. The analysis revealed the deformed and irregularized morphology of R. solani mycelia and Xoo cells after VOC treatments. The microscopic analysis showed that the rapid inhibition was due to severe oxidative productions inside the R. solani mycelia and Xoo cells. By using molecular docking, it was determined that the synthetic VOCs entered the active binding site of trehalase and NADH dehydrogenase proteins, causing R. solani and Xoo cells to die prematurely and an accumulation of ROS. In the greenhouse experiment, FZB42, NMTD17, and LLTC93-VOCs significantly reduced the lesions of R. solani 8, 7, and 6 cm, and Xoo 7, 6, and 6 cm, respectively, then control. The synthetic VOCs demonstrated that the PDC, BDH, 1,2-BIT, and MIX-VOCs significantly reduced R. solani lesions on leaves 6, 6, 6, and 5 cm and Xoo 6, 5, 5, and 4 cm, respectively, as compared to control. Furthermore, plant defence-related genes and antioxidant enzymes were upregulated in rice plants. These findings provide novel mechanisms by which Bacillus antimicrobial VOCs control plant diseases.

Green Tea Catechins as Potential Drug Scaffolding Molecules in Structural Studies with Diverse Protein Targets

Abstract: Previous studies provide substantial evidence that catechins, polyphenol bioactive compounds, exhibit medicinal benefits. These polyphenols are found in abundance in green teas, including a combination of the four major types of catechins: (-)-Epicatechin (EC), (-)-Epicatechin-3-gallate (ECG), (-)- Epigallocatechin (EGC), and (-)- Epigallocatechin-3-gallate (EGCG). Although all four exhibit medicinal benefits, the catechin cited in the literature the most is EGCG, so derivatives of this catechin were selected for these studies. Literature searches identified catechins as biologically active compounds for a diverse set of diseases ranging from cancer, metabolism, neurological, and neuromuscular ailments. A diverse set of potential protein targets for docking with catechin derivatives was first identified as a list (n = 48). The targets were then selected based on the presence of 3D protein coordinates for these targets provided by the Rutgers Consortium for Structural Biology (RCSB) Protein Data Bank (PDB) (n = 10). The surfaces of the 3D protein targets were evaluated with computational methods to identify potential binding sites for the EGCG catechin derivatives. Static and flexible docking was done using target protein binding sites performed with the catechin derivatives followed by molecular dynamics (MD). MD protocols were run to confirm binding in the physiological range and environment. In summary, the results of computational protocols confirmed predicted binding by docking with MD of several catechin derivatives to be used as scaffolds once validated in lab-based assays. Possible changes to these scaffolding molecules that could result in tighter, more specific binding is discussed.

In Vitro and In Vivo Effects of Synthesis Novel Phenoxyacetamide Derivatives as Potent Apoptotic Inducer against HepG2 Cells through PARP-1 Inhibition.

To discover potential cytotoxic agents, new semi-synthetic phenoxy acetamide derivatives, compound I and compound II, were synthesized, characterized, and screened for their cytotoxic activity against breast cancer (MCF-7) and liver cancer (HepG2) cell lines. The two compounds were more promising against HepG2 than the MCF-7 cell line according to IC50 values. When tested against the HepG2 cell line, compound I, and compound II both had significantly increased cytotoxic activity when compared to the reference medication 5-Fluorouracil (5-FU), with IC50 values of 1.43 M, 5.32 M, and 6.52 M for compound 1, 5-FU and compound II, respectively. Also, compound I displayed a degree of selectivity towards cancer cells compared to normal cells. Compound I significantly enhanced HepG2 total apoptotic cell death by about a 24.51-fold increase. According to cell cycle analysis, compound I induced the arrest of the cell cycle phases G1/S and blocked the progression of the HepG2 cells. Applying the RT-PCR technique achieved a highly significant upregulation in pro-apoptotic genes. The anti-apoptotic gene was significantly downregulated. There was an intrinsic and extrinsic pathway, but the intrinsic pathway was the dominant one. Tumor growth suppression as measured by tumor weight and volume and other hematological, biochemical, and histopathological analyses confirmed the efficacy of compound I as an anticancer agent in vivo examination. Finally, the molecular docking study revealed that compound I was properly docked inside the binding site of PARP-1 protein with stable binding energies and interactive binding modes. Therefore, compound I shows promise as a selective anti-cancer derivative for the treatment of liver cancer after more investigations and clinical studies. This selectivity is a favorable characteristic in the developing cytotoxic agents for cancer treatment, as it indicates a potential for reduced harm to health tissues.

High-throughput virtual screening approach and dynamic simulation of natural compounds as target inhibitors of BACE1 in Alzheimer's disease

Background Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) protein holds significance in the development of Alzheimer’s disease (AD). This protein is highly expressed in the central nervous system, playing a significant role in the conversion of amyloid precursor protein (APP) into amyloid-beta peptides. The primary objective of the current study was to perform in silico inhibition investigations on this protein, utilizing computational methodologies such as molecular docking and molecular dynamics, to identify novel inhibitors with potential against BACE1. Our focus was on targeting the active site of BACE1, aiming to discover optimal interactions between the ligands and key residues of the target protein. Methods The natural compounds inhibitors data was carefully reviewed from the literature review articles. Molecular docking was conducted using AutoDock Vina with an active site defined by CB-Dock2. We assessed drug-likeness and toxicity using Lipinski’s rule of fives via SwissADME and the ProTox-II web server. Interaction visualization was facilitated using Discovery Studio, while molecular dynamics simulations were performed for 100 nanoseconds with hinokiflavone and BACE1 using Schrodinger LLC Desmond software. Results The molecular docking results showed promising binding affinities and the best binding free energy values were selected, and after conducting Lipinski’s rule of five using SwissADME as well as predicting toxicity using ProTox-II, only one molecule, hinokiflavone was filtered and succeeded in all the analyses to be a potential candidate. Molecular docking results were supported by molecular dynamics simulation. These results demonstrate the stability of the compound in the target protein binding site. Conclusions Finally, these obtained outcomes represent a strong lead to developing promising new natural compound inhibitors against BACE1. For future works, it is essential to concentrate on further experimental validation to ensure the effectiveness of the proposed approach.

Computational screening of potential bromodomain-containing protein 2 inhibitors for blocking SARS-CoV-2 infection through pharmacophore modeling, molecular docking and molecular dynamics simulation

Recently, human bromodomain-containing protein 2 (BRD2) has been reported as a potential drug target in host cells to combat SARS-CoV-2 as well as to treat COVID-19. In this study, we aimed to screen potential BRD2 inhibitors as anti- SARS-CoV-2 agents through computational approach. A pharmacophore model was generated with crystal structure of BRD2 in complex with RVX208 using ZINCPharmer server, and 10,842 compounds were screened from ZINC15 database against the pharmacophore model. Next, 102 compounds exhibited more affinity to BRD2 than control compound (RVX208) were screened using molecular docking. The top five hits (ZINC20417563, ZINC12322175, ZINC12391479, ZINC13098192, and ZINC20407881), which showed docking affinity to BRD2 with −10, −9.9, −9.8, −9.7 and −9.7 kcal/mol, respectively, were subjected to 100 ns molecular dynamics simulation to evaluate the stability of docked complexes and their interaction mechanisms. The appropriate stability of these complexes was displayed, and these compounds were found to be closely adhered to the binding site of the BRD2 protein based on the global dynamics analysis with RMSD, RMSF, Rg, SASA, DSSP and H-bond. The essential dynamics analysis with principal component analysis, dynamic cross-correlation matrix, and free energy landscape showed BRD2 binding of the screened compounds remained the stable conformation throughout molecular dynamics simulation. In addition, the pharmacokinetic and ADMET properties of the five compounds showed these compounds could be potential drug candidates. The findings of this study may contribute to further examinational validation and rational design of novel BRD2 inhibitors as anti-SARS-CoV-2 agents.

Harnessing a 4-Formyl-Aniline Handle to Tune the Stability of a DNA Aptamer-Protein Complex via Fluorescent Surrogates.

Interactions between DNA aptamers and protein targets hold promise for the development of pharmaceuticals and diagnostics. As such, the utilization of fluorescent nucleobase surrogates in studying aptamer-protein interactions is a powerful tool due to their ability to provide site-specific information through turn-on fluorescence. Unfortunately, previously described turn-on probes serving as nucleobase replacements have only been strongly disruptive to the affinity of aptamer-protein interactions. Herein, we present a modified TBA15 aptamer for thrombin containing a fluorescent surrogate that provides site-specific turn-on emission with low nanomolar affinity. The modification, referred to as AnBtz, was substituted at position T3 and provided strong turn-on emission (Irel ≈ 4) and brightness (ε·Φ > 20 000 cm-1 M-1) with an apparent dissociation constant (Kd) of 15 nM to afford a limit of detection (LOD) of 10 nM for thrombin in 20% human serum. The probe was selected through a modular "on-strand" synthesis process that utilized a 4-formyl-aniline (4FA) handle. Using this platform, we were able to enhance the affinity of the final aptamer conjugate by ∼30-fold in comparison with the initial conjugate design. Molecular dynamics simulations provide insight into the structural basis for this phenomenon and highlight the importance of targeting hydrophobic protein binding sites with fluorescent nucleobase surrogates to create new contacts with protein targets.

The novel RNA-RNA activation of H19 on MyoD transcripts promoting myogenic differentiation of goat muscle satellite cells

The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth.

Syndapin and GTPase RAP-1 control endocytic recycling via RHO-1 and non-muscle myosin II

After endocytosis, many plasma membrane components are recycled via membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that Syndapin/PACSIN-family protein SDPN-1 is required invivo for basolateral endocytic recycling in the C.elegans intestine. Here, we document an interaction between the SDPN-1 SH3 domain and a target sequence in PXF-1/PDZ-GEF1/RAPGEF2, a known exchange factor for Rap-GTPases. We found that endogenous mutations engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling invivo, as does the loss of the PXF-1 target RAP-1. In some contexts, Rap-GTPases negatively regulate RhoA activity, suggesting a potential for Syndapin to regulate RhoA. Our results indicate that in the C.elegans intestine, RHO-1/RhoA is enriched on SDPN-1- and RAP-1-positive endosomes, and the loss of SDPN-1 or RAP-1 elevates RHO-1(GTP) levels on intestinal endosomes. Furthermore, we found that depletion of RHO-1 suppressed sdpn-1 mutant recycling defects, indicating that control of RHO-1 activity is a key mechanism by which SDPN-1 acts to promote endocytic recycling. RHO-1/RhoA is well known for controlling actomyosin contraction cycles, although little is known about the effects of non-muscle myosin II on endosomes. Our analysis found that non-muscle myosin II is enriched on SDPN-1-positive endosomes, with two non-muscle myosin II heavy-chain isoforms acting in apparent opposition. Depletion of nmy-2 inhibited recycling like sdpn-1 mutants, whereas depletion of nmy-1 suppressed sdpn-1 mutant recycling defects, indicating that actomyosin contractility controls recycling endosome function.

FL-circAS: an integrative resource and analysis for full-length sequences and alternative splicing of circular RNAs with nanopore sequencing.

Circular RNAs (circRNAs) are RNA molecules with a continuous loop structure characterized by back-splice junctions (BSJs). While analyses of short-read RNA sequencing have identified millions of BSJ events, it is inherently challenging to determine exact full-length sequences and alternatively spliced (AS) isoforms of circRNAs. Recent advances in nanopore long-read sequencing with circRNA enrichment bring an unprecedented opportunity for investigating the issues. Here, we developed FL-circAS (https://cosbi.ee.ncku.edu.tw/FL-circAS/), which collected such long-read sequencing data of 20 cell lines/tissues and thereby identified 884636 BSJs with 1853692 full-length circRNA isoforms in human and 115173 BSJs with 135617 full-length circRNA isoforms in mouse. FL-circAS also provides multiple circRNA features. For circRNA expression, FL-circAS calculates expression levels for each circRNA isoform, cell line/tissue specificity at both the BSJ and isoform levels, and AS entropy for each BSJ across samples. For circRNA biogenesis, FL-circAS identifies reverse complementary sequences and RNA binding protein (RBP) binding sites residing in flanking sequences of BSJs. For functional patterns, FL-circAS identifies potential microRNA/RBP binding sites and several types of evidence for circRNA translation on each full-length circRNA isoform. FL-circAS provides user-friendly interfaces for browsing, searching, analyzing, and downloading data, serving as the first resource for discovering full-length circRNAs at the isoform level.

Induction of apoptotic cell death of cholangiocarcinoma cells by tiliacorinine from Tiliacora triandra: A mechanistic insight

BackgroundCholangiocarcinoma (CCA) exhibits poor response to the present chemotherapeutic agents and frequently develops drug resistance. Finding novel anticancer drugs might enhance patient outcomes. Tiliacorinine, a bisbenzylisoquinoline alkaloid from the Thai medicinal plant Tiliacora triandra, effectively induced apoptosis of human CCA cell lines and inhibited tumor growth in mice. Here, we elucidate further the molecular mechanisms underlining the cytotoxicity of tiliacorinine and its implication in overcoming gemcitabine-resistance of CCA cells. MethodsCytotoxicity of tiliacorinine against CCA cell lines was assessed using MTT assay. The molecular signaling was determined using Western blot analysis. Molecular docking simulations were applied to predict the binding affinity and orientation of tiliacorinine to the possible binding site(s) of the target proteins. ResultsTiliacorinine induced apoptotic cell death of CCA cells in a dose- and time-dependent manner. Tiliacorinine significantly suppressed the expression of anti-apoptotic proteins, Bcl-xL and XIAP; activated apoptotic machinery proteins, caspase-3, caspase-9, and PARP; and decreased the levels of pAkt and pSTAT3. EGF/EGFR activation model and molecular docking simulations revealed EGFR, Akt, and STAT3 as potent targets of tiliacorinine. Molecular docking simulations indicated a strong binding affinity of tiliacorinine to the ATP-binding pockets of EGFR, PI3K, Akt, JAK2, and SH2 domain of STAT3. Tiliacorinine could synergize with gemcitabine and restore the cytotoxicity of gemcitabine against gemcitabine-resistant CCA cells. ConclusionTiliacorinine effectively induced apoptosis via binding and blocking the actions of EGFR, Akt, and STAT3. General significanceTiliacorinine is a novel multi-kinase inhibitor and possibly a potent anti-cancer agent, in cancers with high activation of EGFR.

Identification of potential flavonoid compounds as antibacterial therapeutics against Klebsiella pneumoniae infection using structure-based virtual screening and molecular dynamics simulation.

Klebsiella pneumoniae, which is among the top three pathogens on WHO's priority list, is one of the gram-negative bacteria that doctors and researchers around the world have fought for decades.Capsular polysaccharide (CPS) protein is extensively recognized as an importantK. pneumoniaevirulence factor. Thus, CPS has become the most characterized target for the discovery of novel drug candidates. The ineffectiveness of currently existing antibiotics urgesthe search for potent antimicrobial compounds. Flavonoidsare a group of plant metabolites that have antibacterial potential and can enhance the present medications to elicit improved results against diverse diseases without adverse reactions. Henceforth, the present study aims to illustrate the inhibitory potential of flavonoids with varying pharmacological properties, targeting the CPS protein of K. pneumoniae by in silico approaches. The flavonoid compounds (n = 169) were retrieved from the PubChem database and screened using the structure-based virtual screening approach. Compounds with the highest binding score were estimated through their pharmacokinetic effects by ADMET descriptors. Finally, four potential inhibitors with PubChem CID: (4301534, 5213, 5481948, and 637080) were selected after molecular docking and drug-likeness analysis. All four lead compounds were employed for the MDS analysis of a 100ns time period. Various studies were undertaken to assess the stability of the protein-ligand complexes. The binding free energy was computed using MM-PBSA, and the outcomes indicated that the molecules are having stable interactions with the binding site of the target protein. The results revealed that all four compounds can be employed as potential therapeutics against K. pneumoniae.

Nucleotide-level prediction of CircRNA-protein binding based on fully convolutional neural network.

Introduction: CircRNA-protein binding plays a critical role in complex biological activity and disease. Various deep learning-based algorithms have been proposed to identify CircRNA-protein binding sites. These methods predict whether the CircRNA sequence includes protein binding sites from the sequence level, and primarily concentrate on analysing the sequence specificity of CircRNA-protein binding. For model performance, these methods are unsatisfactory in accurately predicting motif sites that have special functions in gene expression. Methods: In this study, based on the deep learning models that implement pixel-level binary classification prediction in computer vision, we viewed the CircRNA-protein binding sites prediction as a nucleotide-level binary classification task, and use a fully convolutional neural networks to identify CircRNA-protein binding motif sites (CPBFCN). Results: CPBFCN provides a new path to predict CircRNA motifs. Based on the MEME tool, the existing CircRNA-related and protein-related database, we analysed the motif functions discovered by CPBFCN. We also investigated the correlation between CircRNA sponge and motif distribution. Furthermore, by comparing the motif distribution with different input sequence lengths, we found that some motifs in the flanking sequences of CircRNA-protein binding region may contribute to CircRNA-protein binding. Conclusion: This study contributes to identify circRNA-protein binding and provides help in understanding the role of circRNA-protein binding in gene expression regulation.