Molecular Dynamics Simulations on the Complexes of Glucoamylase II (471) from Aspergillus awamori var. X100 with 1-Deoxynojirimycin and Lentiginosine

Molecular dynamics (MD) simulations on the complexes of glucoamylase II (471) from Aspergillus awamori var. X100 with two powerful inhibitors, 1-deoxynojirimycin and (+)-lentiginosine, have been performed, in order to build a model for these complexes in solution and to clarify the structure-activity relationship. MD calculations were carried out for 105 ps, over a 15 Å sphere centered on the inhibitors. A 8 Å residue-based cut-off was used, and the calculations were performed with explicit inclusion of solvent molecules. The MD structure of the complex 1-deoxynojirimycin-glucoamylase shows only minor deviations from the available X-ray structure. The MD structure of the complex of (+)-lentiginosine-glucoamylase, obtained by docking the inhibitor into the active site, suggests us a suitable orientation for the molecule into the enzyme cavity, which can rationalize the high inhibition activity found for (+)-lentiginosine towards amyloglucosidase from A. niger.     

Molecular mechanics PBSA ligand binding energy and interaction of Efavirenz derivatives with HIV-1 reverse transcriptase

In order to evaluate the properties of several HIV-1 reverse transcripase(RT) inhibitors, Efavirenz (SUSTIVA) and a set of its derivatives (benzoxazinones) have been placed into the nonnucleoside analogue binding site of the enzyme by molecular docking. The resulting geometries were used for a molecular dynamics simulation and binding energy calculations. The enzyme-inhibitor binding energies were estimated from experimental inhibitory activities (IC90). The correlation of the predicted and experimental binding energies were satisfactory acceptable as indicated by r2 = 0.865. Based on MD simulations, the obtained results indicate that the tight association of the ligand to the HIV-1 RT binding pocket was based on hydrogen bonding between Efavirenz's N1 and the oxygen of the backbone of Lys 101, with an estimated average distance of 1.88 A. Moreover, electrostatic interaction was mainly contributed by two amino acid residues in the binding site; Lys 101 and His 235. MD simulations open the possibility to study the reaction of the flexible enzyme to those substances as well as the overall affinity.     

Steered molecular dynamics simulations reveal important mechanisms in reversible monoamine oxidase B inhibition

The monotopic membrane protein monoamine oxidase B (MAO B) is an important drug target for Parkinson's disease. In order to design more specific, and thereby more effective, inhibitors for this enzyme, it is necessary to determine what factors govern inhibitor specificity and the inhibitor binding process, including the roles of the lipid bilayer, the active site loop, and several key residues within the binding pocket. Atomistic molecular dynamics simulations of MAO B either embedded in a lipid bilayer or free in solution have been performed. The simulations suggest that the bilayer controls the availability of the active site cavity by regulating the degree of fluctuation in two key loops that form the greater part of the active site entrance (residues 85-110 and 155-165). In turn, the enzyme itself causes local thinning and a decrease in area per lipid of the surrounding bilayer environment. Additional MD simulations of MAO B in complex with seven different reversible inhibitors followed by nonequilibrium steered MD simulations of the inhibitor unbinding have also been performed. The simulations demonstrate that the average energy of interaction between inhibitor and MAO B residues during inhibitor egress is an effective indicator of inhibitor strength and is also useful for identifying key residues that govern inhibitor specificity. These data provide researchers with valuable tools for designing effective MAO B inhibitors as well as outline a method that can be translated to the study of other enzyme-inhibitor complexes.     

In silico docking and molecular dynamics simulation of 3-dehydroquinate synthase (DHQS) from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The shikimate pathway is as an attractive target because it is present in bacteria, algae, fungi, and plants but does not occur in mammals. In Mycobacterium tuberculosis (MTB), the shikimate pathway is integral to the biosynthesis of naphthoquinones, menaquinones, and mycobactin. In these study, novel inhibitors of 3-dehydroquinate synthase (DHQS), an enzyme that catalyzes the second step of the shikimate pathway in MTB, were determined. 12,165 compounds were selected from two public databases through virtual screening and molecular docking analysis using PyRx 8.0 and Autodock 4.2, respectively. A total of 18 compounds with the best binding energies (?13.23 to ?8.22 kcal/mol) were then selected and screened for absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, and nine of those compounds were found to satisfy all of the ADME and toxicity criteria. Among those nine, the three compounds—ZINC633887?(binding energy =??10.29 kcal/mol), ZINC08983432?(?9.34 kcal/mol), and PubChem73393?(?8.61 kcal/mol)—with the best binding energies were further selected for molecular dynamics (MD) simulation analysis. The results of the 50-ns MD simulations showed that the two compounds ZINC633887 and PubChem73393 formed stable complexes with DHQS and that the structures of those two ligands remained largely unchanged at the ligand-binding site during the simulations. These two compounds identified through docking and MD simulation are potential candidates for the treatment of TB, and should undergo validation in vivo and in vitro.     

Theoretical calculations of the catalytic triad in short-chain alcohol dehydrogenases/reductases

Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD⁺ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.     

An In Silico Analysis of the Binding Modes and Binding Affinities of Small Molecule Modulators of PDZ-Peptide Interactions

Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson’s disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K_i or K_d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD) simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K_i or K_d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors of PDZ-peptide complexes.     

Identification of Novel Aldose Reductase Inhibitors from Spices: A Molecular Docking and Simulation Study

Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR), the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from Zingiber officinale (ginger), Curcuma longa (turmeric) Allium sativum (garlic) and Trigonella foenum graecum (fenugreek). Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors.     

Identification of new Hsp90 inhibitors by structure-based virtual screening

Structure-based virtual screening identified pyrimidine-2,4,6-trione and 4H-1,2,4-triazole-3-thiol as novel scaffolds of Hsp90 ATPase inhibitors. Their binding modes in the ATP-binding pocket of Hsp90 were analyzed using AutoDoc program combined with molecular dynamics (MD) simulations.     

Binding affinity of hydroxamate inhibitors of matrix metalloproteinase-2

Here we report molecular dynamics (MD) and free energy perturbation (FEP) simulations applied to hydroxamate-matrix metalloproteinase-2 (MMP-2) complex systems. We have developed some new force field parameters for the hydroxamate functional group that were not included in the AMBER94 force field but were necessary in our simulations. For the representation of the active zinc center, a bonded model was adopted in which restrained electrostatic potential fitting (RESP) charges were used as the electrostatic representation of this model. Using the resulted bonded model, FEP simulations predict the relative binding free energy in good agreement with the experimental value. By analyzing the molecular dynamics (MD) trajectories of the two complex systems, we can provide an explanation of why one of the two inhibitors is favored over the other. The results provide a chemical insight into the interactions between inhibitor and enzyme, and can indicate changes in the inhibitor that would enhance inhibitor–enzyme interactions.Figure The scheme of the binding site     

Binding free energies for nicotine analogs inhibiting cytochrome P450 2A6 by a combined use of molecular dynamics simulations and QM/MM-PBSA calculations

Molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations have been performed to explore the dynamic behaviors of cytochrome P450 2A6 (CYP2A6) binding with nicotine analogs (that are typical inhibitors) and to calculate their binding free energies in combination with Poisson–Boltzmann surface area (PBSA) calculations. The combined MD simulations and QM/MM-PBSA calculations reveal that the most important structural parameters affecting the CYP2A6-inhibitor binding affinity are two crucial internuclear distances, that is, the distance between the heme iron atom of CYP2A6 and the coordinating atom of the inhibitor, and the hydrogen-bonding distance between the N297 side chain of CYP2A6 and the pyridine nitrogen of the inhibitor. The combined MD simulations and QM/MM-PBSA calculations have led to dynamic CYP2A6-inhibitor binding structures that are consistent with the observed dynamic behaviors and structural features of CYP2A6-inhibitor binding, and led to the binding free energies that are in good agreement with the experimentally-derived binding free energies. The agreement between the calculated binding free energies and the experimentally-derived binding free energies suggests that the combined MD and QM/MM-PBSA approach may be used as a valuable tool to accurately predict the CYP2A6-inhibitor binding affinities in future computational design of new, potent and selective CYP2A6 inhibitors.     

Successful molecular dynamics simulation of the zinc-bound farnesyltransferase using the cationic dummy atom approach

Farnesyltransferase (FT) inhibitors can suppress tumor cell proliferation without substantially interfering with normal cell growth, thus holding promise for cancer treatment. A structure-based approach to the design of improved FT inhibitors relies on knowledge of the conformational flexibility of the zinc-containing active site of FT. Although several X-ray structures of FT have been reported, detailed information regarding the active site conformational flexibility of the enzyme is still not available. Molecular dynamics (MD) simulations of FT can offer the requisite information, but have not been applied due to a lack of effective methods for simulating the four-ligand coordination of zinc in proteins. Here, we report in detail the problems that occurred in the conventional MD simulations of the zinc-bound FT and a solution to these problems by employing a simple method that uses cationic dummy atoms to impose orientational requirement for zinc ligands. A successful 1.0 ns (1.0 fs time step) MD simulation of zinc-bound FT suggests that nine conserved residues (Asn127alpha, Gln162alpha, Asn165alpha, Gln195alpha, His248beta, Lys294beta, Leu295beta, Lys353beta, and Ser357beta) in the active site of mammalian FT are relatively mobile. Some of these residues might be involved in the ligand-induced active site conformational rearrangement upon binding and deserve attention in screening and design of improved FT inhibitors for cancer chemotherapy.     

Structural insight into Mycobacterium tuberculosis maltosyl transferase inhibitors: pharmacophore-based virtual screening,docking, and molecular dynamics simulations

Pharmacophore-based virtual screening, subsequent docking, and molecular dynamics (MD) simulations have been done to identify potential inhibitors of maltosyl transferase of Mycobacterium tuberculosis (mtb GlgE). Ligand and structure-based pharmacophore models representing its primary binding site (pbs) and unique secondary binding site 2 (sbs2), respectively, were constructed based on the three dimensional structure of mtb GlgE. These pharmacophore models were further used for screening of ZINC and antituberculosis compounds database (ATD). Virtually screened molecules satisfying Lipinski’s rule of five were then analyzed using docking studies and have identified 23 molecules with better binding affinity than its natural substrate, maltose. Four top scoring ligands from ZINC and ATD that either binds to pbs or sbs2 have been subjected to 10 ns each MD simulations and binding free energy calculations. Results of these studies have confirmed stable protein ligand binding. Results reported in the article are likely to be helpful in antitubercular therapeutic development research.     

Intermolecular insights into allosteric inhibition of histone lysine-specific demethylase 1

BackgroundHistone lysine-specific demethylase 1 (LSD1) has become a potential anticancer target for the novel drug discovery. Recent reports have shown that SP2509 and its derivatives strongly inhibit LSD1 as allosteric inhibitors. However, the binding mechanism of these allosteric inhibitors in the allosteric site of LSD1 is not known yet.MethodsThe stability and binding mechanism of allosteric inhibitors in the binding site of LSD1 were evaluated by molecular docking, ligand-based pharmacophore, molecular dynamics (MD) simulations, molecular mechanics generalized born surface area (MM/GBSA) analysis, quantum mechanics/molecular mechanics (QM/MM) calculation and Hirshfeld surface analysis.ResultsThe conformational geometry and the intermolecular interactions of allosteric inhibitors showed high binding affinity towards allosteric site of LSD1 with the neighboring amino acids (Gly358, Cys360, Leu362, Asp375 and Glu379). Meanwhile, MD simulations and MM/GBSA analysis were performed on selected allosteric inhibitors in complex with LSD1 protein, which confirmed the high stability and binding affinity of these inhibitors in the allosteric site of LSD1.ConclusionThe simulation results revealed the crucial factors accounting for allosteric inhibitors of LSD1, including different protein–ligand interactions, the positions and conformations of key residues, and the ligands flexibilities. Meanwhile, a halogen bond interaction between chlorine atom of ligand and key residues Trp531 and His532 was recurrent in our analysis confirming its importance.General significanceOverall, our research analyzed in depth the binding modes of allosteric inhibitors with LSD1 and could provide useful information for the design of novel allosteric inhibitors.     

Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts.

Molecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such as d-phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 A centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 A sphere of water molecules. MD calculations were carried out for 45-60 ps. There is no large deviation from the available X-ray structures of native and the dPhe adduct for the MD structures. Average MD structures were calculated starting from the X-ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed.     

Interaction of wild type, G68R and L125M isoforms of the arylamine-N-acetyltransferase from Mycobacterium tuberculosis with isoniazid: a computational study on a new possible mechanism of resistance

Isoniazid (INH) is a front-line drug used in the treatment of tuberculosis (TB), a disease that remains a major cause of death worldwide. Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase-peroxidase (CP) enzyme. Recent studies have suggested that acetylation of INH by the arylamine-N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) may be a possible cause of inactivation of the drug thus resulting in resistant strains. In this study, computational techniques were applied to investigate the binding of isoniazid to three TBNAT isoforms: wild type, G68R and L125M. Since there is no experimental structure available, molecular dynamics (MD) simulations were initially used for the refinement of TBNAT homology models. Distinct conformations of the models were selected during the production stage of MD simulations for molecular docking experiments with the drug. Finally, each mode of binding was refined by new molecular MD simulations. Essential dynamics (ED) analysis and linear interaction energy calculations (LIE) were used to evaluate the impact of amino acid substitutions on the structural and binding properties of the enzymes. The results suggest that the wild type and the G68R TBNATs have a similar pattern of affinity to INH. On the other hand, the calculated enzyme-INH dissociation constant (KD) was estimated 33 times lower for L125M isoform in comparison with wild type enzyme. This last finding is consistent with the hypothesis that isolated mutations in the tbnat gene can produce M. tuberculosis strains resistant to isoniazid.     

Subtle functional collective motions in pancreatic-like ribonucleases: from ribonuclease A to angiogenin

The analysis of the dynamic behavior of enzymes is fundamental to structural biology. A direct relationship between protein flexibility and biological function has been shown for bovine pancreatic ribonuclease (RNase A) (Rasmussen et al., Nature 1992;357:423-424). More recently, crystallographic studies have shown that functional motions in RNase A involve the enzyme beta-sheet regions that move concertedly on substrate binding and release (Vitagliano et al., Proteins 2002;46:97-104). These motions have been shown to correspond to intrinsic dynamic properties of the native enzyme by molecular dynamics (MD) simulations. To unveil the occurrence of these collective motions in other members of pancreatic-like superfamily, we carried out MD simulations on human angiogenin (Ang). Essential dynamics (ED) analyses performed on the trajectories reveal that Ang exhibits collective motions similar to RNase A, despite the limited sequence identity (33%) of the two proteins. Furthermore, we show that these collective motions are also present in ensembles of experimentally determined structures of both Ang and RNase A. Finally, these subtle concerted beta-sheet motions were also observed for other two members of the pancreatic-like superfamily by comparing the ligand-bound and ligand-free structures of these enzymes. Taken together, these findings suggest that pancreatic-like ribonucleases share an evolutionary conserved dynamic behavior consisting of subtle beta-sheet motions, which are essential for substrate binding and release.     

Shape-based virtual screening,docking, and molecular dynamics simulations to identify Mtb-ASADH inhibitors

Aspartate β-semialdehyde dehydrogenase (ASADH) is a key enzyme for the biosynthesis of essential amino acids and several important metabolites in microbes. Inhibition of ASADH enzyme is a promising drug target strategy against Mycobacterium tuberculosis (Mtb). In this work, in silico approach was used to identify potent inhibitors of Mtb-ASADH. Aspartyl β-difluorophosphonate (β-AFP), a known lead compound, was used to understand the molecular recognition interactions (using molecular docking and molecular dynamics analysis). This analysis helped in validating the computational protocol and established the participation of Arg99, Glu224, Cys130, Arg249, and His256 amino acids as the key amino acids in stabilizing ligand–enzyme interactions for effective binding, an essential feature is H-bonding interactions with the two arginyl residues at the two ends of the ligand. Best binding conformation of β-AFP was selected as a template for shape-based virtual screening (ZINC and NCI databases) to identify compounds that competitively inhibit the Mtb-ASADH. The top rank hits were further subjected to ADME and toxicity filters. Final filter was based on molecular docking analysis. Each screened molecule carries the characteristics of the highly electronegative groups on both sides separated by an average distance of 6?Å. Finally, the best predicted 20 compounds exhibited minimum three H-bonding interactions with Arg99 and Arg249. These identified hits can be further used for designing the more potent inhibitors against ASADH family. MD simulations were also performed on two selected compounds (NSC4862 and ZINC02534243) for further validation. During the MD simulations, both compounds showed same H-bonding interactions and remained bound to key active residues of Mtb-ASADH.     

Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei

RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.     

Tunnel dynamics of quinone derivatives and its coupling to protein conformational rearrangements in respiratory complex I

Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 Å long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.     

Discovery of novel nonpeptide small-molecule NRP1 antagonists: Virtual screening,molecular simulation and structural modification

Multifaceted roles of vascular endothelial growth factor (VEGF)-neuropilin-1 (NRP1) interaction have been implicated in cancer, but reports on small-molecule inhibitors of VEGF-NRP1 interaction are scarce. Herein, we describe the identification of 1, a novel nonpeptide small-molecule NRP1 antagonist with moderate activity via structure-based virtual screening. Ensemble docking and molecular dynamics (MD) simulations of 1 were carried out and an interesting binding model was obtained. We found that the “aromatic box” enclosed by Tyr297, Trp301 and Tyr353 of NRP1 is critical for NRP1-1 binding. Further structure modification of 1 based on the binding model derived from MD simulations resulted in the identification of 12a with significantly improved activity.