Molecular modeling to investigate the binding of Congo red toward GNNQQNY protofibril and in silico virtual screening for the identification of new aggregation inhibitors

Understanding the nature of the recognition between amyloid protofibrils and dye molecules at the molecular level is essential to improving instructive guides for designing novel molecular probes or new inhibitors. However, the atomic details of the binding between dyes and amyloid fibrils are still not fully understood. In this study, molecular docking, consensus scoring, molecular dynamics (MD), and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analyses were integrated to investigate the binding between Congo red (CR) and the GNNQQNY protofibril from yeast prion protein Sup35 and to further evaluate their binding stabilities and affinities. Our results reveal that there are four CR binding sites located on GNNQQNY protofibril surface. These four CR binding sites adopt dual binding modes by which CR binding with its long axis parallel and perpendicular to the long axis of the protofibril. In addition, CR was also found to bind to the edge of the protofibril via hydrophobic/aromatic and hydrogen-bonding interactions, which is inferred as the possible inhibition mechanism to prevent the elongation of the protofibril from the addition of incoming peptides. Virtual screening from National Cancer Institute (NCI) database obtained three hit compounds with higher binding affinity than CR to the edge of the protofibril due to the fact that the central parts of these compounds are able to form additional hydrogen bonds with the protofibril. The results of the study could be useful for the development of new molecular probes or inhibitors for clinical applications.

Discovery of novel low-molecular-weight HIV-1 inhibitors interacting with cyclophilin A using in silico screening and biological evaluations

Cyclophilin A has attracted attention recently as a new target of anti-human immunodeficiency virus type 1 (HIV-1) drugs. However, so far no drug against HIV-1 infection exhibiting this mechanism of action has been approved. To identify new potent candidates for inhibitors, we performed in silico screening of a commercial database of more than 1,300 drug-like compounds by using receptor-based docking studies. The candidates selected from docking studies were subsequently tested using biological assays to assess anti-HIV activities. As a result, two compounds were identified as the most active. Specifically, both exhibited anti-HIV activity against viral replication at a low concentration and relatively low cytotoxicity at the effective concentration inhibiting viral growth by 50 %. Further modification of these molecules may lead to the elucidation of potent inhibitors of HIV-1.

Retrospective molecular docking study of WY-25105 ligand to β-secretase and bias of the three-dimensional structure flexibility

β-Secretase (BACE) is a very promising target in the search for a treatment for Alzheimer’s disease using a protein–ligand inhibition approach. Given the many published X-ray structures of BACE protein, structure-based drug design has been used extensively to support new inhibitor discovery programs. Due to the high flexibility and large catalytic site of this protein, sampling of the huge conformational space of the binding site is the big challenge to overcome and is the main limitation of the most widely used docking programs. Incorrect treatment of these pitfalls can introduce bias into ligand docking and could affect the results. This is especially the case with the WY-25105 compound reported by the Wyeth Corporation as a BACE ligand that did not fit into any of the known crystal structures. In the present retrospective study, a set of available X-ray enzyme structures was selected and molecular dynamics simulations were conducted to generate more diverse representative BACE protein conformations. These conformations were then used for a docking study of the WY-25105 compound. The results confirmed the need to use an ensemble of structures in protein–ligand docking for identification of new binding modes in structure-based drug design of BACE inhibitors.

Discovery of potent inhibitors for interleukin-2-inducible T-cell kinase: structure-based virtual screening and molecular dynamics simulation approaches

In our study, a structure-based virtual screening study was conducted to identify potent ITK inhibitors, as ITK is considered to play an important role in the treatment of inflammatory diseases. We developed a structure-based pharmacophore model using the crystal structure (PDB ID: 3MJ2) of ITK complexed with BMS-50944. The most predictive model, SB-Hypo1, consisted of six features: three hydrogen-bond acceptors (HBA), one hydrogen-bond donor (HBD), one ring aromatic (RA), and one hydrophobic (HY). The statistical significance of SB-Hypo1 was validated using wide range of test set molecules and a decoy set. The resulting well-validated model could then be confidently used as a 3D query to screen for drug-like molecules in a database, in order to retrieve new chemical scaffolds that may be potent ITK inhibitors. The hits retrieved from this search were filtered based on the maximum fit value, drug-likeness, and ADMET properties, and the hits that were retained were used in a molecular docking study to find the binding mode and molecular interactions with crucial residues at the active site of the protein. These hits were then fed into a molecular dynamics simulation to study the flexibility of the activation loop of ITK upon ligand binding. This combination of methodologies is a valuable tool for identifying structurally diverse molecules with desired biological activities, and for designing new classes of selective ITK inhibitors.

Development of a theoretical model for the inhibition of nsP3 protease of Chikungunya virus using pyranooxazoles

Abstract

Chikungunya virus (CHIKV) causes Chikungunya fever (CHIKF) and till date no effective medicine for its cure is available in market. Different research groups find various possible interactions between small molecules and non-structural proteins, viz. nsP3, one of the most important viral elements in CHIKV. In this work, authors have studied the interactions of nsP3 protease of CHIKV with pyranooxazoles. Initially, a one-pot three-component reaction was designed using oxazolidine-2,4-dione, benzaldehyde and cyanoethylacetate to get a proposed biological active molecule, i.e. based on pyranooxazoles. The mechanism for the synthesis of the product based on pyranooxazole was studied through density functional theory (DFT) using Gaussian. Then, a library of the obtained pyranooxazole was created through computational tools by varying the substituents. Further, virtual screening of the designed library of pyranooxazoles (200 compounds) against nsP3 protease of CHIKV was performed. Herein, CMPD 104 showed strongest binding affinity toward the targeted nsP3 protease of CHIKV, based on the least binding energy obtained from docking. Based on docking results, the pharmacological, toxicity, biological score and Lipinski’s filters were studied. Further, DFT studies of top five compounds were done using Gaussian. Molecular dynamics (MD) simulation of nsP3 protease of CHIKV with and without 104 was performed using AMBER18 utilizing ff14SB force field in three steps (minimization, equilibration and production). This work is emphasized to designing of one-pot three-component synthesis and to develop a theoretical model to inhibit the nsP3 protease of CHIKV. Abbreviations CHIKF Chikungunya fever

CHIKV Chikungunya virus

DFT density functional theory

DS Discovery Studio

MD molecular dynamics

MM-GBSA molecular mechanics-generalized born surface area

MMV Molegro molecular viewer

Communicated by Ramaswamy H. Sarma     

Rational design of the survivin/CDK4 complex by combining protein–protein docking and molecular dynamics simulations

Survivin, the smallest inhibitor of apoptosis protein (IAP), is a valid target for cancer research. It mediates both the apoptosis pathway and the cell cycle and has been proposed to form a complex with the cyclin-dependent kinase protein CDK4. The resulting complex transports CDK4 from the cytosol to the nucleus, where CDK4 participates in cell division. Survivin has been recognized as a node protein that interacts with several partners; disruption of the formed complexes can lead to new anticancer compounds. We propose a rational model of the survivin/CDK4 complex that fulfills the experimental evidence and that can be used for structure-based design of inhibitors modifying its interface recognition. In particular, the suggested complex involves the alpha helical domain of survivin and resembles the mode of binding of survivin in the survivin/borealin X-ray structure. The proposed model has been obtained by combining protein–protein docking, fractal-based shape complementarity, electrostatics studies and extensive molecular dynamics simulations.

Determination of key receptor–ligand interactions of dopaminergic arylpiperazines and the dopamine D2 receptor homology model

Interest in structure-based G-protein-coupled receptor (GPCR) ligand discovery is huge, given that almost 30 % of all approved drugs belong to this category of active compounds. The GPCR family includes the dopamine receptor subtype D2 (D2DR), but unfortunately—as is true of most GPCRs—no experimental structures are available for these receptors. In this publication, we present the molecular model of D2DR based on the previously published crystal structure of the dopamine D3 receptor (D3DR). A molecular modeling study using homology modeling and docking simulation provided a rational explanation for the behavior of the arylpiperazine ligand. The observed binding modes and receptor–ligand interactions provided us with fresh clues about how to optimize selectivity for D2DR receptors.

NMR study of non-structural proteins–part III: <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and side-chain resonance assignment of macro domain from <Emphasis Type="Italic">Chikungunya</Emphasis> virus (CHIKV)

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130–190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.     

Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

Background

Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.

Methods

Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.

Results

Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.

Conclusion

Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.     

In vitro inhibitory profile of NDGA against AChE and its in silico structural modifications based on ADME profile

Acetylcholinesterase (AChE) inhibitors are currently in focus for the pharmacotherapy of Alzheimer’s disease (AD). These inhibitors increase the level of acetylcholine in the brain and facilitate cholinergic neurotransmission. AChE inhibitors such as rivastigmine, galantamine, physostigmine and huperzine are obtained from plants, indicating that plants can serve as a potential source for novel AChE inhibitors. We have performed a virtual screening of diverse natural products with distinct chemical structure against AChE. NDGA was one among the top scored compounds and was selected for enzyme kinetic studies. The IC₅₀ of NDGA on AChE was 46.2 μM. However, NDGA showed very poor central nervous system (CNS) activity and blood–brain barrier (BBB) penetration. In silico structural modification on NDGA was carried out in order to obtain derivatives with better CNS activity as well as BBB penetration. The studies revealed that some of the designed compounds can be used as lead molecules for the development of drugs against AD

Structure-based design of nitrogen-linked macrocyclic kinase inhibitors leading to the clinical candidate SB1317/TG02, a potent inhibitor of cyclin dependant kinases (CDKs), Janus kinase 2 (JAK2), and Fms-like tyrosine kinase-3 (FLT3)

A high-throughput screen against Aurora A kinase revealed several promising submicromolar pyrimidine-aniline leads. The bioactive conformation found by docking these leads into the Aurora A ATP-binding site had a semicircular shape. Macrocycle formation was proposed to achieve novelty and selectivity via ring-closing metathesis of a diene precursor. The nature of the optimal linker and its size was directed by docking. In a kinase panel screen, selected macrocycles were active on other kinase targets, mainly FLT3, JAK2, and CDKs. These compounds then became leads in a CDK/FLT3/JAK2 inhibitor project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. Interaction with this residue explains the observed selectivity. The Asp86 residue is conserved in most CDKs, resulting in potent pan-CDK inhibition by these compounds. Optimized macrocycles generally have good DMPK properties, and are efficacious in mouse models of cancer. Compound 5 (SB1317/TG02), a pan-CDK/FLT3/JAK2 inhibitor, was selected for preclinical development, and is now in phase 1 clinical trials.

A flexible-protein molecular docking study of the binding of ruthenium complex compounds to PIM1, GSK-3β, and CDK2/Cyclin A protein kinases

We employ ensemble docking simulations to characterize the interactions of two enantiomeric forms of a Ru-complex compound (1-R and 1-S) with three protein kinases, namely PIM1, GSK-3β, and CDK2/cyclin A. We show that our ensemble docking computational protocol adequately models the structural features of these interactions and discriminates between competing conformational clusters of ligand-bound protein structures. Using the determined X-ray crystal structure of PIM1 complexed to the compound 1-R as a control, we discuss the importance of including the protein flexibility inherent in the ensemble docking protocol, for the accuracy of the structure prediction of the bound state. A comparison of our ensemble docking results suggests that PIM1 and GSK-3β bind the two enantiomers in similar fashion, through two primary binding modes: conformation I, which is very similar to the conformation presented in the existing PIM1/compound 1-R crystal structure; conformation II, which represents a 180° flip about an axis through the NH group of the pyridocarbazole moiety, relative to conformation I. In contrast, the binding of the enantiomers to CDK2 is found to have a different structural profile including a suggested bound conformation, which lacks the conserved hydrogen bond between the kinase and the ligand (i.e., ATP, staurosporine, Ru-complex compound). The top scoring conformation of the inhibitor bound to CDK2 is not present among the top-scoring conformations of the inhibitor bound to either PIM1 or GSK-3β and vice-versa. Collectively, our results help provide atomic-level insights into inhibitor selectivity among the three kinases.

Cytotoxic effect and molecular docking of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide—a novel topoisomerase II inhibitor

The preliminary cytotoxic effect of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide hydrochloride (1)—a potent topoisomerase II inhibitor—was measured using a MTT assay. It was found that the compound decreased the number of viable cells in both estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231breast cancer cells, with IC₅₀ values of 146?±?2 and 132?±?2 μM, respectively. To clarify the molecular basis of the inhibitory action of 1, molecular docking studies were carried out. The results suggest that 1 targets the ATP binding pocket.

A knowledge-based halogen bonding scoring function for predicting protein-ligand interactions

Halogen bonding, a non-covalent interaction between the halogen σ-hole and Lewis bases, could not be properly characterized by majority of current scoring functions. In this study, a knowledge-based halogen bonding scoring function, termed XBPMF, was developed by an iterative method for predicting protein-ligand interactions. Three sets of pairwise potentials were derived from two training sets of protein-ligand complexes from the Protein Data Bank. It was found that two-dimensional pairwise potentials could characterize appropriately the distance and angle profiles of halogen bonding, which is superior to one-dimensional pairwise potentials. With comparison to six widely used scoring functions, XBPMF was evaluated to have moderate power for predicting protein-ligand interactions in terms of “docking power”, “ranking power” and “scoring power”. Especially, it has a rather satisfactory performance for the systems with typical halogen bonds. To the best of our knowledge, XBPMF is the first halogen bonding scoring function that is not dependent on any dummy atom, and is practical for high-throughput virtual screening. Therefore, this scoring function should be useful for the study and application of halogen bonding interactions like molecular docking and lead optimization.

Computational evaluation of factors governing catalytic 2-keto acid decarboxylation

Lymphatic filariasis (LF) is a vector borne infectious disease caused by the nematode Wuchereria bancrofti, Brugia malayi, and Brugia timori. Over 120 million people are affected by LF in the world, of which two-thirds are in Asia. The infection restricts the normal flow of lymph from the infected area resulting in swelling of the extremities and causing permanent disability. As the available drugs for the treatment of LF are becoming ineffective due to the development of resistance, there is an urgent need to find new leads for drug development. In this study, asparaginyl-tRNA synthetase (AsnRS; PDB ID: 2XGT) essential for the protein bio-synthesis in the filarial nematode was used to carry out virtual screening (VS) of plant constituents from traditional Chinese medicine (TCM) database. Docking as well as E-pharmacophore based VS were carried out to identify the hits. The top scoring hits, Agri 1 (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one-3-O-beta-D-glucopyranoside) and Agri 2 (5,7-dihydroxy-2-propylchromone 7-O-beta-D-glucopyranoside), constituents of Agrimonia pilosa, were selected for molecular dynamics (MD) simulation study for 10 ns. MD simulation showed that both the glycosides Agri 1 and Agri 2 were forming stable interactions with the target protein. Moreover, docking and MD simulation of the lead A (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one; Mol. Wt.: 304.25; CLogP: 3.07) and lead B (5,7-dihydroxy-2-propylchromone; Mol. Wt.: 220.22; CLogP: 3.02), the aglycones of Agri 1 and Agri 2, respectively, were carried out with the target AsnRS. The in silico investigations of the aglycones suggest that the lead B could be a suitable fragment-like lead molecule for anti-filarial drug discovery.

In-silico screening of cancer associated mutation on PLK1 protein and its structural consequences

The Polo-like kinases (Plks) are a conserved subfamily of serine-threonine protein kinases that have significant roles in cell proliferation. The serine/threonine protein kinases or polo-like kinase 1 (PLK1) exist in centrosome during interphase and is an important regulatory enzyme in cell cycle progression during M phase. Mutations in mammalian PLK1 were found to be over expressed in various human cancers and it is disrupting the binding ability of polo box domain with target peptide. In this analysis we implemented a computational approach to filter the most deleterious and cancer associated mutation on PLK1 protein. We found W414F as the most deleterious and cancer associated by Polyphen 2.0, SIFT, I-mutant 3.0, PANTHER, PhD-SNP, SNP&GO, Mutpred and Dr Cancer tools. Molecular docking and molecular dynamics simulation (MDS) approach was used to investigate the structural and functional behavior of PLK1 protein upon mutation. MDS and docking results showed stability loss in mutant PLK1 protein. Due to mutation, PLK1 protein became more flexible and alters the dynamic property of protein which might affect the interaction with target peptide and leads to cell proliferation. Our study provided a well designed computational methodology to examine the cancer associated nsSNPs and their molecular mechanism. It further helps scientists to develop a drug therapy against PLK1 cancer-associated diseases.

Why tazobactam and sulbactam have different intermediates population with SHV-1 β-lactamase: a molecular dynamics study

The imine intermediates of tazobactam and sulbactam bound to SHV-1 β-lactamase were investigated by molecular dynamics (MD) simulation respectively. Hydrogen bond networks around active site were found different between tazobactam and sulbactam acyl-enzymes. In tazobactam imine intermediate, it was observed that the triazolyl ring formed stable hydrogen bonds with Asn170 and Thr167. The results suggest that conformation of imine determined the population of intermediates. In imine intermediate of tazobactam, the triazolyl ring is trapped in Thr_Asn pocket, and it restricts the rotation of C5-C6 bond so that tazobactam can only generate trans enamine intermediate. Further, conformational cluster analyses are performed to substantiate the results. These findings provide an explanation for the corresponding experimental results, and will be potentially useful in the development of new inhibitors.

Entropy versus aromaticity in the conformational dynamics of aromatic rings

Comparison of the results of Car-Parrinello molecular dynamics simulations of isolated benzene, pyrimidine and 1,2,4-triazine molecules reveals that the unusually low population of planar geometry of the benzene ring is caused by entropy effects despite its high aromaticity. The decrease in symmetry of the molecule results in smaller changes in entropy and Gibbs free energy due to out-of-plane deformations of the ring, leading to an increase in the population of planar geometry of the ring. This leads to differences in the topology of potential energy and Gibbs free energy surfaces.

Molecular dynamics analysis of a series of 22 potential farnesyltransferase substrates containing a CaaX-motif

Protein farnesyltransferase (FTase) is an important target in many research fields, more markedly so in cancer investigation since several proteins known to be involved in human cancer development are thought to serve as substrates for FTase and to require farnesylation for proper biological activity. Several FTase inhibitors (FTIs) have advanced into clinical testing. Nevertheless, despite the progress in the field several functional and mechanistic doubts on the FTase catalytic activity have persisted. This work provides some crucial information on this important enzyme by describing the application of molecular dynamics simulations using specifically designed molecular mechanical parameters for a variety of 22 CaaX peptides known to work as natural substrates or inhibitors for this enzyme. The study involves a comparative analysis of several important molecular aspects, at the mechanistic level, of the behavior of substrates and inhibitors at the dynamic level, including the behavior of the enzyme and peptides, as well as their interaction, together with the effect of the solvent. Properties evaluated include the radial distribution function of the water molecules around the catalytically important zinc metal atom and cysteine sulfur of CaaX, the conformations of the substrate and inhibitor and the corresponding RMSF values, critical hydrogen bonds, and several catalytically relevant distances. These results are discussed in light of recent experimental and computational evidence that provides new insights into the activity of this enzyme.

Insights into the structural determinants for selective inhibition of nitric oxide synthase isoforms

Selective inhibition of the nitric oxide synthase isoforms (NOS) is a promising approach for the treatment of various disorders. However, given the high active site conservation among all NOS isoforms, the design of selective inhibitors is a challenging task. Analysis of the X-ray crystal structures of the NOS isoforms complexed with known inhibitors most often gives no clues about the structural determinants behind the selective inhibition since the inhibitors share the same binding conformation. Aimed at a better understanding of the structural factors responsible for selective inhibition of NOS isoforms we have performed MD simulations for iNOS, nNOS and eNOS complexed with N^ω-NO₂-L-Arg (1), and with the aminopyridine derivatives 2 and 3. The slightly better selectivity of 1 for nNOS may be assigned to the presence of extra charge–charge interactions due to its “extended” conformation. While the high affinity of 2 for iNOS can be explained by the formation of an iNOS-specific subpocket upon binding, the lack of affinity for eNOS is associated to a conformational change in Glu363. The strong van der Waals and electrostatic interactions between 3 and the active site of nNOS are most likely responsible for its higher affinity for this isoform. Owing to the elongated and narrow binding pocket of iNOS, the correct positioning of 3 over the heme group is difficult, which may account for its lower affinity toward this isoform. Brought together, our results might help to rationalize the design of selective NOS inhibitors.