Mechanistic insights into sodium ion-mediated ligand binding affinity and modulation of 5-HT2B GPCR activity: implications for drug discovery and development.

PURPOSE: The G-protein coupled receptor (GPCR) family, implicated in neurological disorders and drug targets, includes the sensitive serotonin receptor subtype, 5-HT2B. The influence of sodium ions on ligand binding at the receptor's allosteric region is being increasingly studied for its impact on receptor structure. METHODS: High-throughput virtual screening of three libraries, specifically the Asinex-GPCR library, which contains 8,532 compounds and FDA-approved (2466 compounds) and investigational compounds (2731)) against the modeled receptor [4IB4-5HT2BRM] using the standard agonist/antagonist (Ergotamine/Methysergide), as previously selected from our studies based on ADMET profiling, and further on basis of binding free energy a single compound - dihydroergotamine is chosen. RESULTS: This compound displayed strong interactions with the conserved active site. Ions influence ligand binding, with stronger interactions (3-H-bonds and 1-π-bond around 3.35 Å) observed when an agonist and ions are present. Ions entry is guided by conserved motifs in helices III, IV, and VII, which regulate the receptor. Dihydroergotamine, the selected drug, showed binding variance based on ions presence/absence, affecting amino acid residues in these motifs. DCCM and PCA confirmed the stabilization of ligands, with a greater correlation (∼46.6%-PC1) observed with ions. Dihydroergotamine-modified interaction sites within the receptor necessary for activation, serving as a potential 5HT2BRM agonist. RDF analysis showed the sodium ions density around the active site during dihydroergotamine binding. CONCLUSION: Our study provides insights into sodium ion mobility's role in controlling ligand binding affinity in 5HT2BR, offering therapeutic development insights.

Designing the 5HT2BR structure and its modulation as a therapeutic target for repurposing approach in drug-resistant epilepsy.

The study intends to repurpose FDA drugs and investigate the mechanism of (5HT2BR) activation by comprehending inter-residue interactions. The 5HT2BR is a novel thread, and its role in reducing seizures in Dravet syndrome is emerging. The crystal structure (5HT2BR) is a chimera with mutations; hence 3D-structure is modeled (4IB4: 5HT2BRM). The structure is cross-validated to simulate the human receptor using enrichment analysis (ROC: 0.79) and SAVESv6.0. Virtual screening of 2456 approved drugs yielded the best hits that are subjected to MM/GBSA and molecular dynamic (MD) simulations. The 2 top drugs Cabergoline (-53.44 kcal/mol) and Methylergonovine (-40.42 kcal/mol), display strong binding affinity, and ADMET/SAR analysis also suggests their non-mutagenic or non-carcinogenic nature. Methylergonovine has a weaker binding affinity and lower potency than standards [Ergotamine (agonist) and Methysergide (antagonist)] due to its higher Ki (1.32 M) and Kd (6.44 ×10-8 M) values. Compared to standards, Cabergoline has moderate binding affinity and potency [Ki = 0.85 M and Kd = 5.53 × 10-8 M]. The top 2 drugs primarily interact with conserved residues (ASP135, LEU209, GLY221, ALA225, and THR140) as in agonists, unlike the antagonist. The top 2 drugs, upon binding to the 5HT2BRM, modify the helices VI, V, and III and shift the RMSD 2.48 Å and 3.07 Å. LEU209 forms a latch with residues 207-214 (forms a lid) in the 5HT2BRM receptor, which enhances agonist binding and prevents drug escape. Methylergonovine and Cabergoline interact more stongly with ALA225 than the antagonist. The post-MD analysis of Cabergoline suggests a better MM/GBSA value (-89.21 kcal/mol) than Methylergonovine (-63.54 kcal/mol). In this study, Cabergoline and Methylergonovine's agonistic mechanism and solid binding properties suggest their strong role in regulating the 5HT2BR and might target drug-resistant epilepsy.

An Atomic Level Investigation of Sodium Ions Regulating Agonist and Antagonist Binding in the Active Site of a Novel Target 5HT2BR Against Drug-Resistant Epilepsy.

The study investigates the movement of sodium ions inside the ligand-binding pocket of the class-A GPCR serotonin receptor (5HT2BR), a primary target for modern drugs. The available PDBs are mutant chimeras, so a 3D structure is modeled and validated by structural similarity (84.05%), Ramachandran favorable residues (93.01%), and clash score. Using MD simulations (500 ns), the ion active site is tracked in the presence and absence of ions and ligands. The ions enter the active site along helices III, VI, and VII, and the primary residue (ASP3.32) interacts with ions via H-bond (stronger- ~2.4 Å). The radial distribution function around ASP3.32 rises promptly at intermediate distances (2 Å < r < 4 Å), suggesting a higher probability of finding sodium ions at these distances. The ions stabilize the receptor at a better RMSD and promote stronger interactions (3-H-bonds, 1-π-bond~3.35 Å) with the agonist, and not the antagonist (no H-bond). Simulating unrestrained ligands further confirms this pattern, suggesting that ions might promote agonist binding but not be a prerequisite for antagonist action. The study highlights the mechanistic evaluation of sodium ions mobility in 5HT2BR modulation and ligand binding, showing potential in drug development.

Allosteric modulation of conserved motifs and helices in 5HT2BR: Advances drug discovery and therapeutic approach towards drug resistant epilepsy.

The 5HT2BR, class-A GPCR is a new target, and its significance for seizure reduction in Dravet syndrome is just now gaining interest, suggesting its specific role in epileptic seizure management. Homology modeling of human 5HT2BR (P41595), was performed using a template 4IB4, the modeled structure was cross-validated (stereo chemical hindrance, Ramachandran plot, enrichment analysis) to mimic a closer native structure. Virtual screening (8532 compounds), drug-likeliness, mutagenicity, and carcinogenicity profiling prioritized six compounds for molecular dynamics (500 ns), Rgyr, DCCM. The receptor's C-alpha fluctuation upon bound agonist (6.91 Å), known antagonist (7.03 Å), and LAS 52115629 (5.83 Å) binding varies, leading to receptor stabilization. The residues C-alpha side-chain in active site strongly interacts (hydrogen bonds) with bound agonist (100% interaction: ASP135), known antagonist (95%:ASP135), and LAS 52115629 (100%:ASP135). The Rgyr for receptor-ligand complex, LAS 52115629 (25.68 Å), lies close to bound agonist-Ergotamine, and DCCM analysis also shows strong positive correlations for LAS 52115629 as compared to known drugs. LAS 52115629 is less likely to cause toxicity than known drugs. The structural parameters in the modeled receptor's conserved motifs (DRY, PIF, NPY) were altered for receptor activation upon ligand-binding, which otherwise was in the in-activated state. The ligand (LAS 52115629)-binding further alters the helices-III, V, VI (G-protein bound), and VII, which form potential interacting sites with the receptor and are proven necessary for activating the receptor. Therefore, LAS 52115629 can act as a potential 5HT2BR agonist, targeting drug-resistant epilepsy.Communicated by Ramaswamy H. Sarma.

Terpenoid analogues as putative therapeutic agents towards glutathione peroxidase (GPX4) in neurodegenerative disorders: a dynamic computational approach.

Carvacrol, a monoterpenoid phenolic phytochemical, a potent antioxidant, and neuroprotective agent is an emerging neuroprotective agent for neurodegenerative diseases (NDDs). Considering scarce information on carvacrol analogues, we hypothesized an in silico investigation emphasizing their preferential binding towards glutathione peroxidase (GPX4) as a target across different species for evaluating through preclinical to clinical studies (2OBI and 6HN3 for Homo sapiens; 5L71 for Mus musculus). Enrichment analysis suggests that ROC (0.59) and AUC (0.61) values have higher sensitivity and significant number of ranked actives. Extra Precision (XP) of 59 compounds was conducted, followed by molecular dynamics and trajectory analysis. Top three hits were chosen for each target i.e., 101203408, 101419546, 59294 (2OBI); 101419546, 100938426, and 28092 (6HN3); and 12059, 52434, 335 (5L71) implying high docking score. 101419546 is common among 2OBI and 6HN3 targets, indicating a multi-target approach. Trajectory analysis of hits provides a permissible range of RMSD, RMSF, Rgyr (∼1.3-2 Å, ∼0.84-1.09 Å, ∼15.05-15.29 Å). Overlapped dynamically simulated 3D-structures of Apo and complexes display significant conformational changes in RMSD of the complexes (∼1.40-2.0 Å) in contrast to Apo (∼1.3-1.8 Å), suggesting structural stability and compactness of the complexes within 45-90 ns. DCCM and PCA analysis shows positive correlation and residual clustering among residues of complexes. The establishment of firm H-bonding, favorable aromaticity and ADMET profile makes them promising drugs across various GPX4 targets among the species. Studies considering the targets across different species aids in anticipating and discovering a common compound for future NDDs therapeutics from bench to bedside.Communicated by Ramaswamy H. Sarma.

Structure and dynamic simulation-based interactions of benzenoids, pyrroles and organooxygen compounds for effective targeting of GPX4 in ischemic stroke.

The discovery of a novel drug for ischemic stroke is plagued by expensive and unsuccessful outcomes. FDA-approved drugs could be a viable repurposing strategy for stroke therapy. Emerging evidence suggests the regulating role of Glutathione peroxidase (GPX4) in stroke and attracts as a potential target. To overcome limited therapeutic interventions, a drug repurposing in silico investigation of FDA-approved drugs is proposed for the GPX4 receptor in distinctive species (Homo sapiens and Mus musculus). The GPX4 UniProt wild type ids, that is, P36969 (Homo sapiens), P36970 (Rattus norvegicus) and O70325 (Mus musculus) are Swiss modelled, and resultant templates are 2OBI and 6HN3 for Homo sapiens, and 5L71 for Mus musculus with a sequence identity of ∼88%. Enrichment analysis reveals high sensitivity and ranked actives with ROC and AUC values of 0.59 and 0.61, respectively. Virtual screening at extra precision resulted hit Acarbosum, is similar between 2OBI and 6HN3, demonstrating a multiple-target specificity and Iopromide, targeting 2OBI. MD simulation at 100 ns following trajectory analysis provides RMSD (∼1.2-1.8Å), RMSF (∼1.6-2.7Å), Rgyr (∼15-15.6Å) depicting stabilisation of receptor-ligand complexes. Furthermore, average B-factor value of 2OBI, 6HN3 and 5L71 is 25Å, 24Å and 60Å with a defined resolution of 1.55Å, 1.01Å and 1.80Å, respectively, depicting the thermodynamic stability of the protein structures. The dynamic cross-correlation and principal component analysis of residual fluctuations reveal more positive correlation, high atomic displacements and greater residual clustering of residues from atomic coordinates. Therefore, Acarbosum, an FDA-approved drug, could act as a potential repurposing drug with a multi-target approach translating from preclinical to clinical stages.Communicated by Ramaswamy H. Sarma.

Integrative Expression, Survival Analysis and Cellular miR-2909 Molecular Interplay in MRN Complex Check Point Sensor Genes (MRN-CSG) Involved in Breast Cancer.

BACKGROUND: Breast cancer, an emerging global challenge, is evidenced by recent studies of miRNAs involvement in DNA repair gene variants (MRE11, RAD50, and NBN as checkpoint sensor genes (CSG) - MRN-CSG). The identification of various mutations in MRN-CSG and their interactions with miRNAs is still not understood. The emerging studies of miR-2909 involvement in other cancers led us to explore its role as molecular mechanistic marker in breast cancer. MATERIALS AND METHODS: The genomic and proteomic data of MRN-CSG of breast cancer patients (8426 samples) was evaluated to identify the mutation types linked with the patient's survival rate. Additionally, molecular, 3D-structural and functional analysis was performed to identify miR-2909 as regulator of MRN-CSG. RESULTS: The genomic and proteomic data analysis shows genetic alterations with majority of missense mutations [RAD50 (0.7%), MRE11 (1.5%), and NBN (11%)], though with highest MRE11 mRNA expression in invasive ductal breast carcinoma as compared to other breast cancer types. The Kaplan-Meier survival curves suggest higher survival rate for unaltered groups as compared to the altered group. Network analysis and disease association of miR-2909 and MRN-CSG shows strong interactions with other partners. The molecular hybridization between miR-2909-RAD50 and miR-2909-MRE11 complexes showed thermodynamically stable structures. Further, argonaute protein, involved in RNA silencing, docking studies with miR-MRE11-mRNA and miR-RAD50-mRNA hybridized complexes showed strong binding affinity. CONCLUSION: The results suggest that miR-2909 forms strong thermodynamically stable molecular hybridized complexes with MRE11 and RAD50 mRNAs which further strongly interacts with argonaute protein to show potential molecular mechanistic role in breast cancer.

Structural, molecular hybridization and network based identification of miR-373-3p and miR-520e-3p as regulators of NR4A2 human gene involved in neurodegeneration.

MicroRNAs (miRNAs) are short non-coding RNAs with a 22 nucleotide sequence length and docks to the 3'UTR/5'UTR of the gene to regulate their mRNA translation to play a vital role in neurodegenerative diseases. The Nuclear Receptor gene (NR4A2), a transcription factor, and a steroid-thyroid hormone retinoid receptor is involved in neural development, memory formation, dopaminergic neurotransmission, and cellular protection from inflammatory damage. Therefore, recognizing the miRNAs is essential to efficiently target the 3'UTR/5'UTR of the NR4A2 gene and regulate neurodegeneration. Highly stabilized top miRNA-mRNA hybridized structures, their homologs, and identification of the best structures based on their least free energy were evaluated using in silico techniques. The miR-gene, gene-gene network analysis, miR-disease association, and transcription factor binding sites were also investigated. Results suggest top 166 miRNAs targeting the NR4A2 mRNA, but with a total of 10 miRNAs bindings with 100% seed sequence identity (both at 3' and 5'UTR) at the same position on the NR4A2 mRNA region. The miR-373-3p and miR-520e-3p are considered the best candidate miRNAs hybridizing with high efficiency at both 3' and 5'UTR of NR4A2 mRNA. This could be due to the most significant seed sequence length complementary, supplementary pairing, and absence of non-canonical base pairs. Furthermore, the miR-gene network, target gene-gene interaction analysis, and miR-disease association provide an understanding of the molecular, cellular, and biological processes involved in various pathways regulated by four transcription factors (PPARG, ZNF740, NRF1, and RREB1). Therefore, miR-373-3p, 520e-3p, and four transcription factors can regulate the NR4A2 gene involved in the neurodegenerative process.

Screening and identification of phytochemical drug molecules against mutant BRCA1 receptor of breast cancer using computational approaches.

The American Cancer Society claims that breast cancer is the second most significant cause of cancer-related death, with over one million women diagnosed each year. Breast cancer linked to the BRCA1 gene has a significant risk of mortality and recurrence and is susceptible to alteration or over-expression, which can lead to hereditary breast cancer. Given the shortage of effective and possibly curative treatments for breast cancer, the present study combined molecular and computational analysis to find prospective phytochemical substances that can suppress the mutant gene (BRCA1) that causes the disease. Virtual screening and Molecular docking approaches are utilized to find probable phytochemicals from the ZINC database. The 3D structure of mutant BRCA1 protein with the id 3PXB was extracted from the NCBI-PDB. Top 10 phytochemical compounds shortlisted based on molecular docking score between - 11.6 and - 13.0. Following the ADMET properties, only three (ZINC000085490903 = - 12.50, ZINC000085490832 = - 12.44, and ZINC000070454071 = - 11.681) of the 10 selected compounds have drug-like properties. The molecular dynamic simulation study of the top three potential phytochemicals showed stabilized RMSD and RMSF values as compared to the APO form of the BRCA1 receptor. Further, trajectory analysis revealed that approximately similar radius of gyration score tends to the compactness of complex structure, and principal component and cross-correlation analysis suggest that the residues move in a strong correlation. Thermostability of the target complex (B-factor) provides information on the stable energy minimized structure. The findings suggest that the top three ligands show potential as breast cancer inhibitors.

An insight into the simulation directed understanding of the mechanism in SARS CoV-2 N-CTD, dimer integrity, and RNA-binding: Identifying potential antiviral inhibitors.

Coronavirus 2019 is a transmissible disease and has caused havoc throughout the world. The present study identifies the novel potential antiviral inhibitors against the nucleocapsid C-terminal domain that aids in RNA-binding and replication. A total of 485,629 compounds were screened, and MD was performed. The trajectory analysis (DCCM & PCA), structural integrity, and degree of compaction depicted the protein-ligand complex stability (PDB-PISA and Rgyr). Results obtained from screening shortlists 13 compounds possessing high Docking score. Further, seven compounds had a permissible RMSD limit (3 Å), with robust RMSF. Post-MD analysis of the top two compounds (204 and 502), DCCM & PCA analysis show a positive atomic displacements correlation among residues of active sites-dimer (Chain A and Chain B) & residual clustering. The ΔGint of RNA-bound (-83.5 kcal/mol) and drug-bound N-CTD-204 (-40.8 kcal/mol) and 502(-39.7 kcal/mol) as compared to Apo (-35.95 kcal/mol) suggests stabilization of protein, with less RNA-binding possibility. The Rgyr values depict the loss of compactness on RNA-binding when compared to the drug-bound N-CTD complex. Further, overlapping the protein complexes (0 ns and 100 ns) display significant changes in RMSD of the protein (204-2.07 Å and 502-1.89 Å) as compared to the Apo (1.72 Å) and RNA-bound form (1.76 Å), suggesting strong interaction for compound 204 as compared to 502. ADMET profiling indicates that these compounds can be used for further experiments (in vitro and pre-clinical). Compound 204 could be a promising candidate for targeting the N-protein-RNA assembly and viral replication.

Identification of homologous human miRNAs as antivirals towards COVID-19 genome.

The COVID-19 fatality rate is ~57% worldwide. The investigation of possible antiviral therapy using host microRNA (miRNA) to inhibit viral replication and transmission is the need of the hour. Computational techniques were used to predict the hairpin precursor miRNA (pre-miRNAs) of COVID-19 genome with high homology towards human (host) miRNA. Top 21 host miRNAs with >80% homology towards 18 viral pre miRNAs were identified. The Gibbs free energy (ΔG) between host miRNAs and viral pre-miRNAs hybridization resulted in the best 5 host miRNAs having the highest base-pair complementarity. miR-4476 had the strongest binding with viral pre-miRNA (ΔG = -21.8 kcal/mol) due to maximum base pairing in the seed sequence. Pre-miR-651 secondary structure was most stable due to the (1) least minimum free energy (ΔG = -24.4 kcal/mol), energy frequency, and noncanonical base pairing and (2) maximum number of stem base pairing and small loop size. Host miRNAs-viral mRNAs interaction can effectively inhibit viral transmission and replication. Furthermore, miRNAs gene network and gene-ontology studies indicate top 5 host miRNAs interaction with host genes involved in transmembrane-receptor signaling, cell migration, RNA splicing, nervous system formation, and tumor necrosis factor-mediated signaling in respiratory diseases. This study identifies host miRNA/virus pre-miRNAs strong interaction, structural stability, and their gene-network analysis provides strong evidence of host miRNAs as antiviral COVID-19 agents.