Precise tuning of bacterial translation initiation by non-equilibrium 5'-UTR unfolding observed in single mRNAs.

Noncoding, structured 5'-untranslated regions (5'-UTRs) of bacterial messenger RNAs (mRNAs) can control translation efficiency by forming structures that either recruit or repel the ribosome. Here we exploit a 5'-UTR embedded preQ1-sensing, pseudoknotted translational riboswitch to probe how binding of a small ligand controls recruitment of the bacterial ribosome to the partially overlapping Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5'-UTR during mRNA accommodation into the mRNA binding cleft. Ligand binding to the riboswitch stabilizes the structure to both antagonize 30S recruitment and accelerate 30S dissociation. Proximity of the 5'-UTR and stability of the SD:anti-SD interaction both play important roles in modulating the initial 30S-mRNA interaction. Finally, depletion of small ribosomal subunit protein S1, known to help resolve structured 5'-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5'-UTR during accommodation provides a mechanistic understanding of how translation efficiency is governed by riboswitches and other dynamic structure motifs embedded upstream of the translation initiation site of bacterial mRNAs.

Design and development of novel potential inhibitors of the human USP21 enzyme using a pharmacophore-based virtual screening technique.

An overexpression and increase have been observed in the concentration and activity of the ubiquitin-specific protease 21 (USP21) enzyme in many cancers, necessitating the need for the development of new inhibitor drugs against the same. The current study attempts to discover one such novel potential inhibitor of USP21 by the application of various bioinformatics techniques which include molecular modeling, pharmacophore mapping, pharmacophore-based virtual screening, molecular docking, and ADMET prediction followed by molecular dynamics simulations. Following this inverted funnel-like approach, we finally ended up with one ligand-ZINC02422616 which displays a very high binding affinity toward the USP21 domain. This ligand contains all the pharmacophoric features displayed by the compounds that are potential inhibitors of the USP21 domain. Moreover, it shows a favorable pharmacokinetic, pharmacodynamic, and ADMET profile, along with strong hydrophobic interaction and hydrogen bonding with the domain. Simulation studies showed that the complex remains stable over time, with the bound protein displaying a more constrained motion in the conformational space compared to the unbound form. The ligand showed a highly favorable free energy landscape/surface, forming several energy minima's in contrast to the unbound domain in which most conformations occupied a relatively higher energy state. Moreover, the ligand also displayed a Kd of 422.8 nM and a free energy of binding ΔG of -8.6 kcal/mol, both of which indicate a very high affinity toward the target domain. This potential drug candidate can then be used as a viable treatment method for many types of cancers caused by USP21.

An in-silico scaffold- hopping approach to design novel inhibitors against gp130: A potential therapeutic application in cancer and Covid-19.

An upregulation of the gp130-signalling cascade has been reported in multiple cancers, making gp130 an attractive target for the development of anticancer drugs. An inverted-funnel-like approach was utilised along with various structure-based drug designing strategies to discover and optimise novel potential inhibitors of gp130. The study resulted in the discovery of 2 ligands- 435 and 510, both of which exhibit a very high-binding affinity towards the gp130 D1 domain which controls cytokine recognition and interaction thus being involved in complexation. The two resulting complexes remained stable over time with the ligands maintaining a steady interaction with the target. This inference is drawn from their RMSD, Rg, SASA and RMSF analysis. We also tested the protein folding patterns based on their principal component analysis, energy of surface and landscape. The leads also displayed a more favourable ADMET profile than their parent compounds. The two lead candidates show a better therapeutic profile in comparison to the two existing drugs- bazedoxifene and raloxifene. Both these potential leads can be addressed for their activity in-vitro and can be used as a potential anti-cancer treatment as well as to combat Covid-19 related cytokine storm.

Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination.

Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by 'snap-locking' of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

Molecular Design of Novel Inhibitor by Targeting IL-6Rα using Combined Pharmacophore and Experimentally Verified Plant Products with Scaffold-Hopping Techniques: A Dual Therapeutic Strategy for COVID-19 and Cancer.

The IL-6/IL-6R/gp130 complex serves as a significant indicator of cytokine release syndrome in COVID-19 and chronic inflammation, increasing the risk of cancer. Therefore, we identified IL-6Rα as a potential target to block gp130 interaction. Notably, there has been no reception of approval for an orally available drug to serve this purpose, to date. In this study, we targeted IL-6Rα to inhibit IL-6Rα/gp130 interaction. The selection of the lead candidate L821 involved the amalgamation of three drug discovery approaches. This library was screened employing tertiary structure-based pharmacophore models followed by molecular docking models, scaffold-hopping, MM/PBSA as well as MM/GBSA analysis, and assessments of pKi and ADMET properties. After evaluating the binding interactions with key amino acids, 15 potential ligands were chosen, with the top ligand undergoing further investigation by means of molecular dynamics simulations. Considering the stability of the complexes, the strong interactions observed between ligand and residues of IL-6Rα/gp130, and the favorable binding free energy calculations, L821 emerged as the prime candidate for inhibiting IL-6Rα. Notably, L821 exhibited a docking-based binding affinity of -9.5 kcal/mol. Our study presents L821 as a promising inhibitor for future in vitro analysis, potentially combatting SARS-CoV-2-related cytokine storms and serving as an oncogenic drug therapy.

In silico screening and exploration into phenotypic alterations of deleterious oncogenic single nucleotide polymorphisms in HSPB1 gene.

A small heat shock protein, HSP27, encoded by HSPB1 gene strongly favors survival, proliferation and metastasis of cancer cells and its expression is dependent on post-translational modifications like phosphorylation. This study performed an extensive in silico screening of 20 deleterious non-synonymous SNPs in the coding region of HSPB1 gene, among which four were identified to be cancer associated. The SNP variant I181S introduced a new phosphorylation site in position 181, which might elevate the protein's activation potential. Emergence of other post-translational modifications was also observed in SNP variants: L144P and E130K.Significant conformational changes were observed in I181S, L144P and E130K SNP variants with respect to wild-type HSP27. These SNPs appear in one among 105 individuals, making them more susceptible towards cancer. This study would therefore, instigate development of novel biomarkers for cancer risk detection and would provide a detailed understanding towards varied cancer susceptibility of human population.

Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World.

The emergence of single-molecule (SM) fluorescence techniques has opened up a vast new toolbox for exploring the molecular basis of life. The ability to monitor individual biomolecules in real time enables complex, dynamic folding pathways to be interrogated without the averaging effect of ensemble measurements. In parallel, modern biology has been revolutionized by our emerging understanding of the many functions of RNA. In this comprehensive review, we survey SM fluorescence approaches and discuss how the application of these tools to RNA and RNA-containing macromolecular complexes in vitro has yielded significant insights into the underlying biology. Topics covered include the three-dimensional folding landscapes of a plethora of isolated RNA molecules, their assembly and interactions in RNA-protein complexes, and the relation of these properties to their biological functions. In all of these examples, the use of SM fluorescence methods has revealed critical information beyond the reach of ensemble averages.

Kinetics coming into focus: single-molecule microscopy of riboswitch dynamics.

Riboswitches are dynamic RNA motifs that are mostly embedded in the 5'-untranslated regions of bacterial mRNAs, where they regulate gene expression transcriptionally or translationally by undergoing conformational changes upon binding of a small metabolite or ion. Due to the small size of typical ligands, relatively little free energy is available from ligand binding to overcome the often high energetic barrier of reshaping RNA structure. Instead, most riboswitches appear to take advantage of the directional and hierarchical folding of RNA by employing the ligand as a structural 'linchpin' to adjust the kinetic partitioning between alternate folds. In this model, even small, local structural and kinetic effects of ligand binding can cascade into global RNA conformational changes affecting gene expression. Single-molecule (SM) microscopy tools are uniquely suited to study such kinetically controlled RNA folding since they avoid the ensemble averaging of bulk techniques that loses sight of unsynchronized, transient, and/or multi-state kinetic behavior. This review summarizes how SM methods have begun to unravel riboswitch-mediated gene regulation.

Quantum Phase Transition in Few-Layer NbSe_{2} Probed through Quantized Conductance Fluctuations.

We present the first observation of dynamically modulated quantum phase transition between two distinct charge density wave (CDW) phases in two-dimensional 2H-NbSe_{2}. There is recent spectroscopic evidence for the presence of these two quantum phases, but its evidence in bulk measurements remained elusive. We studied suspended, ultrathin 2H-NbSe_{2} devices fabricated on piezoelectric substrates-with tunable flakes thickness, disorder level, and strain. We find a surprising evolution of the conductance fluctuation spectra across the CDW temperature: the conductance fluctuates between two precise values, separated by a quantum of conductance. These quantized fluctuations disappear for disordered and on-substrate devices. With the help of mean-field calculations, these observations can be explained as to arise from dynamical phase transition between the two CDW states. To affirm this idea, we vary the lateral strain across the device via piezoelectric medium and map out the phase diagram near the quantum critical point. The results resolve a long-standing mystery of the anomalously large spectroscopic gap in NbSe_{2}.

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding.

Human telomeres terminate with a single-stranded 3' G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K(+), POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na(+), in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.

RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP.

Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.

G-Quadruplex-Enabling Sequence within the Human Tyrosine Hydroxylase Promoter Differentially Regulates Transcription.

G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3'-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.

Molecular level biodegradation of phenol and its derivatives through dmp operon of Pseudomonas putida: A bio-molecular modeling and docking analysis.

Participation of Pseudomonas putida-derived methyl phenol (dmp) operon and DmpR protein in the biodegradation of phenol or other harmful, organic, toxic pollutants was investigated at a molecular level. Documentation documents that P. putida has DmpR protein which positively regulates dmp operon in the presence of inducers; like phenols. From the operon, phenol hydroxylase encoded by dmpN gene, participates in degrading phenols after dmp operon is expressed. For the purpose, the 3-D models of the four domains from DmpR protein and of the DNA sequences from the two Upstream Activation Sequences (UAS) present at the promoter region of the operon were demonstrated using discrete molecular modeling techniques. The best modeled structures satisfying their stereo-chemical properties were selected in each of the cases. To stabilize the individual structures, energy optimization was performed. In the presence of inducers, probable interactions among domains and then the two independent DNA structures with the fourth domain were perused by manifold molecular docking simulations. The complex structures were made to be stable by minimizing their overall energy. Responsible amino acid residues, nucleotide bases and binding patterns for the biodegradation, were examined. In the presence of the inducers, the biodegradation process is initiated by the interaction of phe50 from the first protein domain with the inducers. Only after the interaction of the last domain with the DNA sequences individually, the operon is expressed. This novel residue level study is paramount for initiating transcription in the operon; thereby leading to expression of phenol hydroxylase followed by phenol biodegradation.

Mutations in LIFR rewire the JAK/STAT signaling pathway: A study unveiling mechanistic details of Stüve-Wiedemann syndrome.

Stüve-Wiedemann syndrome (SWS), a rare autosomal recessive disorder, characterized by diminutive size, curvature of the elongated bones, bent fingers, episodes of heightened body temperature, respiratory distress or periods of breath-holding, and challenges with feeding, especially causes fatality in infants. SWS is an outcome of potential missense mutations in the leukemia inhibitory factor receptor gene reflected as numerous amino acid mutations at protein level. Employing in silico tools and techniques like mutational screening with Pred_MutHTP, I-Mutant2.0, PANTHER.db, PolyPhen, to classify mutations as deleterious/destabilizing, in conjunction with experimental data analysis, P136A and S279P emerged as 'effect'-causing mutations. Pre-existing knowledge suggests, SWS progression is effectuated conformationally altered and dysfunctional LIFR, unable to bind to LIF and further form the LIF/LIFR/gp130 signalling complex. To gain functional insights into the effect of the said mutations on the wild type protein, an all-atom, explicit, solvent molecular dynamics simulation was performed following docking approaches. Consequently, referring to the RMSD, RMSF, protein dynamic network analysis, energy landscape plots and domain motion analysis, it was revealed that unbound LIFR_WT was more prone to LIF binding as usual whereas the mutants exhibited considerable domain closure to inhibit LIF binding. We conducted binding affinity analysis via MM/GBSA and dissociation constant estimation after LIFR-LIF docking and found the WT_complex to be more stable and compact as a whole when compared to the flexible mutant complexes thus being associated with SWS. Our study offers a route for understanding molecular level implications upon LIFR mutations which opens an avenue for therapeutic interventions.

A rhythmically pulsing leaf-spring DNA-origami nanoengine that drives a passive follower.

Molecular engineering seeks to create functional entities for modular use in the bottom-up design of nanoassemblies that can perform complex tasks. Such systems require fuel-consuming nanomotors that can actively drive downstream passive followers. Most artificial molecular motors are driven by Brownian motion, in which, with few exceptions, the generated forces are non-directed and insufficient for efficient transfer to passive second-level components. Consequently, efficient chemical-fuel-driven nanoscale driver-follower systems have not yet been realized. Here we present a DNA nanomachine (70 nm × 70 nm × 12 nm) driven by the chemical energy of DNA-templated RNA-transcription-consuming nucleoside triphosphates as fuel to generate a rhythmic pulsating motion of two rigid DNA-origami arms. Furthermore, we demonstrate actuation control and the simple coupling of the active nanomachine with a passive follower, to which it then transmits its motion, forming a true driver-follower pair.

RPA-mediated unfolding of systematically varying G-quadruplex structures.

G-quadruplex (GQ) is a noncanonical nucleic acid structure that is formed by guanine rich sequences. Unless it is destabilized by proteins such as replication protein A (RPA), GQ could interfere with DNA metabolic functions, such as replication or repair. We studied RPA-mediated GQ unfolding using single-molecule FRET on two groups of GQ structures that have different loop lengths and different numbers of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Those with shorter loops (less than three nucleotides long) or a greater number of layers (more than three layers) maintained a significant folded population even at physiological RPA concentration (≈1 µM), raising the possibility of physiological viability of such GQ structures. Finally, we measured the transition time between the start and end of the RPA-mediated GQ unfolding process to be 0.35 ± 0.10 s for all GQ constructs we studied, despite significant differences in their steady-state stabilities. We propose a two-step RPA-mediated GQ unfolding mechanism that is consistent with our observations.

An in-silico study to understand the effect of lineage diversity on cold shock response: unveiling protein-RNA interactions among paralogous CSPs of E. coli.

Cold shock proteins (CSPs) are small, cytoplasmic, ubiquitous and acidic proteins. They have a single nucleic acid-binding domain and pose as "RNA chaperones" by binding to ssRNA in a low sequence specificity and cooperative manner. They are found in a family of nine homologous CSPs in E. coli. CspA, CspB, CspG and CspI are immensely cold inducible, CspE and CspC are consistently released at usual physiological temperatures and CspD is also induced under nutrient stress. The paralogous protein pairs CSPA/CSPB, CSPC/CSPE, CSPG/CSPI and CSPF/CSPH were first identified. The eight proteins were subjected to molecular modelling and simulation to obtain the most stable conformation in correspondence to their equilibrated RMSD and RMSF graph. The results were compared and it was observed that CSPB, CSPE, CSPF and CSPI were more stable than their paralogous partner conforming to their near equilibrated RMSD curve and low fluctuating RMSF graph. The paralogous proteins were docked with ssRNA and simultaneously binding affinity, interaction types, electrostatic surface potential, hydrophobicity, conformational analysis and SASA were calculated to minutely study and understand the molecular mechanism initiated by these proteins. It was found that CSPB, CSPC, CSPH and CSPI displayed higher affinity towards ssRNA than their paralogous partner. The results further corroborated with ΔGmmgbsa and ΔGfold energy. Between the paralogous pairs CSPC, CSPH and CSPI exhibited higher binding free energy than their partner. Further, CSPB, CSPC and CSPI exhibited higher folding free energy than their paralogous pair. CSPH exhibited highest ΔGmmgbsa of - 522.2 kcal/mol and lowest was displayed by CSPG of around - 309.3 kcal/mol. Highest number of mutations were recognised in CSPF/CSPH and CSPG/CSPI pair. Difference in interaction pattern was maximum in CSPF/CSPH owing to their high number of non-synonymous substitutions. Maximum difference in surface electrostatic potential was observed in case of CSPA, CSPG and CSPF. This research work emphasizes on discerning the molecular mechanism initiated by these proteins with a structural, mutational and functional approach. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03656-2.

Traversing DNA-Protein Interactions Between Mesophilic and Thermophilic Bacteria: Implications from Their Cold Shock Response.

Cold shock proteins (CSPs) are small, acidic proteins which contain a conserved nucleic acid-binding domain. These perform mRNA translation acting as "RNA chaperones" when triggered by low temperatures initiating their cold shock response. CSP- RNA interactions have been predominantly studied. Our focus will be CSP-DNA interaction examination, to analyse the diverse interaction patterns such as electrostatic, hydrogen and hydrophobic bonding in both thermophilic and mesophilic bacteria. The differences in the molecular mechanism of these contrasting bacterial proteins are studied. Computational techniques such as modelling, energy refinement, simulation and docking were operated to obtain data for comparative analysis. The thermostability factors which stabilise a thermophilic bacterium and their effect on their molecular regulation is investigated. Conformational deviation, atomic residual fluctuations, binding affinity, Electrostatic energy and Solvent Accessibility energy were determined during stimulation along with their conformational study. The study revealed that mesophilic bacteria E. coli CSP have higher binding affinity to DNA than thermophilic G. stearothermophilus. This was further evident by low conformation deviation and atomic fluctuations during simulation.

A comprehensive analysis of NAC gene family in Oryza sativa japonica: a structural and functional genomics approach.

NAC gene family regulates diverse aspects of plant growth and developmental processes. The NAC DNA binding domains together with cis-acting elements play inter-related roles in regulating gene expression. In this study, an in silico approach for genome wide analysis of NAC gene in Oryza sativa japonica lead to an identification of 11 NAC genes, distributed over 12 chromosomes. A detailed analysis of phylogenetic relationship, motifs, gene structure, duplication patterns, positive-selection pressure and cis-elements of 11 OsNAC genes were performed. Three pairs of NAC genes with a high degree of homology in terminal nodes were observed and were inferred to be paralogous pairs. One conserved NAC domain was analyzed in all the NAC proteins. Only one gene was studied to be intronless and the majority had 2 introns. Segmental gene duplication pattern was predominant in 11 NAC genes. Ka/Ks ratio of 3 pairs of segmentally duplicated gene was substantially lower than 1, suggesting that the OsNAC sequences are under strong purifying selection pressure. NAC74 and NAC71 gene showed the maximum responsiveness for several factors. The paralogous genes, NAC2 and NAC67 were found to have maximum mya values, respectively. They showed maximum difference amongst themselves in all the categories of responsiveness. Responsiveness towards abscisic acid was observed to be absent in NAC67, but present in NAC2, while responsiveness to meristem inducibility was observed to remain absent in NAC2 but present in NAC67. These results would provide a platform for the future identification and analysis of NAC genes in Oryza sativa japonica.Communicated by Ramaswamy H. Sarma.

An in-silico receptor-pharmacophore based multistep molecular docking and simulation study to evaluate the inhibitory potentials against NS1 of DENV-2.

DENV-2 strain is the most fatal and infectious of the five dengue virus serotypes. The non-structural protein NS1 encoded by its genome is the most significant protein required for viral pathogenesis and replication inside the host body. Thus, targeting the NS1 protein and designing an inhibitor to limit its stability and secretion is a propitious attempt in our fight against dengue. Four novel inhibitors are designed to target the conserved cysteine residues (C55, C313, C316, and C329) and glycosylation sites (N130 and N207) of the NS1 protein in an attempt to halt the spread of the dengue infection in the host body altogether. Numerous computer-aided drug designing techniques including molecular docking, molecular dynamics simulation, virtual screening, principal component analysis, and dynamic cross-correlation matrix were employed to determine the structural and functional activity of the NS1-inhibitor complexes. From our analysis, it was evident that the extent of structural and atomic level fluctuations of the ligand-bound protein exhibited a declining trend in contrast to unbound protein which was prominently noticeable through the RMSD, RMSF, Rg, and SASA graphs. The ADMET analysis of the four ligands revealed a promising pharmacokinetics and pharmacodynamic profile, along with good bioavailability and toxicity properties. The proposed drugs when bound to the targeted cavities resulted in stable conformations in comparison to their unbound state, implying they have good affinity promising effective drug action. Thus, they can be tested in vitro and used as potential anti-dengue drugs.Communicated by Ramaswamy H. Sarma.