A lesson for the maestro of the replication fork: Targeting the protein-binding interface of proliferating cell nuclear antigen for anticancer therapy.

The proliferating cell nuclear antigen (PCNA) has emerged as a promising candidate for the development of novel cancer therapeutics. PCNA is a nononcogenic mediator of DNA replication that regulates a diverse range of cellular functions and pathways through a comprehensive list of protein-protein interactions. The hydrophobic binding pocket on PCNA offers an opportunity for the development of inhibitors to target various types of cancers and modulate protein-protein interactions. In the present study, we explored the binding modes and affinity of molecule I1 (standard molecule) with the previously suggested dimer interface pocket and the hydrophobic pocket present on the frontal side of the PCNA monomer. We also identified potential lead molecules from the library of in-house synthesized 3-methylenisoindolin-1-one based molecules to inhibit the protein-protein interactions of PCNA. Our results were based on robust computational methods, including molecular docking, conventional, steered, and umbrella sampling molecular dynamics simulations. Our results suggested that the standard inhibitor I1 interacts with the hydrophobic pocket of PCNA with a higher affinity than the previously suggested binding site. Also, the proposed molecules showed better or comparable binding free energies as calculated by the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach and further validated by enhanced umbrella sampling simulations. In vitro and in vivo methods could test the computationally suggested molecules for advancement in the drug discovery pipeline.

Identification of acridinedione scaffolds as potential inhibitor of DENV-2 C protein: An in silico strategy to combat dengue.

Dengue is a prominent viral disease transmitted by mosquitoes to humans that affects mainly tropical and subtropical countries worldwide. The global spread of dengue virus (DENV) is mainly occurred by Aedes aegypti and Aedes albopictus mosquitoes. The dengue virus serotypes-2 (DENV-2) is a widely prevalent serotype of DENV, that causes the hemorrhagic fever and bleeding in the mucosa, which can be fatal. In the life cycle of DENV-2, a structural capsid (DENV-2 C) protein forms the nucleocapsid assembly and bind to the viral progeny RNA. For DENV-2 maturation, the nucleocapsid is a vital component. We used virtual ligand screening to filter out the best in-house synthesized acridinedione analogs (DSPD molecules) that could efficiently bind to DENV-2 C protein. The molecular docking and dynamics simulations studies were performed to analyze the effect of DSPD molecules on DENV-2 C protein after binding. Our findings showed that DSPD molecules strongly interacted with DENV-2 C protein, as evident from molecular interactions and several time-dependent molecular dynamics-driven analyses. Moreover, this study was also supported by the thermodynamic binding free energy and steered molecular dynamics simulations. Therefore, we intend to suggest that the DSPD3 molecule could be used as a potential therapeutic molecule against dengue complications as compared to the cocrystallized inhibitor ST-148. However, further studies are required to demonstrate the ability of DSPD3 to induce DENV-2 C tetramer formation.

Inhibition of nonstructural protein 15 of SARS-CoV-2 by golden spice: A computational insight.

The quick widespread of the coronavirus and speedy upsurge in the tally of cases demand the fast development of effective drugs. The uridine-directed endoribonuclease activity of nonstructural protein 15 (Nsp15) of the coronavirus is responsible for the invasion of the host immune system. Therefore, developing potential inhibitors against Nsp15 is a promising strategy. In this concern, the in silico approach can play a significant role, as it is fast and cost-effective in comparison to the trial and error approaches of experimental investigations. In this study, six turmeric derivatives (curcuminoids) were chosen for in silico analysis. The molecular interactions, pharmacokinetics, and drug-likeness of all the curcuminoids were measured. Further, the stability of Nsp15-curcuminoids complexes was appraised by employing molecular dynamics (MD) simulations and MM-PBSA approaches. All the molecules were affirmed to have strong interactions and pharmacokinetic profile. The MD simulations data stated that the Nsp15-curcuminoids complexes were stable during simulations. All the curcuminoids showed stable and high binding affinity, and these curcuminoids could be admitted as potential modulators for Nsp15 inhibition.

Computational targeting of allosteric site of MEK1 by quinoline-based molecules.

MEK1 is an attractive target due to its role in selective extracellular-signal-regulated kinase phosphorylation, which plays a pivotal role in regulating cell proliferation. Another benefit of targeting the MEK protein is its unique hydrophobic pocket that can accommodate highly selective allosteric inhibitors. To date, various MEK1 inhibitors have reached clinical trials against several cancers, but they were discarded due to their severe toxicity and low efficacy. Thus, the development of allosteric inhibitors for MEK1 is the demand of the hour. In this in-silico study, molecular docking, long-term molecular dynamics (5 µs), and molecular mechanics Poisson-Boltzmann surface area analysis were undertaken to address the potential of quinolines as allosteric inhibitors. We selected four reference MEK1 inhibitors for the comparative analysis. The drug-likeness and toxicity of these molecules were also examined based on their ADMET and Toxicity Prediction by Komputer Assisted Technology profiles. The outcome of the analysis revealed that the quinolines (4m, 4o, 4s, and 4n) exhibited better stability and binding affinity while being nontoxic compared to reference inhibitors. We have reached the conclusion that these quinoline molecules could be checked by experimental studies to validate their use as allosteric inhibitors against MEK1.

Discovery and in silico evaluation of aminoarylbenzosuberene molecules as novel checkpoint kinase 1 inhibitor determinants.

Checkpoint kinase 1 (CHK1) is an essential kinase with a critical function in cell cycle arrest. Several potent inhibitors targeting CHK1 have been published, but most of them have failed in clinical trials. Acknowledging the emerging consequence of CHK1 inhibitors in medication of cancer, there is a demand for widening the chemical range of CHK1 inhibitors. In this research, we considered a set of in-house plant based semi-synthetic aminoarylbenzosuberene molecules as potential CHK1 inhibitors. Based on a combined computational research that consolidates molecular docking and binding free energy computations we recognized the crucial determinants for their receptor binding. The drug likeness of these molecules were also scrutinized based on their toxicity and bioavailibilty profile. The computational strategy indicates that the Bch10 could be regarded as a potential CHK1 inhibitor in comparison with top five co-crystallize molecules. Bch10 signifies a promising outlet for the development of potent inhibitors for CHK1.

Plant-based analogues identified as potential inhibitor against tobacco mosaic virus: A biosimulation approach.

Benzosuberene compounds with a pyrrolone group adhered to it are compounds extracted from the oils of Cedrus deodara plant, that bear inhibitory capabilities. Tobacco mosaic virus is known to affect crop production every year. The currently known inhibitors against TMV have a weak inhibition effect and also tend to be toxic towards non-target living organisms as well as the environment. Thus, the requirement of non-toxic potent inhibitors is the need of the hour, which led us to test our benzosuberene molecules on the binding site of TMV and check their affinity as well as stability. The non-toxic nature of these molecules has already been experimentally established. Through in-silico analysis involving docking and simulation experiments, we compared the interaction pattern of these ligand molecules with the already present inhibitors. Our investigation proved that the reported ligands (ligands 3, 7, 9, and 17 obtained -177.103, -228.632, -184.134, and - 188.075 kJ/mol binding energies, respectively) interacted with the binding site of TMV much efficiently than the known inhibitors (Ribavirin and Zhao et al. 2020 obtained 121.561 and - 221.393 kJ/mol binding energies, respectively). Moreover, they acquired a stable conformation inside the binding pocket, where a higher number of binding site residues contributed towards interaction. Thus, their structural framework can be optimized for the exploration of their antiviral properties to develop potent botanical viricides against plant virus infection.

Structural changes induced by substitution of amino acid 129 in the coat protein of Cucumber mosaic virus.

Cucumber mosaic virus infection leads to mosaic symptoms on a broad range of crop plants. Mutation at positions 129 in the coat protein of virus causes alterations in the severity of symptoms caused by the viral infection. In our investigation, we performed long term molecular dynamics simulations to elucidate the effect of different amino acid substitutes (infectious and non-infectious) at position 129 in the coat protein of Cucumber mosaic virus using various structural parameters. We found that the contagious mutants displayed more flexibility at loops ßE-αEF (129-136) and ßF-ßG loop (155-163) as compared to the non-infectious and native structures. This specific study at the atomic level yields innovative ideas for designing new therapeutic agents against the pathogen, which would further pave the path for researchers to control this devastating plant virus.

Elimination of bitter-off taste of stevioside through structure modification and computational interventions.

Stevioside is a natural non-caloric sweetener obtained from Stevia rebaudiana Bertoni plant. The major challenge in the commercialization of stevioside as a natural sweetener is its bitter-off taste. In this study, we prepared molecular models of potential taste receptors of stevioside, both sweet and bitter. With appropriate modifications on the stevioside backbone, we performed molecular docking of prepared ligands with both sweet and bitter taste receptors. Based on binding energy, we found that one of the potential substituents, Kamiya-8, shows a good affinity towards sweet taste and a weak affinity for bitter receptors. Further, we selected Kamiya-8 for molecular dynamics simulations to improve the prediction of binding energy and to check the binding strength of Kamiya-8 with taste receptors. Moreover, we also performed MM-PBSA calculation for calculating the end state free energies of molecules in solvent and found that Kamiya-8 gives a 2-fold effect as it interacts with sweet receptors (T1R2, T1R3) with lowest binding energy conformation (-285.265 kcal/mol, -571.481 kcal/mol). Secondly, it gives high binding energy (-273.319 kcal/mol, -355.500 kcal/mol) with bitter taste receptors (T2R4, T2R14) as compared to stevioside. Based on this study, we found that Kamiya-8 can be the potential substituent that can improve the palatability of stevioside.

A comparative study on inclusion complex formation between formononetin and ß-cyclodextrin derivatives through multiscale classical and umbrella sampling simulations.

Formononetin, a naturally occurring isoflavone exhibits a wide range of therapeutic applications including antioxidant, anti-tumor, antiviral, anti-diabetic and neuroprotective activities. However, the low hydro-solubility of formononetin has limited its prospective use in cosmetic, neutraceutical and pharmaceutical industries. Cyclodextrins (CDs), especially ß-CD and its derivatives have emerged as promising agents to improve the water solubility of poorly hydrosoluble compounds by the formation of inclusion complexes. We employed multiscale (1000 ns) explicit solvent and umbrella sampling molecular dynamics (MD) simulations to study the interactions and thermodynamic parameters of inclusion complex formation between formononetin and five most commonly used ß-CD derivatives. Classical MD simulations revealed two possible binding conformations of formononetin inside the central cavity of hydroxypropyl-ß-CD (HP-ß-CD), randomly methylated-ß-CD (ME-ß-CD), and sulfobutylether-ß-CD (SBE-ß-CD). The binding conformation with the benzopyrone ring of formononetin inside the central cavity of ß-CD derivatives was more frequent than the phenyl group occupying the hydrophobic cavity. These interactions were supported by a variety of non-bonded contacts including hydrogen bonds, pi-lone pair, pi-sigma, and pi-alkyl interactions. Formononetin showed favorable end-state MD-driven thermodynamic binding free energies with all the selected ß-CD derivatives, except succinyl-ß-CD (S-ß-CD). Furthermore, umbrella sampling simulations were used to investigate the interactions and thermodynamic parameters of the host-guest inclusion complexes. The SBE-ß-CD/formononetin inclusion complex showed the lowest binding energy signifying the highest affinity among all the selected host-guest inclusion complexes. Our study could be used as a standard for analyzing and comparing the ability of different ß-CD derivatives to enhance the hydro-solubility of poorly soluble molecules.

Structure restoration and aggregate inhibition of V30M mutant transthyretin protein by potential quinoline molecules.

Transthyretin (TTR) is a tetrameric protein found in human plasma and cerebrospinal fluid that functions as a transporter of thyroxine (T4) and retinol. A mutation resulting in the substitution of valine to methionine at position 30 (V30M) is the most common mutation that destabilizes the tetramer structure of TTR protein resulting in a fatal neuropathy known as TTR amyloidosis. The V30M TTR-induced neuropathy can be inhibited through stabilization of the TTR tetramer by the binding of small molecules. We accessed the potential of in-house synthesized quinoline molecules to stabilize the V30M TTR structure and analyzed the impact of protein-ligand interactions through molecular docking, molecular dynamics (MD) simulations, steered MD, and umbrella sampling simulations. This study revealed that the binding of quinoline molecules reverted back the structural changes including the residual flexibility, changes in secondary structural elements, and also restored the alterations in the electrostatic surface potential induced by the V30M mutation. Further, the top-most 4G and 4R molecules were compared with an FDA-approved drug (Tafamidis) and a reference quinoline molecule 14C. Here, we intend to suggest that the quinoline molecules could revert the structural changes, cease tetramer dissociation, prevent abnormal oligomerization and therefore could be developed as an effective therapeutics against TTR amyloidosis.

Dispersion-corrected DFT calculations and umbrella sampling simulations to investigate stability of Chrysin-cyclodextrin inclusion complexes.

The study of inclusion complexes of Chrysin (ChR) with three forms of cyclodextrins (CDs) α-, ß-, and γ-CD was accomplished to examine the stability of ChR inside the central cavities of CDs. The aim of study was to identify the most suitable form of CD to improve the hydro-solubility of poorly soluble ChR bioactive molecule. Microsecond timescale molecular dynamics (MD) simulations were performed on four inclusion complexes (α-CD/ChR, ß-CD/ChR, and two conformations of γ-CD/ChR) to examine the dynamics of ChR inside the cavity of CDs. The first conformation of γ-CD/ChR inclusion complex (γ-CD1/ChR) was identified to possess the highest affinity between host and guest molecule on the basis of binding energy calculated by employing Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) and umbrella sampling simulations. To further strengthen the claims of classical and biased MD studies, Our own N-layered Integrated molecular Orbital and Molecular mechanics (ONIOM) (wB97XD/6-311+g(d,p):pm7) calculations were performed on the selected inclusion complexes. The ONIOM based complexation energy reaffirmed that ChR had highest affinity for the γ-CD1 host molecule. Further, the non-covalent interaction analysis was conducted using Multiwfn software on QM-optimized inclusion complexes with wB97XD/6-311+G(d,p) model chemistry, revealing non-covalent interactions between ChR and CDs. This atomic level information helped us to gain better insights into critical atoms of ChR and CD that participated in intermolecular interactions and identify γ-CD as a suitable host molecule for improving the hydro-solubulity of ChR. The structural insights would help to derive new derivatives of γ-CD with better host capacity.

Mechanistic behavior and subtle key events during DNA clamp opening and closing in T4 bacteriophage.

Clamp loaders ensure processive DNA replication by loading the toroidal shaped sliding clamps onto the DNA. The sliding clamps serve as a platform for the attachment of polymerases and several other proteins associated with the regulation of various cellular processes. Clamp loaders are fascinating as nanomachines that engage in protein-protein and protein-DNA interactions. The loading mechanism of the clamp around dsDNA at the atomic level has not yet been fully explored. We performed microsecond timescale molecular dynamics simulations to reveal the dynamics of two different intermediate complexes involved in loading of the clamps around DNA. We conducted various time-dependent MD-driven analyses including the highly robust Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations to observe changes in the structural elements of the clamp loader-clamp-DNA complexes in open and closed states. Our studies revealed the structural consequences of ATP hydrolysis events at different subunits of the clamp loader. This study would help in a better understanding of the clamp loading mechanism and would allow tackling various complications that might arise due to irregularities in this process.

Identification of 11ß-HSD1 inhibitors through enhanced sampling methods.

Aminoarylbenzosuberene (AAB) molecules were chosen for in silico analysis to develop effective and more competent 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) protein inhibitors. The AAB4 molecule was shown to have stronger interactions and binding affinity than standard inhibitors (co-crystallized molecules). These results were based on conventional, steered and enhanced umbrella sampling simulations.

In-silico evaluation of bioactive compounds from tea as potential SARS-CoV-2 nonstructural protein 16 inhibitors.

BACKGROUND AND AIM: A novel coronavirus, called the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been found to cause COVID-19 in humans and some other mammals. The nonstructural protein 16 (NSP16) of SARS-CoV-2 plays a significant part in the replication of viruses and suppresses the ability of innate immune system to detect the virus. Therefore, inhibiting NSP16 can be a secure path towards identifying a potent medication against SARS-CoV-2. Tea (Camellia sinensis) polyphenols have been reported to exhibit potential treatment options against various viral diseases. METHODS: We conducted molecular docking and structural dynamics studies with a set of 65 Tea bioactive compounds to illustrate their ability to inhibit NSP16 of SARS-CoV-2. Moreover, post-simulations end state thermodynamic free energy calculations were estimated to strengthen our results. RESULTS AND CONCLUSION: Six bioactive tea molecules showed better docking scores than the standard molecule sinefungin. These results were further validated by MD simulations, where Theaflavin compound demonstrated lower binding free energy in comparison to the standard molecule sinefungin. The compound theaflavin could be considered as a novel lead compound for further evaluation by in-vitro and in-vivo studies.

Identification of selective cyclin-dependent kinase 2 inhibitor from the library of pyrrolone-fused benzosuberene compounds: an in silico exploration.

The over-expression of cyclin-dependent kinase 2 is related to multiple cancers, which has led them to be a widely researched topic for nearly two decades. The prime focus of the present research is to design new potent and specific inhibitors against CDK2 to suppress cancer cell proliferation. In this study, we have chosen Flavopiridol, SU9516, and CVT-313 as standard inhibitors to compare with in-house synthesized pyrrolone-fused benzosuberene (PBS) compounds. We scrutinized Ligand2 as a selective inhibitor of CDK2 without off-target binding (CDK1 and CDK9) based on ligand efficiency and binding affinity. Interpretation of dynamic simulations and binding free energy studies unveiled that Ligand2 has a stable and equivalent free energy to standard inhibitors. These outcomes led towards positioning a potential natural molecule as selective inhibitor for CDK2 with low side effects.

Improving the catalytic efficiency and dimeric stability of Cu,Zn superoxide dismutase by combining structure-guided consensus approach with site-directed mutagenesis.

Superoxide dismutase (SOD) leads the front line of defense against injuries mediated by the reactive oxygen species (ROS). The SOD from a high-altitude plant Potentilla atrosanguinea is a unique thermostable enzyme. In this study, we applied a structure-guided consensus approach on Cu,Zn SOD from Potentilla atrosanguinea plant, to improve its enzymatic properties. The polar uncharged amino acid (threonine) at position 97 of wild-type (WT) SOD was selected as a target residue for substitution by aspartate (T97D) through site-directed mutagenesis. The WT and T97D were examined by a combinative approach consisting of robust computational and experimental tools. The in-silico analysis indicated improved dimeric stability in T97D as compared to the WT. The strong interactions between the monomers were related to improved dimerization and enhanced catalytic efficiency of T97D. These results were validated by in-vitro assays showing improved dimer stability and catalytic efficiency in T97D than WT. Moreover, the mutation also improved the thermostability of the enzyme. The combined structural and functional data described the basis for improved specific activity and thermostability. This study could expand the scope of interface residue to be explored as targets for designing of SODs with improved kinetics.

Benchmarking the ability of novel compounds to inhibit SARS-CoV-2 main protease using steered molecular dynamics simulations.

BACKGROUND: The SARS-CoV-2 main protease (Mpro) is an attractive target in the COVID-19 drug development process. It catalyzes the polyprotein's translation from viral RNA and specifies a particular cleavage site. Due to the absence of identical cleavage specificity in human cell proteases, targeting Mpro with chemical compounds can obstruct the replication of the virus. METHODS: To explore the potential binding mechanisms of 1,2,3-triazole scaffolds in comparison to co-crystallized inhibitors 11a and 11b towards Mpro, we herein utilized molecular dynamics and enhanced sampling simulation studies. RESULTS AND CONCLUSION: All the 1,2,3-triazole scaffolds interacted with catalytic residues (Cys145 and His41) and binding pocket residues of Mpro involving Met165, Glu166, Ser144, Gln189, His163, and Met49. Furthermore, the adequate binding free energy and potential mean force of the topmost compound 3h was comparable to the experimental inhibitors 11a and 11b of Mpro. Overall, the current analysis could be beneficial in developing the SARS-CoV-2 Mpro potential inhibitors.

A ricin-based peptide BRIP from Hordeum vulgare inhibits Mpro of SARS-CoV-2.

COVID-19 pandemic caused by SARS-CoV-2 led to the research aiming to find the inhibitors of this virus. Towards this world problem, an attempt was made to identify SARS-CoV-2 main protease (Mpro) inhibitory peptides from ricin domains. The ricin-based peptide from barley (BRIP) was able to inhibit Mpro in vitro with an IC50 of 0.52 nM. Its low and no cytotoxicity upto 50 µM suggested its therapeutic potential against SARS-CoV-2. The most favorable binding site on Mpro was identified by molecular docking and steered molecular dynamics (MD) simulations. The Mpro-BRIP interactions were further investigated by evaluating the trajectories for microsecond timescale MD simulations. The structural parameters of Mpro-BRIP complex were stable, and the presence of oppositely charged surfaces on the binding interface of BRIP and Mpro complex further contributed to the overall stability of the protein-peptide complex. Among the components of thermodynamic binding free energy, Van der Waals and electrostatic contributions were most favorable for complex formation. Our findings provide novel insight into the area of inhibitor development against COVID-19.

Theaflavin 3-gallate inhibits the main protease (Mpro) of SARS-CoV-2 and reduces its count in vitro.

The main protease (Mpro) of SARS-CoV-2 has been recognized as an attractive drug target because of its central role in viral replication. Our previous preliminary molecular docking studies showed that theaflavin 3-gallate (a natural bioactive molecule derived from theaflavin and found in high abundance in black tea) exhibited better docking scores than repurposed drugs (Atazanavir, Darunavir, Lopinavir). In this study, conventional and steered MD-simulations analyses revealed stronger interactions of theaflavin 3-gallate with the active site residues of Mpro than theaflavin and a standard molecule GC373 (a known inhibitor of Mpro and novel broad-spectrum anti-viral agent). Theaflavin 3-gallate inhibited Mpro protein of SARS-CoV-2 with an IC50 value of 18.48 ± 1.29 µM. Treatment of SARS-CoV-2 (Indian/a3i clade/2020 isolate) with 200 µM of theaflavin 3-gallate in vitro using Vero cells and quantifying viral transcripts demonstrated reduction of viral count by 75% (viral particles reduced from Log106.7 to Log106.1). Overall, our findings suggest that theaflavin 3-gallate effectively targets the Mpro thus limiting the replication of the SARS-CoV-2 virus in vitro.

Targeting the protein-protein interface pocket of Aurora-A-TPX2 complex: rational drug design and validation.

Aurora-A is a novel therapeutic target that belongs to the serine/threonine kinase family of proteins. Several cancers are associated with gene amplification or over-expression of Aurora-A. The somatic mutation (S155R) in Aurora-A results in a loss of interaction with its binding partner TPX2. The S155R mutation thus leads to ectopic expression of Aurora-A, resulting in centrosome amplification, chromosomal instability, aneuploidy, and oncogenic transformations. In order to restore the interaction between mutant Aurora-A and TPX2, we predicted a binding pocket in the interface of the Aurora-A-TPX2 complex. We performed molecular docking of potential bioactive molecules of the Himalayan region at the predicted site. Alantolactone and Dactylose-A were selected as potential molecules that could bind to the interface pocket and restore the lost interaction between mutant Aurora-A and TPX2. The molecular docking results were validated by performing explicit long term molecular dynamics simulations (4.0 µs) and MM-PBSA analysis. The molecular dynamics results confirmed that both the selected molecules could act as potent drugs to tackle the abnormal expression of Aurora-A manifested due to the somatic mutation (S155R). [Formula: see text] Communicated by Ramaswamy H. Sarma.