Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2‐3)Gal: a study by in silico mutations and molecular dynamics simulations

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM‐PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.     

Refinement of X-ray data on dual cosubstrate specificity of CK2 kinase by free energy calculations based on molecular dynamics simulation

Free energy differences of binding of adenosine triphosphate (ATP) and guanine triphosphate (GTP) to the protein kinase CK2 (casein kinase 2) were calculated, using molecular dynamics (MD) simulations and the thermodynamic cycle approach. Good agreement with experimental data was obtained. Simulations confirm observations based on crystallographic data that specifically interacting water molecules in the binding site region of CK2 kinase play a key role in its ability to use ATP or GTP as equally efficient phosphate donors. We point out that to obtain quantitatively reasonable results, it was necessary to modify original X-ray data by assuming the presence of an additional water molecule in the CK2 binding site structure with GTP.     

Investigating the disorder–order transition of calmodulin binding domain upon binding calmodulin using molecular dynamics simulation

We have studied the conformational transition of the calmodulin binding domains (CBD) in several calmodulin‐binding kinases, in which CBD changes from the disordered state to the ordered state when binding with calmodulin (CaM). Targeted molecular dynamics simulation was used to investigate the binding process of CaM and CBD of CaM‐dependent kinase I (CaMKI–CBD). The results show that CaMKI–CBD began to form an α‐helix and the interaction free energy between CaM and CaMKI–CBD increased once CaM fully encompassed CaMKI–CBD. Two series of CaM/CBD complex systems, including the complexes of CaM with the initially disordered and the final ordered CBD, were constructed to study the interaction using molecular dynamics simulations. Our analyses suggest that the VDW interaction plays a dominant role in CaM/CBD binding and is a key factor in the disorder–order transition of CBD. Additionally, the entropy effect is not in favor of the formation of the CaM/CBD complex, which is consistent with the experimental evidence. Based on the results, it appears that the CBD conformational change from a non‐compact extended structure to compact α‐helix is critical in gaining a favorable VDW interaction and interaction free energy. Copyright © 2009 John Wiley & Sons, Ltd.     

A comparative study of trypsin specificity based on QM/MM molecular dynamics simulation and QM/MM GBSA calculation

Hydrogen bonding and polar interactions play a key role in identification of protein-inhibitor binding specificity. Quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations combined with DFT and semi-empirical Hamiltonian (AM1d, RM1, PM3, and PM6) methods were performed to study the hydrogen bonding and polar interactions of two inhibitors BEN and BEN1 with trypsin. The results show that the accuracy of treating the hydrogen bonding and polar interactions using QM/MM MD simulation of PM6 can reach the one obtained by the DFT QM/MM MD simulation. Quantum mechanics/molecular mechanics generalized Born surface area (QM/MM-GBSA) method was applied to calculate binding affinities of inhibitors to trypsin and the results suggest that the accuracy of binding affinity prediction can be significantly affected by the accurate treatment of the hydrogen bonding and polar interactions. In addition, the calculated results also reveal the binding specificity of trypsin: (1) the amidinium groups of two inhibitors generate favorable salt bridge interaction with Asp189 and form hydrogen bonding interactions with Ser190 and Gly214, (2) the phenyl of inhibitors can produce favorable van der Waals interactions with the residues His58, Cys191, Gln192, Trp211, Gly212, and Cys215. This systematic and comparative study can provide guidance for the choice of QM/MM MD methods and the designs of new potent inhibitors targeting trypsin.     

Investigation on the binding mechanism of loratinib with the c-ros oncogene 1 (ROS1) receptor tyrosine kinase via molecular dynamics simulation and binding free energy calculations

The c-ros oncogene 1 (ROS1) has proven to be an important cancer target for the treatment of various human cancers. The anaplastic lymphoma kinase inhibitor crizotinib has been granted approval for the treatment of patients with ROS1 positive metastatic non-small-cell lung cancer by the Food and Drug Administration on 2016. However, serious resistance due to the secondary mutation of glycine 2032 to arginine (G2032R) was developed in clinical studies. Loratinib (PF-06463922), a macrocyclic analog of crizotinib, showed significantly improved inhibitory activity against wild–type (WT) ROS1 and ROS1^G2032R mutant. To provide insights into the inhibition mechanism, molecular dynamics simulations and free energy calculations were carried out for the complexes of loratinib with WT and G2032R mutated ROS1. The apo-ROS1^WT and apo-ROS1^G2032R systems showed similar RMSF distributions, while ROS1^G2032R-loratinib showed significantly higher than that of WT ROS1-loratinib, which revealed that the binding of loratinib to ROS1^G2032R significantly interfered the ?uctuation of protein. Calculations of binding free energies indicate that G2032R mutation significantly reduces the binding affinity of loratinib for ROS1, which arose mostly from the increase of conformation entropy and the decrease of solvation energy. Furthermore, detailed per-residue binding free energies highlighted the increased and decreased contributions of some residues in the G2032R mutated systems. The present study revealed the detailed inhibitory mechanism of loratinib as potent WT and G2032R mutated ROS1 inhibitor, which was expected to provide a basis for rational drug design.     

Evaluating the suitability of RNA intervention mechanism exerted by some flavonoid molecules against dengue virus MTase RNA capping site: a molecular docking,molecular dynamics simulation,and binding free energy study

Abstract

Dengue virus (DENV) is one of the most dangerous mosquito-borne human pathogens known to the mankind. Currently, no vaccines or standard therapy is avaliable to treate DENV infection. This makes the drug development against DENV more significant and challenging. The MTase domain of DENV RNA RdRp NS5 is a promising drug target, because this domain hosts the RNA capping process of DENV RNA to escape from human immune system. In the present study, we have analysed the RNA intervention mechanism exerted by flavoniod molecules against NS5 MTase RNA capping site by using molecular docking, molecular dynamics simulation and the binding free energy calculations. The results from the docking analysis confirmed that the RNA intervention mecanism is exerted by the quercetagetin (QGN) molecule with all necessary intermolecular interactions and high binding affinity. Notably, QGN forms strong hydrogen bonding interactions with Asn18, Leu20 and Ser150 residues and π???π stacking interaction with Phe25 residue. The apo and QGN bound NS5 MTase and QGN-NS5 MTase complex were used for MD simulation. The results of MD simulation reveal that the RMSD and RMSF values of QGN-MTase complex have increased on comparing the apo protein due to the effect of ligand binding. The binding free energy calulation includes prediction of total binding free energy of ligand-protein complex and per-residue free energy decomposition. The QGN binding to NS5 MTase affects it’s native motion, this result is found from Principal component analysis.

Communicated by Ramaswamy H. Sarma     

Computational insights into the binding pattern of mitochondrial calcium uniporter inhibitor through homology modeling,molecular dynamics simulation,binding free energy prediction and density functional theory calculation

Abstract

The mitochondrial calcium uniporter (MCU) is the critical protein of the inner mitochondrial membrane that is the primary mediator for calcium uptake into the mitochondrial matrix. Herein we built the optimal homology model of human MCU which was refined through all-atom molecular dynamics simulation. Then, the binding mode of known inhibitor was predicted through molecular docking method, along with molecular dynamics simulation and binding free energy calculation to verify the docking result and stability of the protein-inhibitor complex. Finally, density functional theory (DFT) calculation enhanced our understanding of the molecular interaction of MCU inhibitor. Our research would provide a deeper insight into the interactions between human MCU and its inhibitor, which boosts to develop novel therapy against MCU related disease.

Communicated by Ramaswamy H. Sarma     

Targeted molecular dynamics simulation studies of binding and conformational changes in E. coli MurD

Enzymes involved in the biosynthesis of bacterial peptidoglycan, an essential cell wall polymer unique to prokaryotic cells, represent a highly interesting target for antibacterial drug design. Structural studies of E. coli MurD, a three-domain ATP hydrolysis driven muramyl ligase revealed two inactive open conformations of the enzyme with a distinct C-terminal domain position. It was hypothesized that the rigid body rotation of this domain brings the enzyme to its closed active conformation, a structure, which was also determined experimentally. Targeted molecular dynamics 1 ns-length simulations were performed in order to examine the substrate binding process and gain insight into structural changes in the enzyme that occur during the conformational transitions into the active conformation. The key interactions essential for the conformational transitions and substrate binding were identified. The results of such studies provide an important step toward more powerful exploitation of experimental protein structures in structure-based inhibitor design.     

Multi‐spectroscopic and molecular dynamics simulations investigation of the binding mechanism of polybrominated diphenyl ethers to hen egg white lysozyme

Three PBDEs (BDE25, BDE47, and BDE154) were selected to investigate the interactions between PBDEs and hen egg white lysozyme (HEWL) by molecular modeling, fluorescence spectroscopy, and FT‐IR spectra. The docking results showed that hydrogen bonds were formed between BDE25 and residue TRP63 and between BDE47 and TRP63 with bond lengths of 2.178 Å and 2.146 Å, respectively. The molecular dynamics simulations indicated that van der Waals forces played a predominant role in the binding of three PBDEs to HEWL. The observed fluorescence quenching can be attributed to the formation of complexes between HEWL and PBDEs, and the quenching mechanism is a static quenching. According to Förster's non‐radiative energy transfer theory, the binding distances r were < 7 nm, indicating a high probability of energy transfer from HEWL to the three PBDEs. The synchronous fluorescence showed that the emission maximum wavelength of tryptophan (TRP) residues emerged a red‐shift. FT‐IR spectra indicated that BDE25, BDE47 and BDE154 induced the α‐helix percentage of HEWL decreased from 32.70% ± 1.64% to 28.27% ± 1.41%, 27.50% ± 1.38% and 29.78% ± 1.49%, respectively, whereas the percentage of random coil increased from 26.67% ± 1.33% to 27.60% ± 1.38%, 29.18% ± 1.46% and 30.59% ± 1.53%, respectively.     

A computational analysis of pyrazole-based inhibitors binding to Hsp90 using molecular dynamics simulation and the MM-GBSA method

Owing to the key role of heat-shock protein 90 (Hsp90) in the evolution, development and disease pathogenesis of cancer, it has been an important target for anti-cancer chemotherapy over the years. A five-nanosecond molecular dynamics simulation combined with the calculation of the binding free energy was carried out to investigate the binding mechanisms of three Hsp90 inhibitors 4BH, 2E1 and 2D9 to Hsp90. The binding free energy of each complex was computed using the molecular mechanics–generalised Born surface area method. Detailed binding free energies between each inhibitor and residues of Hsp90 were calculated using a per-residue basis decomposition method. The detailed inhibitor–residue interaction provides insights into binding mechanisms and in-depth understanding of the structure–affinity relationship. This study suggests that van der Waals energy is primarily responsible for driving the binding of the inhibitors to Hsp90, and the three inhibitors bind to Hsp90 in a similar binding mode. However, a substituent in 2D9 leads to higher binding free energy than the other two inhibitors. These data may assist in designing new potent drugs to combat cancer.     

Insight into binding mechanisms of inhibitors MKP56, MKP73, MKP86, and MKP97 to HIV-1 protease by using molecular dynamics simulation

HIV-1 protease (PR) has been a significant target for design of potent inhibitors curing acquired immunodeficiency syndrome. Molecular dynamics simulations coupled with molecular mechanics Poisson–Boltzmann surface area method were performed to study interaction modes of four inhibitors MKP56, MKP73, MKP86, and MKP97 with PR. The results suggest that the main force controlling interactions of inhibitors with PR should be contributed by van der Waals interactions between inhibitors and PR. The cross-correlation analyses based on MD trajectories show that inhibitor binding produces significant effect on the flap dynamics of PR. Hydrogen bond analyses indicate that inhibitors can form stable hydrogen bonding interactions with the residues from the catalytic strands of PR. The contributions of separate residues to inhibitor bindings are evaluated by using residue-based free energy decomposition method and the results demonstrate that the CH–π and CH–CH interactions between the hydrophobic groups of inhibitors with residues drive the associations of inhibitors with PR. We expect that this study can provide a significant theoretical aid for design of potent inhibitors targeting PR.     

Predicting binding modes of reversible peptide‐based inhibitors of falcipain‐2 consistent with structure–activity relationships

Falcipain‐2 (FP‐2) is a major hemoglobinase of Plasmodium falciparum, considered an important drug target for the development of antimalarials. A previous study reported a novel series of 20 reversible peptide‐based inhibitors of FP‐2. However, the lack of tridimensional structures of the complexes hinders further optimization strategies to enhance the inhibitory activity of the compounds. Here we report the prediction of the binding modes of the aforementioned inhibitors to FP‐2. A computational approach combining previous knowledge on the determinants of binding to the enzyme, docking, and postdocking refinement steps, is employed. The latter steps comprise molecular dynamics simulations and free energy calculations. Remarkably, this approach leads to the identification of near‐native ligand conformations when applied to a validation set of protein‐ligand structures. Overall, we proposed substrate‐like binding modes of the studied compounds fulfilling the structural requirements for FP‐2 binding and yielding free energy values that correlated well with the experimental data. Proteins 2017; 85:1666–1683. © 2017 Wiley Periodicals, Inc.     

Quercetin derivatives as non-nucleoside inhibitors for dengue polymerase: molecular docking,molecular dynamics simulation,and binding free energy calculation

Dengue is an important public health problem in tropical and subtropical regions of the world. Neither vaccine nor an antiviral medication is available to treat dengue. This insists the need of drug discovery for dengue. In order to find a potent lead molecule, RNA-dependent RNA polymerase which is essential for dengue viral replication is chosen as a drug target. As Quercetin showed antiviral activity against several viruses, quercetin derivatives developed by combinatorial library synthesis and mined from PubChem databases were screened for a potent anti-dengue viral agent. Our study predicted Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a dengue polymerase inhibitor. The results were validated by molecular dynamics simulation studies which reveal water bridges and hydrogen bonds as major contributors for the stability of the polymerase-lead complex. Interactions formed by this compound with residues Trp795, Arg792 and Glu351 are found to be essential for the stability of the polymerase-lead complex. Our study demonstrates Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a potent non-nucleoside inhibitor for dengue polymerase.     

Targeted molecular dynamics simulation studies of calcium binding and conformational change in the C-terminal half of gelsolin

Gelsolin consists of six related domains (G1-G6) and the C-terminal half (G4-G6) acts as a calcium sensor during the activation of the whole molecule, a process that involves large domain movements. In this study, we used targeted molecular dynamics simulations to elucidate the conformational transitions of G4-G6 at an atomic level. Domains G4 and G6 are initially ruptured, followed by a rotation of G6 by approximately 90 degrees , which is the dominant conformational change. During this period, local conformational changes occur at the G4 and G5 calcium-binding sites, facilitating large changes in interdomain distances. Alterations in the binding affinities of the calcium ions in these three domains appear to be related to local conformational changes at their binding sites. Analysis of the relative stabilities of the G4-G6-bound calcium ions suggests that they bind first to G6, then to G4, and finally to G5.     

An explorative study on Staphylococcus aureus MurE inhibitor: induced fit docking,binding free energy calculation,and molecular dynamics

Staphylococcus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the central domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔG_vdW, ?57.30?kcal/mol) and electrostatic solvation (ΔG_solv, ?36.86?kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.     

Pharmacophore modeling,multiple docking,and molecular dynamics studies on Wee1 kinase inhibitors

Wee1-like protein kinase (Wee1) is a tyrosine kinase that regulates the G₂ checkpoint and prevents entry into mitosis in response to DNA damage. Based on a series of signaling pathways initiated by Wee1, Wee1 has been recognized as a potential target for cancer therapy. To discover potent Wee1 inhibitors with novel scaffolds, ligand-based pharmacophore model has been built based on 101 known Wee1 inhibitors. Then the best pharmacophore model, AADRRR.340, with good partial least square (PLS) statistics (R²?=?0.9212, Q²?=?0.7457), was selected and validated. The validated model was used as a three-dimensional (3D) search query for databases virtual screening. The filtered molecules were further analyzed and refined by Lipinski’s rule of 5, multiple docking procedures (high throughput virtual screening (HTVS), standard precision (SP), genetic optimization for ligand docking (GOLD), extra precision (XP), and unique quantum polarized ligand docking (QPLD)); absorption, distribution, metabolism, excretion, and toxicity (ADMET) screening; and the Prime/molecular mechanics generalized born surface area (MM-GBSA) method binding free energy calculations. Eight leads were identified as potential Wee1 inhibitors, and a 50?ns molecular dynamics (MD) simulation was carried out for top four inhibitors to predict the stability of ligand–protein complex. Molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) based on MD simulation and the energy contribution per residue to the binding energy were calculated. In the end, three hits with good stabilization and affinity to protein were identified.

Communicated by Ramaswamy H. Sarma     

3D-QSAR-aided design of potent c-Met inhibitors using molecular dynamics simulation and binding free energy calculation

Abstract

Mesenchymal-epithelial transition factor (c-Met) is a member of receptor tyrosine kinase. It involves in various cellular signaling pathways which includes proliferation, motility, migration, and invasion. Over-expression of c-Met has been reported in various cancers. Hence, it is an ideal therapeutic target for cancer. The main objective of the study is to identify crucial residues involved in the inhibition of c-Met kinase and to design a series of potent imidazo [4,5-b] pyrazine derivatives as c-Met inhibitors. Docking was used to identify important active site residues involved in the inhibition of c-Met kinase which was further validated by 100 ns of molecular dynamics simulation and free energy calculation using molecular mechanics generalized born surface area. Furthermore, binding energy decomposition identified that residues Tyr1230, Met1211, Asp1222, Tyr1159, Met1160, Val1092, Ala1108, and Leu1157 contributed favorably to the binding stability of compound 32. Receptor-guided Comparative Molecular Field Analysis (CoMFA) (q² = 0.751, NOC = 6, r² = 0.933) and Comparative Molecular Similarity Indices Analysis (COMSIA) (q² = 0.744, NOC = 6, r² = 0.950) models were generated based on the docked conformation of the most active compound 32. The robustness of these models was tested using various validation techniques and found to be predictive. The results of CoMFA and CoMSIA contour maps exposed the regions favorable to enhance the activity. Based on this information, 27 novel c-Met inhibitors were designed. These designed compounds exhibited potent activity than the most active compound of the existing dataset.

Communicated by Ramaswamy H. Sarma     

Investigating the dynamics and polyanion binding sites of fibroblast growth factor‐1 using hydrogen‐deuterium exchange mass spectrometry

In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF‐1) as well as the binding sites of various polyanions including poly‐sulfates (heparin and low MW heparin) and poly‐phosphates (phytic acid and ATP) using hydrogen‐deuterium exchange mass spectrometry (HX‐MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B‐factors and compared with a residue level heat map generated from HX‐MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C‐terminal strands 8, 9, and 10 appear to be more rigid which is also consistent with crystallographic B‐factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF‐1 in complex with heparin have shown that heparin binds to N‐terminal Asn18 and to C‐terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127, and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX‐MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF‐1. Using HX‐MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C‐terminal region of FGF‐1 along with two distinct N‐terminal regions that contains lysines and arginines/histidines. This confirms the interactions between FGF‐1 and polyanions are primary directed by electrostatics.