In-silico evaluation of Oroxylum indicum vent compounds in the plausible treatment and prevention of nasopharyngeal cancer.

BACKGROUND: Shyonaka (Oroxylum indicum Vent) is widely used in Ayurveda and in ethnomedical practice for the treatment of inflammation, pain, diarrhea, non-healing ulcers, and cancer. Owing to the high prevalence of Epstein-Barr virus (EBV) infection in Nasopharyngeal carcinoma (NPC) patients, simultaneous targeting of proteins involved in both EBV replication and NPC proliferation might help to manage the disease effectively. OBJECTIVES: This study is designed to identify potential dual targeting inhibitors from Oroxylum indicum having the potential to inhibit both EBV and NPC. This study also attempted quantitative analysis of Shyonaka Bark Decoction (SBD) to confirm the presence of Baicalein and Chrysin which are predominant marker compounds of Shyonaka. METHODOLOGY: The HPLC analysis of stem bark and root bark of Oroxylum indicum was done to estimate the presence of marker compounds Baicalein and Chrysalin. The in-silico analysis included ADMET analysis followed by molecular docking of known compounds from Oroxylum indicum (retrieved from IMPPAT database) onto the target proteins of EBV (BHRF1, NEC1, dUTPase, Uracil DNA glycosylase) and NPC (COX-2, EGFR, and MDM2) using DOCK6 tool. Further validations were done using the molecular dynamics simulations of top screened molecules onto the selected target proteins using AMBER20 package and their corresponding MMGBSA binding free-energy values were calculated. RESULTS: The molecular docking revealed that the key molecules from the plant, scutellarein 7-rutinoside (S7R), scutellarin (SCU) and 6-hydroxyluteolin, Baicalein and 5,7-Dihydroxy-2-phenyl-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one (57D) are effectively intervening with the target proteins of EBV, one of the key causative factors of NPC and the NPC specific targets which have the potential to reduce tumor size and other consequences of NPC. The molecular dynamics simulations of S7R, Baicalein and 57D, Baicalein with MDM-2 protein and dUTPase protein, respectively, showed stable interactions between them which were further assessed by the binding energy calculations. CONCLUSION: Overall, the in-silico evaluation of these phytochemicals with target proteins indicates their potential to inhibit both EBV and NPC which needs further in-vitro and in-vivo validations.

Studying early structural changes in SOS1 mediated KRAS activation mechanism.

KRAS activation is known to be modulated by a guanine nucleotide exchange factor (GEF), namely, Son of Sevenless1 (SOS1). SOS1 facilitates the exchange of GDP to GTP thereby leading to activation of KRAS. The binding of GDP/GTP to KRAS at the REM/allosteric site of SOS1 regulates the activation of KRAS at CDC25/catalytic site by facilitating its exchange. Different aspects of the allosteric activation of KRAS through SOS1 are still being explored. To understand the SOS1 mediated activation of KRAS, molecular dynamics simulations for a total of nine SOS1 complexes (KRAS-SOS1-KRAS) were performed. These nine systems comprised different combinations of KRAS-bound nucleotides (GTP/GDP) at REM and CDC25 sites of SOS1. Various conformational and thermodynamic parameters were analyzed for these simulation systems. MMPBSA free energy analysis revealed that binding at CDC25 site of SOS1 was significantly low for GDP-bound KRAS as compared to that of GTP-bound KRAS. It was observed that presence of either GDP/GTP bound KRAS at the REM site of SOS1 affected the activation related changes in the KRAS present at CDC25 site. The conformational changes at the catalytic site of SOS1 resulting from GDP/GTP-bound KRAS at the allosteric changes may hint at KRAS activation through different pathways (slow/fast/rare). The allosteric effect on activation of KRAS at CDC25 site may be due to conformations adopted by switch-I, switch-II, beta2 regions of KRAS at REM site. The effect of structural rearrangements occurring at allosteric KRAS may have led to increased interactions between SOS1 and KRAS at both the sites. The SOS1 residues involved in these important interactions with KRAS at the REM site were R694, S732 and K735. Whereas the ones interacting with KRAS at CDC25 site were S807, W809 and K814. This may suggest the crucial role of these residues in guiding the allosteric activation of KRAS at CDC25 site. The conformational shifts observed in the switch-I, switch-II and alpha3 regions of KRAS at CDC25 site may be attributed to be a part of allosteric activation. The binding affinities, interacting residues and conformational dynamics may provide an insight into development of inhibitors targeting the SOS1 mediated KRAS activation.

Markov State Modeling Analysis Captures Changes in the Temperature-Sensitive N-Terminal and ß-Turn Regions of the p53 DNA-Binding Domain.

The transcription factor p53 is one of the most widely studied cancer proteins. Its temperature-sensitive nature suggests reduction in functionality at physiological temperatures. Temperature-induced conformational variations and their impact on its functional ability still remain unexplored. A total of 20.8 µs molecular dynamics simulations of wildtype p53 in the apo and the DNA-bound states have been performed at 300 K and 310 K. Further, Markov State Modeling (MSM) analyses were performed, considering Cα-Cα distances as reaction coordinates. Filtering of these distances based on correlation with the time-independent components (tICs) resulted in 16 and 32 distances for apo and DNA-bound systems, respectively. Individual MSM analyses using these filtered distances were performed for both p53 systems. These Cα-Cα residue pairs belonged to the N-terminal, S6/7 ß-turn, loop L2, loop L3, and hydrophobic core residues. At physiological temperatures, apo-p53 exhibits exposure of its hydrophobic core, where the temperature-sensitive hotspot residues were also located. This exposure was the result of the S6/7 ß-turn and N-terminal moving apart. In the DNA-bound p53 system, loop L1 attains an open conformation at physiological temperatures, which weakens the DNA binding. It is already known that p53 mutants that lack DNA binding also tend to show similar conformational variations. The S6/7 ß-turn along with the already known functionally important loop L2 may pose as regions to be targeted to overcome the loss in binding of temperature-sensitive wildtype p53. Rescue strategies directed toward these temperature-sensitive regions may be useful to recuperate its strong binding at physiological temperatures.

Structural insight into the binding interactions of NTPs and nucleotide analogues to RNA dependent RNA polymerase of SARS-CoV-2.

RNA dependent RNA polymerase (RdRP) from positive-stranded RNA viruses has always been a hot target for designing of new drugs. Major class of drugs that are targeted against RdRP are nucleotide analogues. Extensive docking and molecular dynamics study describing the binding of natural nucleotides (NTPs) and its analogues leading to significant structural variation in the RdRP has been presented here. RdRP simulations in its apo, NTP-bound, and analogue-bound form have been performed. Nucleotide analogues included in this study were, favipiravir, galidesivir, lamivudine, ribavirin, remdesivir and sofosbuvir. The conformational flexibility of the RdRP molecule has been explored using principal component (PCA) and Markov state modeling (MSM) analysis. PCA inferred the presence of correlated motions among the conserved motifs of RdRP. Inter-domain distances between the finger and thumb subdomain flanking the nascent RNA template entry site sampled open and closed conformations. The ligand and template binding motifs F and G showed negatively correlated motions. K551, R553, and R555, a part of motif F appear to form strong interactions with the ligand molecules. R836, a primer binding residue was observed to strongly bind to the analogues. MSM analysis helped to extract statistically distinct conformations explored by the RdRP. Ensemble docking of the ligands on the Markov states also suggested the involvement of the above residues in ligand interactions. Markov states obtained clearly demarcated the open/closed conformations of the template entry site. These observations on residues from the conserved motifs involved in binding to the ligands may provide an insight into designing new inhibitors.Communicated by Ramaswamy H. Sarma.

Molecular dynamics of hERG channel: insights into understanding the binding of small molecules for detuning cardiotoxicity.

Evaluation of cardiotoxicity potential of new chemical entities (NCEs) has lately become one of the stringent filters in the drug discovery and development process. Cardiotoxicity is caused mainly by the inhibition of human ether-a-go-go related gene (hERG) channel protein. Inhibition of the hERG channel leads to a life-threatening condition known as cardiac arrhythmia. Knowledge of the structural behaviour of the hERG would aid greatly in the design of new drug molecules that do not interact with the protein and add to the safety index. In this study, a computational model for the active-state of hERG was developed. This model was equilibrated by performing the molecular dynamics simulations for 100 ns followed by clustering and selection of a representative structure based on the largest populated cluster. To study the changes in the protein structure on inhibition, three inhibitory ligands, namely, dofetilide, cisapride and terfenadine were docked, followed by molecular dynamics simulations of 200 ns for the apo and each ligand-bound structure. It was observed that docking and simulation studies of the hERG model exhibited noticeable conformational changes in the protein upon ligand-binding. A significant change in the kink of the S6-transmembrane helix was observed. Inter-chain distances between the crucial residues Y652 and F656 (present below the ion-selectivity filter), their side-chain orientation and hydrogen bonding indicated a probable collapse of the pore. These changes may infer the initiation in transition of hERG from an open to an inactive state. Hence, these findings would help in designing compounds devoid of hERG inhibition with reduced cardiotoxicity.Communicated by Ramaswamy H. Sarma.

Understanding the binding affinities between SFRP1CRD, SFRP1Netrin, Wnt5B and frizzled receptors 2, 3 and 7 using MD simulations.

cWnt-signalling plays a crucial role in stem cell maintenance and tissue homeostasis. Secreted frizzled-related proteins(SFRP), Wnt inhibitors consist of the N-terminal cysteine rich domain(CRD) and the C-terminal netrin(NTR) domain. SFRP1 binds to the Wnt ligands and frizzled receptors(FZ) either through its SFRP1CRD or through its SFRP1Netrin domains; however, very little is known on these binding affinities. Here, we attempted to understand the interactions and binding affinities of SFRP1-Wnt5B, SFRP1-FZ(2, 3 & 7) and Wnt5B-FZ(2, 3 & 7) that are mainly expressed in murine hair follicle stem cells. SFRP1CRD, SFRP1Netrin, Wnt5B and FZ(2, 3 & 7) structures were built using homology modelling, followed by their molecular dynamics simulations. SFRP1CRD showed lower fluctuation when in complex with FZ2, FZ3 and FZ7 and Wnt5B as compared to SFRP1Netrin using RMSF and RMSD. However, free energy showed SFRP1Netrin was energetically more stable than SFRP1CRD. SFRP1Netrin formed more number of interactions with FZ as compared to SFRP1CRD. Importantly, SFRP1Netrin favoured binding to the FZ receptors(FZ3 > FZ7 > FZ2) as compared to Wnt5B ligand. Conversely, the SFRP1CRD showed more affinity towards the Wnt5B ligand as compared to FZ receptors. Wnt5B showed the best binding affinity with FZ3 followed by SFRP1CRD and SFRP1Netrin. Therefore, SFRP1Netrin can bind to the FZ3 with higher binding affinity and may inhibit non-canonical Wnt-signalling pathway. Our study provides the comprehensive information on the binding affinities among the Wnt5B, SFRP1CRD/Netrin and FZ(2, 3 & 7). Thus, this information might also help in designing novel strategies to inhibit aberrant Wnt-signalling.Communicated by Ramaswamy H. Sarma.

Natural plant products as potential inhibitors of RNA dependent RNA polymerase of Severe Acute Respiratory Syndrome Coronavirus-2.

Drug repurposing studies targeting inhibition of RNA dependent RNA polymerase (RdRP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have exhibited the potential effect of small molecules. In the present work a detailed interaction study between the phytochemicals from Indian medicinal plants and the RdRP of SARS-CoV-2 has been performed. The top four phytochemicals obtained through molecular docking were, swertiapuniside, cordifolide A, sitoindoside IX, and amarogentin belonging to Swertia chirayita, Tinospora cordifolia and Withania somnifera. These ligands bound to the RdRP were further studied using molecular dynamics simulations. The principal component analysis of these systems showed significant conformational changes in the finger and thumb subdomain of the RdRP. Hydrogen bonding, salt-bridge and water mediated interactions supported by MM-GBSA free energy of binding revealed strong binding of cordifolide A and sitoindoside IX to RdRP. The ligand-interacting residues belonged to either of the seven conserved motifs of the RdRP. These residues were polar and charged amino acids, namely, ARG 553, ARG 555, ASP 618, ASP 760, ASP 761, GLU 811, and SER 814. The glycosidic moieties of the phytochemicals were observed to form favourable interactions with these residues. Hence, these phytochemicals may hold the potential to act as RdRP inhibitors owing to their stability in binding to the druggable site.

An insight into the inhibitory mechanism of phytochemicals and FDA-approved drugs on the ACE2-Spike complex of SARS-CoV-2 using computational methods.

The S-glycoprotein (Spike) of the SARS-CoV-2 forms a complex with the human transmembrane protein angiotensin-converting enzyme 2 (ACE2) during infection. It forms the first line of contact with the human cell. The FDA-approved drugs and phytochemicals from Indian medicinal plants were explored. Molecular docking and simulations of these molecules targeting the ACE2-Spike complex were performed. Rutin DAB10 and Swertiapuniside were obtained as the top-scored drugs as per the docking protocol. The MD simulations of ligand-free, Rutin DAB10-bound, and Swertiapuniside-bound ACE2-Spike complex revealed abrogation of the hydrogen bonding network between the two proteins. The principal component and dynamic cross-correlation analysis pointed out conformational changes in both the proteins unique to the ligand-bound systems. The interface residues, His34, and Lys353 from ACE2 and Arg403, and Tyr495 from the Spike protein formed significant strong interactions with the ligand molecules, inferring the inhibition of ACE2-Spike complex. Few novel interactions specific to Rutin-DAB10 and Swertiapuniside were also identified. The conformational flexibility of the drug-binding pocket was captured using the RMSD-based clustering of the ligand-free simulations. Ensemble docking was performed wherein the FDA-approved database and phytochemical dataset were docked on each of the cluster representatives of the ACE2-Spike. The phytochemicals identified belonged to Withania somnifera, Swertia chirayita, Tinospora cordifolia and Rutin DAB10, fulvestrant, elbasvir from FDA. Supplementary Information: The online version contains supplementary material available at 10.1007/s11696-021-01680-1.

Drug repurposing studies targeting SARS-CoV-2: an ensemble docking approach on drug target 3C-like protease (3CLpro).

The COVID-19 pandemic has been responsible for several deaths worldwide. The causative agent behind this disease is the Severe Acute Respiratory Syndrome - novel Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 belongs to the category of RNA viruses. The main protease, responsible for the cleavage of the viral polyprotein is considered as one of the hot targets for treating COVID-19. Earlier reports suggest the use of HIV anti-viral drugs for targeting the main protease of SARS-CoV, which caused SARS in the year 2002-2003. Hence, drug repurposing approach may prove to be useful in targeting the main protease of SARS-CoV-2. The high-resolution crystal structure of the main protease of SARS-CoV-2 (PDB ID: 6LU7) was used as the target. The Food and Drug Administration approved and SWEETLEAD database of drug molecules were screened. The apo form of the main protease was simulated for a cumulative of 150 ns and 10 µs open-source simulation data was used, to obtain conformations for ensemble docking. The representative structures for docking were selected using RMSD-based clustering and Markov State Modeling analysis. This ensemble docking approach for the main protease helped in exploring the conformational variation in the drug-binding site of the main protease leading to the efficient binding of more relevant drug molecules. The drugs obtained as top hits from the ensemble docking possessed anti-bacterial and anti-viral properties. This in silico ensemble docking approach would support the identification of potential candidates for repurposing against COVID-19.Communicated by Ramaswamy H. Sarma.

Remdesivir-bound and ligand-free simulations reveal the probable mechanism of inhibiting the RNA dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2.

The efforts towards developing a potential drug against the current global pandemic, COVID-19, have increased in the past few months. Drug development strategies to target the RNA dependent RNA polymerase (RdRP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are being tried worldwide. The gene encoding this protein, is known to be conserved amongst positive strand RNA viruses. This enables an avenue to repurpose the drugs designed against earlier reported inhibitors of RdRP. One such strong inhibitor is remdesivir which has been used against EBOLA infections. The binding of remdesivir to RdRP of SARS-CoV-2 has been studied using the classical molecular dynamics and ensemble docking approach. A comparative study of the simulations of RdRP in the apo and remdesivir-bound form revealed blocking of the template entry site in the presence of remdesivir. The conformation changes leading to this event were captured through principal component analysis. The conformational and thermodynamic parameters supported the experimental information available on the involvement of crucial arginine, serine and aspartate residues belonging to the conserved motifs in RdRP functioning. The catalytic site comprising of SER 759, ASP 760, and ASP 761 (SDD) was observed to form strong contacts with remdesivir. The significantly strong interactions of these residues with remdesivir may infer the latter's binding similar to the normal nucleotides thereby remaining unidentified by the exonuclease activity of RdRP. The ensemble docking of remdesivir too, comprehended the involvement of similar residues in interaction with the inhibitor. This information on crucial interactions between conserved residues of RdRP with remdesivir through in silico approaches may be useful in designing inhibitors.

TANGO: A high through-put conformation generation and semiempirical method-based optimization tool for ligand molecules.

Lead optimization is one of the crucial steps in the drug discovery pipeline. After identifying the lead molecule and obtaining its 2D geometry, understanding the best conformation it would attain in 3D still remains one of the most challenging steps in drug discovery. There have been multiple methods and algorithms that are directed toward achieving best conformation for the lead molecules. TANGO focuses on conformation generation and its optimization using semiempirical energy calculations. The conformation generation is based on torsion angle rotation of the exocyclic bonds. The energy calculations are performed using MOPAC. The unique feature of this tool lies in the implementation of Message Passing Interface (MPI) for conformation generation and semiempirical-based optimization. A well-defined architecture handling the input and output generation has been used. The master and slave approach to handle operations involved in torsion angle rotation and energy calculations has helped in load balancing the process of conformation generation. The benchmarking results suggest that TANGO scales significantly well across eight nodes with each node utilizing 16 cores. This tool may prove to very useful in high throughput generation of semiempirically optimized small molecule conformations. The use of semiempirical methods for optimization generates a conformational ensemble thereby helping to obtain stable and alternate stable conformers for a given ligand molecule. © 2018 Wiley Periodicals, Inc.

Investigating DNA Binding and Conformational Variation in Temperature Sensitive p53 Cancer Mutants Using QM-MM Simulations.

The tp53 gene is found to be mutated in 50% of all the cancers. The p53 protein, a product of tp53 gene, is a multi-domain protein. It consists of a core DNA binding domain (DBD) which is responsible for its binding and transcription of downstream target genes. The mutations in p53 protein are responsible for creating cancerous conditions and are found to be occurring at a high frequency in the DBD region of p53. Some of these mutations are also known to be temperature sensitive (ts) in nature. They are known to exhibit partial or strong binding with DNA in the temperature range (298-306 K). Whereas, at 310 K and above they show complete loss in binding. We have analyzed the changes in binding and conformational behavior at 300 K and 310 K for three of the ts-mutants viz., V143A, R249S and R175H. QM-MM simulations have been performed on the wild type and the above mentioned ts-mutants for 30 ns each. The optimal estimate of free energy of binding for a particular number of interface hydrogen bonds was calculated using the maximum likelihood method as described by Chodera et. al (2007). This parameter has been observed to be able to mimic the binding affinity of the p53 ts-mutants at 300 K and 310 K. Thus the correlation between MM-GBSA free energy of binding and hydrogen bonds formed by the interface residues between p53 and DNA has revealed the temperature dependent nature of these mutants. The role of main chain dihedrals was obtained by performing dihedral principal component analysis (PCA). This analysis, suggests that the conformational variations in the main chain dihedrals (ϕ and ψ) of the p53 ts-mutants may have caused reduction in the overall stability of the protein. The solvent exposure of the side chains of the interface residues were found to hamper the binding of the p53 to the DNA. Solvent Accessible Surface Area (SASA) also proved to be a crucial property in distinguishing the conformers obtained at 300 K and 310 K for the three ts-mutants from the wild type at 300 K.

QM-MM simulations on p53-DNA complex: a study of hot spot and rescue mutants.

p53 is a transcription factor involved in the expression of a number of downstream genes in response to genotoxic stress. It is activated through post translation modifications in normal as well as cancerous cells. However, due to mutations occurring in p53 in cancer cells it is not able to perform its function of DNA binding which leads to cell proliferation. It is found to be mutated in 50% of the cancers. These mutations occur at a high frequency in the DNA binding region of the p53. Among the known seven hot spot cancer mutations G245S, R249S, and R273C have been studied here using quantum mechanics and molecular mechanics (QM-MM) simulations. These mutations along with their experimentally proven rescue mutations have also been included in the present work. A comparative study of these cancer mutations along with wild type and their rescue mutations has been performed. A computational measure based on the free energy changes occurring in the binding of the p53 to the DNA has been presented. A correlation between the DNA binding property and important interaction between p53 and DNA has been observed for all the mutants. The keys residues which contribute to the binding of p53 to DNA by forming crucial hydrogen bonds have also been discussed in detail. A 30 ns simulation study was analyzed to observe the local structural changes and DNA binding property of p53 in case of wild type, cancer and rescue mutants.

Insights into the folding pathway of the Engrailed Homeodomain protein using replica exchange molecular dynamics simulations.

Protein folding studies were carried out by performing microsecond time scale simulations on the ultrafast/fast folding protein Engrailed Homeodomain (EnHD) from Drosophila melanogaster. It is a three-helix bundle protein consisting of 54 residues (PDB ID: 1ENH). The positions of the helices are 8-20 (Helix I), 26-36 (Helix II) and 40-53 (Helix III). The second and third helices together form a Helix-Turn-Helix (HTH) motif which belongs to the family of DNA binding proteins. The molecular dynamics (MD) simulations were performed using replica exchange molecular dynamics (REMD). REMD is a method that involves simulating a protein at different temperatures and performing exchanges at regular time intervals. These exchanges were accepted or rejected based on the Metropolis criterion. REMD was performed using the AMBER FF03 force field with the generalised Born solvation model for the temperature range 286-373 K involving 30 replicas. The extended conformation of the protein was used as the starting structure. A simulation of 600 ns per replica was performed resulting in an overall simulation time of 18 µs. The protein was seen to fold close to the native state with backbone root mean square deviation (RMSD) of 3.16 Å. In this low RMSD structure, the Helix I was partially formed with a backbone RMSD of 3.37 Å while HTH motif had an RMSD of 1.81 Å. Analysis suggests that EnHD folds to its native structure via an intermediate in which the HTH motif is formed. The secondary structure development occurs first followed by tertiary packing. The results were in good agreement with the experimental findings.