Targeting AFP-RARß complex formation: a potential strategy for treating AFP-positive hepatocellular carcinoma.

Alpha-fetoprotein (AFP) is a glycoprotein primarily expressed during embryogenesis, with declining levels postnatally. Elevated AFP levels correlate with pathological conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Recent investigations underscore AFP's intracellular role in HCC progression, wherein it forms complexes with proteins like Phosphatase and tensin homolog (PTEN), Caspase 3 (CASP3), and Retinoic acid receptors and Retinoid X receptors (RAR/RXR). RAR and RXR regulate gene expression linked to cell death and tumorigenesis in normal physiology. AFP impedes RAR/RXR dimerization, nuclear translocation, and function, promoting gene expression favoring cancer progression in HCC that provoked us to target AFP as a drug candidate. Despite extensive studies, inhibitors targeting AFP to disrupt complex formation and activities remain scarce. In this study, employing protein-protein docking, amino acid residues involved in AFP-RARß interaction were identified, guiding the definition of AFP's active site for potential inhibitor screening. Currently, kinase inhibitors play a significant role in cancer treatment and, the present study explores the potential of repurposing FDA-approved protein kinase inhibitors to target AFP. Molecular docking with kinase inhibitors revealed Lapatinib as a candidate drug of the AFP-RARß complex. Molecular dynamics simulations and binding energy calculations, employing Mechanic/Poisson-Boltzmann Surface Area (MM-PBSA), confirmed Lapatinib's stability with AFP. The study suggests Lapatinib's potential in disrupting the AFP-RARß complex, providing a promising avenue for treating molecularly stratified AFP-positive HCC or its early stages.

Computational identification of potential bioactive compounds from Triphala against alcoholic liver injury by targeting alcohol dehydrogenase.

Alcoholic liver injury resulting from excessive alcohol consumption is a significant social concern. Alcohol dehydrogenase (ADH) plays a critical role in the conversion of alcohol to acetaldehyde, leading to tissue damage. The management of alcoholic liver injury encompasses nutritional support and, in severe cases liver transplantation, but potential adverse effects exist, and effective medications are currently unavailable. Natural products with their potential benefits and historical use in traditional medicine emerge as promising alternatives. Triphala, a traditional polyherbal formula demonstrates beneficial effects in addressing diverse health concerns, with a notable impact on treating alcoholic liver damage through enhanced liver metabolism. The present study aims to identify potential active phytocompounds in Triphala targeting ADH to prevent alcoholic liver injury. Screening 119 phytocompounds from the Triphala formulation revealed 62 of them showing binding affinity to the active site of the ADH1B protein. Promising lipid-like molecule from Terminalia bellirica, (4aS, 6aR, 6aR, 6bR, 7R, 8aR, 9R, 10R, 11R, 12aR, 14bS)-7, 10, 11-trihydroxy-9-(hydroxymethyl)-2, 2, 6a, 6b, 9, 12a-hexamethyl-1, 3, 4, 5, 6, 6a, 7, 8, 8a, 10, 11, 12, 13, 14b-tetradecahydropicene-4a-carboxylic acid showed high binding efficiency to a competitive ADH inhibitor, 4-Methylpyrazole. Pharmacokinetic analysis further confirmed the drug-likeness and non-hepatotoxicity of the top-ranked compound. Molecular dynamics simulation and MM-PBSA studies revealed the stability of the docked complexes with minimal fluctuation and consistency of the hydrogen bonds throughout the simulation. Together, computational investigations suggest that (4aS, 6aR, 6aR, 6bR, 7R, 8aR, 9R, 10R, 11R, 12aR, 14bS)-7, 10, 11-trihydroxy-9-(hydroxymethyl)-2, 2, 6a, 6b, 9, 12a-hexamethyl-1, 3, 4, 5, 6, 6a, 7, 8, 8a, 10, 11, 12, 13, 14b-tetradecahydropicene-4a-carboxylic acid from the Triphala formulation holds promise as an ADH inhibitor, suggesting an alternative therapy for alcoholic liver injury.

Comparative Metabolomics and Network Pharmacology Analysis Reveal Shared Neuroprotective Mechanisms of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb.

Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., two nootropics, are recognized in Indian Ayurvedic texts. Studies have attempted to understand their action as memory enhancers and neuroprotectants, but many molecular aspects remain unknown. We propose that Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb. share common neuroprotective mechanisms. Mass spectrometry-based untargeted metabolomics and network pharmacology approach were used to identify potential protein targets for the metabolites from each extract. Phytochemical analyses and cell culture validation studies were also used to assess apoptosis and ROS activity using aqueous extracts prepared from both herbal powders. Further, docking studies were also performed using the LibDock protocol. Untargeted metabolomics and network pharmacology approach unveiled 2751 shared metabolites and 3439 and 2928 non-redundant metabolites from Bacopa monnieri and Centella asiatica extracts, respectively, suggesting a potential common neuroprotective mechanism among these extracts. Protein-target prediction highlighted 92.4% similarity among the proteins interacting with metabolites for these extracts. Among them, kinases mapped to MAPK, mTOR, and PI3K-AKT signaling pathways represented a predominant population. Our results highlight a significant similarity in the metabolome of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., and their potential protein targets may be attributed to their common neuroprotective functions.

A global phosphosite-correlated network map of Thousand And One Kinase 1 (TAOK1).

Thousand and one amino acid kinase 1 (TAOK1) is a sterile 20 family Serine/Threonine kinase linked to microtubule dynamics, checkpoint signaling, DNA damage response, and neurological functions. Molecular-level alterations of TAOK1 have been associated with neurodevelopment disorders and cancers. Despite their known involvement in physiological and pathophysiological processes, and as a core member of the hippo signaling pathway, the phosphoregulatory network of TAOK1 has not been visualized. Aimed to explore this network, we first analyzed the predominantly detected and differentially regulated TAOK1 phosphosites in global phosphoproteome datasets across diverse experimental conditions. Based on 709 qualitative and 210 quantitative differential cellular phosphoproteome datasets that were systematically assembled, we identified that phosphorylation at Ser421, Ser9, Ser965, and Ser445 predominantly represented TAOK1 in almost 75% of these datasets. Surprisingly, the functional role of all these phosphosites in TAOK1 remains unexplored. Hence, we employed a robust strategy to extract the phosphosites in proteins that significantly correlated in expression with predominant TAOK1 phosphosites. This led to the first categorization of the phosphosites including those in the currently known and predicted interactors, kinases, and substrates, that positively/negatively correlated with the expression status of each predominant TAOK1 phosphosites. Subsequently, we also analyzed the phosphosites in core proteins of the hippo signaling pathway. Based on the TAOK1 phosphoregulatory network analysis, we inferred the potential role of the predominant TAOK1 phosphosites. Especially, we propose pSer9 as an autophosphorylation and TAOK1 kinase activity-associated phosphosite and pS421, the most frequently detected phosphosite in TAOK1, as a significant regulatory phosphosite involved in the maintenance of genome integrity. Considering that the impact of all phosphosites that predominantly represent each kinase is essential for the efficient interpretation of global phosphoproteome datasets, we believe that the approach undertaken in this study is suitable to be extended to other kinases for accelerated research.

Computational screening of potential anti-inflammatory leads from Jeevaneeya Rasayana plants targeting COX-2 and 5- LOX by molecular docking and dynamic simulation approaches.

Inflammation plays a pivotal role in various pathological processes, ranging from routine injuries and infections to cancer. Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) are two major enzymes involved in the formation of lipid mediators of inflammation, such as prostaglandins and leukotrienes, through the arachidonic acid pathway. Despite the frequent use of nonsteroidal anti-inflammatory drugs for managing inflammatory disorders by inhibiting these enzymes, there is a wide spectrum of adverse effects linked to their usage. Jeevaneeya Rasayana (JR), a polyherbal formulation traditionally used in India, is renowned for its anti-inflammatory properties. The present study aimed to identify the potential phytocompounds in JR plants against COX-2 and 5-LOX, utilizing molecular docking and dynamic simulations. Among the 429 identified phytocompounds retrieved from publicly available data sources, Terrestribisamide and 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine have shown potential binding affinity and favorable interactions with COX-2 and 5-LOX arachidonic acid binding sites. The physicochemical properties and ADMET profiles of these compounds determined their drug-likeness and pharmacokinetics features. Additional validation using molecular dynamics simulations, SASA, Rg, and MM-PBSA binding energy calculations affirmed the stability of the complex formed between those compounds with target proteins. Together, the study identified the effectual binding potential of those bioactive compounds against COX-2 and 5-LOX, providing a viable approach for the development of effective anti-inflammatory medications.

Meta-analysis of the serum/plasma proteome identifies significant associations between COVID-19 with Alzheimer's/Parkinson's diseases.

In recent years, we have seen the widespread devastations and serious health complications manifested by COVID-19 globally. Although we have effectively controlled the pandemic, uncertainties persist regarding its potential long-term effects, including prolonged neurological issues. To gain comprehensive insights, we conducted a meta-analysis of mass spectrometry-based proteomics data retrieved from different studies with a total of 538 COVID-19 patients and 523 healthy controls. The meta-analysis revealed that top-enriched pathways were associated with neurological disorders, including Alzheimer's (AD) and Parkinson's disease (PD). Further analysis confirmed a direct correlation in the expression patterns of 24 proteins involved in Alzheimer's and 23 proteins in Parkinson's disease with COVID-19. Protein-protein interaction network and cluster analysis identified SNCA as a hub protein, a known biomarker for Parkinson's disease, in both AD and PD. To the best of our knowledge, this is the first meta-analysis study providing proteomic profiling evidence linking COVID-19 to neurological complications.

Epstein-Barr Virus: Human Interactome Reveals New Molecular Insights into Viral Pathogenesis for Potential Therapeutics and Antiviral Drug Discovery.

Host-virus Protein-Protein Interactions (PPIs) play pivotal roles in biological processes crucial for viral pathogenesis and by extension, inform antiviral drug discovery and therapeutics innovations. Despite efforts to develop the Epstein-Barr virus (EBV)-host PPI network, there remain significant knowledge gaps and a limited number of interacting human proteins deciphered. Furthermore, understanding the dynamics of the EBV-host PPI network in the distinct lytic and latent viral stages remains elusive. In this study, we report a comprehensive map of the EBV-human protein interactions, encompassing 1752 human and 61 EBV proteins by integrating data from the public repository HPIDB (v3.0) as well as curated high-throughput proteomic data from the literature. To address the stage-specific nature of EBV infection, we generated two detailed subset networks representing the latent and lytic stages, comprising 747 and 481 human proteins, respectively. Functional and pathway enrichment analysis of these subsets uncovered the profound impact of EBV proteins on cancer. The identification of highly connected proteins and the characterization of intrinsically disordered and cancer-related proteins provide valuable insights into potential therapeutic targets. Moreover, the exploration of drug-protein interactions revealed notable associations between hub proteins and anticancer drugs, offering novel perspectives for controlling EBV pathogenesis. This study represents, to the best of our knowledge, the first comprehensive investigation of the two distinct stages of EBV infection using high-throughput datasets. This makes a contribution to our understanding of EBV-host interactions and provides a foundation for future drug discovery and therapeutic interventions.

Targeting the Pseudomonas aeruginosa quorum sensing system to inhibit virulence factors and eradicate biofilm formation using AHL-analogue phytochemicals.

Quorum sensing plays a major role in the expression of virulence and development of biofilm in the human pathogen Pseudomonas aeruginosa. Natural compounds are well-known for their antibacterial characteristics by blocking various metabolic pathways. The goal of this study is to find natural compounds that mimic AHL (Acyl homoserine lactone) and suppress virulence in P. aeruginosa, which is triggered by quorum sensing-dependent pathways as an alternative drug development strategy. To support this rationale, functional network analysis and in silico investigations were carried out to find natural AHL analogues, followed by molecular docking studies. Out of the 16 top-hit AHL analogues derived from phytochemicals, seven ligands were found to bind to the quorum sensing activator proteins. Cassialactone, an AHL analogue, exhibited the highest binding affinity for RhlI, RhlR, and PqsE of P. aeruginosa, with a docking score of -9.4, -8.9, and -8.7 kcal/mol, respectively. 2(5H)-Furanone, a well-known inhibitor, was also docked to compare the docking score and intermolecular interactions between the ligand and the target protein. Furthermore, molecular dynamics simulations and binding free energy calculations were performed to determine the stability of the docked complexes. Additionally, the ADME properties of the analogues were also analyzed to evaluate the pharmacological parameters. Functional network analysis further showed that the interconnectedness of proteins such as RhlI, RhlR, LasI, and PqsE with the virulence and biofilm phenotype of the pathogen could offer potential as a therapeutic target.Communicated by Ramaswamy H. Sarma.

Identification and stability analysis of potential ADP-ribose modification sites on vascular endothelial growth factor (VEGF) through molecular dynamics simulation.

Post-translational modifications (PTMs) are crucial covalent processes that alter protein properties, achieved through proteolytic cleavage or addition of modifying groups like acetyl, phosphoryl, glycosyl, or methyl to amino acids. ADP-ribosylation is a reversible post-translational modification, where ADP-ribose units are covalently attached to target protein side chains. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays a key role in physiological and pathological conditions. Studies have reported that ADP-ribosylation affects VEGF's ability to bind to VEGF receptors, impacting angiogenesis signalling. However, the specific amino acid undergoing ADP-ribosylation on VEGF remained unknown. To understand the mechanism of ADP-ribose addition to VEGF, an in silico study was designed. The study initially checked for the presence of any conserved motif where ADP-ribosylation could potentially occur and identified the presence of the EIE motif in VEGF, a probable site for ADP-ribosylation for many proteins. Subsequently, the amino acids near this motif were selected and their structural properties were analyzed. Surface-exposed amino acids were chosen, and ADP-ribose was then added to their side chains. The results revealed that the amino acids ASP (67) and GLU (70) underwent glycosidic linkage with ADP-ribose, indicating that they are the most probable modification sites. Subsequently, Molecular dynamic simulation analysis such as RMSD, RMSF, Rg, PCA, and FEL, along with MM-PBSA binding free energy calculations were performed to understand the stability of the VEGF-ADP-ribose complexes. The analysis revealed that amino acid at position 67 (ASP67) is the most probable site for ADP-ribosylation in VEGF.Communicated by Ramaswamy H. Sarma.

Temporal phosphoproteomic analysis of VEGF-A signaling in HUVECs: an insight into early signaling events associated with angiogenesis.

Vascular endothelial growth factor-A (VEGF-A) is one of the primary factors promoting angiogenesis in endothelial cells. Although defects in VEGF-A signaling are linked to diverse pathophysiological conditions, the early phosphorylation-dependent signaling events pertinent to VEGF-A signaling remain poorly defined. Hence, a temporal quantitative phosphoproteomic analysis was performed in human umbilical vein endothelial cells (HUVECs) treated with VEGF-A-165 for 1, 5 and 10 min. This led to the identification and quantification of 1971 unique phosphopeptides corresponding to 961 phosphoproteins and 2771 phosphorylation sites in total. Specifically, 69, 153, and 133 phosphopeptides corresponding to 62, 125, and 110 phosphoproteins respectively, were temporally phosphorylated at 1, 5, and 10 min upon addition of VEGF-A. These phosphopeptides included 14 kinases, among others. This study also captured the phosphosignaling events directed through RAC, FAK, PI3K-AKT-MTOR, ERK, and P38 MAPK modules with reference to our previously assembled VEGF-A/VEGFR2 signaling pathway map in HUVECs. Apart from a significant enrichment of biological processes such as cytoskeleton organization and actin filament binding, our results also suggest a role of AAK1-AP2M1 in the regulation of VEGFR endocytosis. Taken together, the temporal quantitative phosphoproteomics analysis of VEGF signaling in HUVECs revealed early signaling events and we believe that this analysis will serve as a starting point for the analysis of differential signaling across VEGF members toward the full elucidation of their role in the angiogenesis processes. Workflow for the identification of early phosphorylation events induced by VEGF-A-165 in HUVEC cells.

Molecular docking and dynamics studies for the identification of Nipah virus glycoprotein inhibitors from Indian medicinal plants.

The infection by Nipah Virus (NiV), a zoonotic paramyxovirus, is fatal and several outbreaks have been reported in humans in various countries. No effective vaccines or drugs are developed till date to control this infection. The NiV-Glycoprotein (NiV-G) is one of the essential proteins for viral entry by binding to the Ephrin-B receptors. The present study screens the potential phytocompounds that can target NiV-G and thereby inhibit the viral entry to human. Computer-aided virtual screening of 1426 phytocompounds from various medicinal plants was carried out to investigate their efficacy as potential therapeutics. Ribavirin, the currently used drug, was also docked to compare the docking score and intermolecular interactions between ligand and target protein. Further, molecular dynamics simulations and MM-PBSA binding free energy calculations were performed to understand the stability of the docked complexes. Radius of gyrations and Solvent Accessible Surface Area were also performed to evaluate the compactness and solvent behaviour of ligand-receptor complexes during the 100 ns simulation. Our analysis revealed that the alkaloid, Serpentinine, has the highest potency to block NiV-G with favourable binding.Communicated by Ramaswamy H. Sarma.

SARS-CoV-2 variants infectivity prediction and therapeutic peptide design using computational approaches.

The outbreak of severe acute respiratory coronavirus 2 (SARS-CoV-2) has created a public health emergency globally. SARS-CoV-2 enters the human cell through the binding of the spike protein to human angiotensin converting enzyme 2 (ACE2) receptor. Significant changes have been reported in the mutational landscape of SARS-CoV-2 in the receptor binding domain (RBD) of S protein, subsequent to evolution of the pandemic. The present study examines the correlation between the binding affinity of mutated S-proteins and the rate of viral infectivity. For this, the binding affinity of SARS-CoV and variants of SARS-CoV-2 towards ACE2 was computationally determined. Subsequently, the RBD mutations were classified on the basis of the number of strains identified with respect to each mutation and the resulting variation in the binding affinity was computationally examined. The molecular docking studies indicated a significant correlation between the Z-Rank score of mutated S proteins and the rate of infectivity, suitable for predicting SARS-CoV-2 infectivity. Accordingly, a 30-mer peptide was designed and the inhibitory properties were computationally analyzed. Single amino acid-wise mutation was performed subsequently to identify the peptide with the highest binding affinity. Molecular dynamics and free energy calculations were then performed to examine the stability of the peptide-protein complexes. Additionally, selected peptides were synthesized and screened using a colorimetric assay. Together, this study developed a model to predict the rate of infectivity of SARS-CoV-2 variants and propose a potential peptide that can be used as an inhibitor for the viral entry to human.Communicated by Ramaswamy H. Sarma.

Potential protease inhibitors and their combinations to block SARS-CoV-2.

COVID-19, which has emerged recently as a pandemic viral infection caused by SARS-coronavirus 2 has spread rapidly around the world, creating a public health emergency. The current situation demands an effective therapeutic strategy to control the disease using drugs that are approved, or by inventing new ones. The present study examines the possible repurposing of existing anti-viral protease inhibitor drugs. For this, the structural features of the viral spike protein, the substrate for host cell protease and main protease of the available SARS CoV-2 isolates were established by comparing with related viruses for which antiviral drugs are effective. The results showed 97% sequence similarity among SARS and SARS-CoV-2 main protease and has same cleavage site positions and ACE2 receptor binding region as in the SARS-CoV spike protein. Though both are N-glycosylated, unlike SARS-CoV, human SARS-CoV-2 S-protein was O-glycosylated as well. Molecular docking studies were done to explore the role of FDA approved protease inhibitors to control SARS-CoV-2 replication. The results indicated that, Ritonavir has the highest potency to block SARS-CoV-2 main protease and human TMPRSS2, a host cell factor that aids viral infection. Other drugs such as Indinavir and Atazanavir also showed favourable binding with Cathepsin B/L that helped viral fusion with the host cell membrane. Further molecular dynamics simulation and MM-PBSA binding free energy calculations confirmed the stability of protein-drug complexes. These results suggest that protease inhibitors particularly Ritonavir, either alone or in combination with other drugs such as Atazanavir, have the potential to treat COVID 19.Communicated by Ramaswamy H. Sarma.

Identification of Hub Genes and Key Pathways Associated with Anti-VEGF Resistant Glioblastoma Using Gene Expression Data Analysis.

Anti-VEGF therapy is considered to be a useful therapeutic approach in many tumors, but the low efficacy and drug resistance limit its therapeutic potential and promote tumor growth through alternative mechanisms. We reanalyzed the gene expression data of xenografts of tumors of bevacizumab-resistant glioblastoma multiforme (GBM) patients, using bioinformatics tools, to understand the molecular mechanisms of this resistance. An analysis of the gene set data from three generations of xenografts, identified as 646, 873 and 1220, differentially expressed genes (DEGs) in the first, fourth and ninth generations, respectively, of the anti-VEGF-resistant GBM cells. Gene Ontology (GO) and pathway enrichment analyses demonstrated that the DEGs were significantly enriched in biological processes such as angiogenesis, cell proliferation, cell migration, and apoptosis. The protein-protein interaction network and module analysis revealed 21 hub genes, which were enriched in cancer pathways, the cell cycle, the HIF1 signaling pathway, and microRNAs in cancer. The VEGF pathway analysis revealed nine upregulated (IL6, EGFR, VEGFA, SRC, CXCL8, PTGS2, IDH1, APP, and SQSTM1) and five downregulated hub genes (POLR2H, RPS3, UBA52, CCNB1, and UBE2C) linked with several of the VEGF signaling pathway components. The survival analysis showed that three upregulated hub genes (CXCL8, VEGFA, and IDH1) were associated with poor survival. The results predict that these hub genes associated with the GBM resistance to bevacizumab may be potential therapeutic targets or can be biomarkers of the anti-VEGF resistance of GBM.

Multiple Targets Directed Multiple Ligands: An In Silico and In Vitro Approach to Evaluating the Effect of Triphala on Angiogenesis.

Angiogenesis is critical in both physiological and pathological conditions and targeting angiogenesis is a promising strategy for the development of therapies against cancer; however, cells develop resistance to anti-angiogenic therapy, necessitating a more effective strategy. Natural medicines have been used in anti-cancer therapy for many years, but the mechanisms behind these have not generally been explored. Triphala churna (THL), an Indian ayurvedic herbal formulation made from the dried fruits of three medicinal plants, is used as a herbal drug for the treatment of various diseases, including cancer. THL contains over fifteen phytochemicals with different pharmacological effects, especially inhibition of tumor progression. In this study, we examined the effect of these compounds against different targets using docking and in vitro studies. Results showed that THL has a prediction efficacy of (-436.7), and it inhibited angiogenesis by blocking multiple components of the VEGF/VEGFR2 signaling pathway. The anti-angiogenic effect was mediated by the combined effect of the two top ranked phytochemicals, punicalagin (-424.8) and chebulagic acid (-414.8). The new approach developed in this study to determine the potential efficacy of herbal formulation could be a useful strategy to assess the efficacy of different herbal formulations.

VEGF-A/VEGFR2 signaling network in endothelial cells relevant to angiogenesis.

Vascular endothelial growth factor-A (VEGF-A) is essential for endothelial cell functions associated with angiogenesis. Signal transduction networks initiated by VEGFA/VEGFR2, the most prominent ligand-receptor complex in the VEGF system, leads to endothelial cell proliferation, migration, survival and new vessel formation involved in angiogenesis. Considering its biomedical importance, we have developed the first comprehensive map of endothelial cell-specific signaling events of VEGFA/VEGFR2 system pertaining to angiogenesis. Screening over 20,000 published research articles and following the post-translational modification (PTM) and site specificity of VEGFR2, we have documented 240 proteins and their diverse PTM-dependent reactions involved in VEGFA/VEGFR2 signal transduction. From the ligand-receptor complex, this map has been extended to the level of major transcriptionally regulated genes for which the signaling cascades leading to their transcription factors are reported. We believe that this map would serve as a novel platform for reference, integration, and representation and more significantly, the progressive analysis of dynamic features of VEGF signaling in endothelial cells including their cross-talks with other ligand-receptor systems involved in angiogenesis.