Structure of Plasmodium vivax dihydrofolate reductase determined by homology modeling and molecular dynamics refinement

The structure of Plasmodium vivax dihydrofolate reductase (PvDHFR), a potentially important target for antimalarial chemotherapy, was determined by means of homology modeling and molecular dynamics refinement. The structure proved to be consistent with DHFRs of known crystal structure. The comparison of the complexes of the antifolate inhibitor pyrimethamine bound at the active sites of PvDHFR and PfDHFR, the related enzyme from Plasmodium falciparum, prospected the possibility of using structure-based drug design to develop inhibitors that are effective against both malarial enzymes. This study constitutes a first step toward understanding of the antifolate-PvDHFR molecular interactions and possible rationalization of resistance in vivax malaria.     

The design, synthesis, and biological evaluation of analogues of the serine-threonine protein phosphatase 1 and 2A selective inhibitor microcystin LA: rational modifications imparting PP1 selectivity

Based on the results from previously reported molecular modeling analyses of the interactions between the inhibitor microcystin and the serine-threonine protein phosphatases 1 and 2A, we have designed analogues of microcystin LA with structural modifications intended to impart PP1 selectivity. The synthesis of several first generation analogues followed by inhibition assays revealed that all three are PP1-selective, as predicted. Although the observed selectivities are modest, one of the designed analogues is more selective for PP1 than any known small molecule inhibitor.     

In silico modelling and molecular dynamics simulation studies of thiazolidine based PTP1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) has been identified as a negative regulator of insulin and leptin signalling pathway; hence, it can be considered as a new therapeutic target of intervention for the treatment of type 2 diabetes. Inhibition of this molecular target takes care of both diabetes and obesity, i.e. diabestiy. In order to get more information on identification and optimization of lead, pharmacophore modelling, atom-based 3D QSAR, docking and molecular dynamics studies were carried out on a set of ligands containing thiazolidine scaffold. A six-point pharmacophore model consisting of three hydrogen bond acceptor (A), one negative ionic (N) and two aromatic rings (R) with discrete geometries as pharmacophoric features were developed for a predictive 3D QSAR model. The probable binding conformation of the ligands within the active site was studied through molecular docking. The molecular interactions and the structural features responsible for PTP1B inhibition and selectivity were further supplemented by molecular dynamics simulation study for a time scale of 30 ns. The present investigation has identified some of the indispensible structural features of thiazolidine analogues which can further be explored to optimize PTP1B inhibitors.     

Antifolate resistance and its circumvention by new analogues.

We have established human leukemia cell lines made resistant to various antifolate drugs and analyzed resistance mechanisms developed in these cells at the cellular and molecular levels. The cells acquired resistance to antifolate drug(s) through: (1) impaired drug uptake via the reduced folate carrier, (2) increased activity of the target enzymes[dihydrofolate reductase(DHFR) or thymidylate synthase(TS)] resulted from a concomitant amplification and overexpression of their gene, (3) induction of a variant DHFR with a low affinity for antifolate drug(s) used for the selection of resistance, and (4) defective polyglutamation. Each resistance mechanism was not necessarily induced at random, but appeared to relate to the biochemical and pharmacological properties of the drug exposed, biological dispositions of the cells, drug-exposure manners to, or culture conditions of the cells. Since it has been shown that a minor modification at the specified position of the folate structure resulted in a drastic change in its pharmacological properties, many new compounds have been rationally designed on the basis of the knowledge of relationships between structure modifications and pharmacological properties. The step-by-step approach to the development of new analogues led to the discoveries of several promising antifolate drugs such as trimetrexate and raltitrexed, which can overcome the acquired and natural resistance to methotrexate, a classical antifolate, and clinical trials of these newer classes of antifolate compounds are currently underway.     

Binding of a C-end rule peptide to the neuropilin-1 receptor: a molecular modeling approach

Neuropilin-1 (NRP-1) is a receptor that plays an essential role in angiogenesis, vascular permeability, and nervous system development. Previous studies have shown that peptides with an N-terminal Arg, especially peptides with the four-residue consensus sequence R/K/XXR/K, bind to NRP-1 cell surfaces. Peptides containing such consensus sequences promote binding and internalization into cells, while blocking the C-terminal Arg (or Lys) prevents the internalization. In this study, we use molecular dynamics simulations to model the structural properties of the NRP-1 complex with a prototypic CendR peptide, RPAR. Our simulations show that RPAR binds NRP-1 through specific interactions of the RPAR C-terminus: three hydrogen bonds and a salt bridge anchor the ligand in the receptor pocket. The modeling results were used as the starting point for a systematic computational study of new RPAR analogues based on chemical modifications of their natural amino acids. Comparison of the structural properties of the new peptide-receptor complexes with the original organization suggests that some of the analogues can increase the binding affinity while reducing the natural sensitivity of RXXR to endogenous proteases.     

2-Benzyl and 2-phenyl-3-hydroxypropyl pivalates as protein kinase C ligands

A series of 2-benzyl and 2-phenyl-3-hydroxypropyl pivalates designed to incorporate the principal pharmacophores of phorbol esters have been synthesized and tested as PKC-alpha ligands. Among the analogues, 13c exhibited the most potent binding affinity with a Ki = 0.7 microM. The synthesized analogues were subjected to molecular modeling analysis based on two alternative models of the phorbol pharmacophore and a docking study of 13c was carried out.     

Crystallographic studies on N-azidoacetyl-beta-D-glucopyranosylamine, an inhibitor of glycogen phosphorylase: comparison with N-acetyl-beta-D-glucopyranosylamine

N-acetyl-beta-D-glucopyranosylamine (NAG) is a potent inhibitor (Ki=32 microM) of glycogen phosphorylase b (GPb), and has been employed as a lead compound for the structure-based design of new analogues, in an effort to utilize its potential as a hypoglycaemic agent. Replacement of the acetamido group by azidoacetamido group resulted in an inhibitor, N-azidoacetyl-beta-D-glucopyranosylamine (azido-NAG), with a Ki value of 48.7 microM, in the direction of glycogen synthesis. In order to elucidate the mechanism of inhibition, we determined the ligand structure in complex with GPb at 2.03 A resolution, and the structure of the fully acetylated derivative in the free form. The molecular packing of the latter is stabilized by a number of bifurcated hydrogen bonds of which the one involving a bifurcated C-H...N...H-C type hydrogen bonding is rather unique in organic azides. Azido-NAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilizes the T-state conformation of the 280 s loop by making several favourable contacts to residues of this loop. The difference observed in the Ki values of the two analogues can be interpreted in terms of desolvation effects, subtle structural changes of protein residues and changes in water structure.     

3D-QSAR, homology modeling, and molecular docking studies on spiropiperidines analogues as agonists of nociceptin/orphanin FQ receptor

The nociceptin/orphanin FQ receptor (NOP) has been implicated in a wide range of biological functions, including pain, anxiety, depression and drug abuse. Especially, its agonists have a great potential to be developed into anxiolytics. However, the crystal structure of NOP is still not available. In the present work, both structure-based and ligand-based modeling methods have been used to achieve a comprehensive understanding on 67N-substituted spiropiperidine analogues as NOP agonists. The comparative molecular-field analysis method was performed to formulate a reasonable 3D-QSAR model (cross-validated coefficient q(2)=0.819 and conventional r(2)=0.950), whose robustness and predictability were further verified by leave-eight-out, Y-randomization, and external test-set validations. The excellent performance of CoMFA to the affinity differences among these compounds was attributed to the contributions of electrostatic/hydrogen-bonding and steric/hydrophobic interactions, which was supported by the Surflex-Dock and CDOCKER molecular-docking simulations based on the 3D model of NOP built by the homology modeling method. The CoMFA contour maps and the molecular docking simulations were integrated to propose a binding mode for the spiropiperidine analogues at the binding site of NOP.     

Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody.

A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.     

Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsin inhibitor (Schistocerca gregaria chymotrypsin inhibitor) were designed and synthesized by convergent peptide synthesis. For each model peptide, the inhibitory constant (Ki) on chymotrypsin and the solution structure were determined. In addition, molecular dynamics calculations were performed for all of them. Two models containing approximately half of the parent inhibitor (17 of 35 residues) were designed and subsequently found to have no substantial inhibitory activity (Ki values in the mM range). The third model composed of 24 amino acid residues proved to be an effective (Ki approximately 10(-7)) inhibitor of bovine chymotrypsin. Both the solution structure properties determined by NMR spectroscopy and the dynamic behaviour of the latter model system are comparable to the native inhibitor. In contrast, the structure and dynamics of the first two related model peptides show characteristic differences. We suggest that the conformation and flexibility of the modelled protease inhibitor are crucial for its biological efficiency. Moreover, the structural and dynamic features of the binding loop (28-33) and those of the rest of the molecule appear to be interdependent. Most importantly, these structural characteristics can be rationally modified, at least partially, by peptide design.     

Molecular dynamics study of the conformations of glycosidic linkages in sialic acid modified ganglioside GM3 analogues

The objective of the present study is to model the analogues of monosialoganglioside (GM3) by making modifications in its sialic acid residue with different substitutions in aqueous environment and to determine their structural stability based upon computational molecular dynamics. Molecular mechanics and molecular dynamics investigation was carried out to study the conformational preferences of the analogues of GM3. Dynamic simulations were carried out on the analogues of GM3 varying in the substituents at C-1, C-4, C-5, C-8 and C-9 positions of their sialic acid or Neuraminic acid (NeuAc) residue. The analogues are soaked in a periodic box of TIP3P water as solvent and subjected to a 10 ns molecular dynamics (MD) simulation using AMBER ff03 and gaff force fields with 30 ps equilibration. The analogue of GM3 with 9-N-succNeuAc (analogue5, C9 substitution) was observed to have the lowest energy of ?6112.5 kcal/mol. Graphical analysis made on the MD trajectory reveals the direct and water mediated hydrogen bonds existing in these sialic acid analogues. The preferable conformations for glycosidic linkages of GM3 analogues found in different minimum energy regions in the conformational maps were identified. This study sheds light on the conformational preferences of GM3 analogues which may be essential for the design of GM3 analogues as inhibitors for different ganglioside specific pathogenic proteins such as bacterial toxins, influenza toxins and neuraminidases.     

Molecular Modeling Study of the Norfloxacin-DNA Complex

Abstract

Molecular modeling and molecular dynamics were performed to investigate the interaction of norfloxacin with the DNA oligonucleotide 5′-d(ATACGTAT)₂. Eight quinolone-DNA binding structures were built by molecular modeling on the basis of experimental results. A 100ps molecular dynamics calculation was carried out on two groove binding models and six partially intercalating models. The resulting average structures were compared with each other and to free DNA structure as a reference. The favorable binding mode of norfloxacin to a DNA substrate was pursued by structural assess including steric hindrance, presence of hydrogen-bonding, non-bonding energies of the complex and presence of abnormal structural distortion. Although two of the intercalative models showed the highest binding energy and the lowest non-bonding interaction energy, they presented structural features which contrast with experimental results. On the other hand, one groove binding model demonstrated the most acceptable structure when the experimental observation was accounted. In this model, hydrogen bonding of the carbonyl and carboxyl group of the norfloxacin rings with the DNA bases was present, and norfloxacin binds to the amine group of the guanine base which protrudes toward the minor groove of B-DNA.     

Molecular modeling study of the norfloxacin-DNA complex

Molecular modeling and molecular dynamics were performed to investigate the interaction of norfloxacin with the DNA oligonucleotide 5'-d(ATACGTAT)(2). Eight quinolone-DNA binding structures were built by molecular modeling on the basis of experimental results. A 100ps molecular dynamics calculation was carried out on two groove binding models and six partially intercalating models. The resulting average structures were compared with each other and to free DNA structure as a reference. The favorable binding mode of norfloxacin to a DNA substrate was pursued by structural assess including steric hindrance, presence of hydrogen-bonding, non-bonding energies of the complex and presence of abnormal structural distortion. Although two of the intercalative models showed the highest binding energy and the lowest non-bonding interaction energy, they presented structural features which contrast with experimental results. On the other hand, one groove binding model demonstrated the most acceptable structure when the experimental observation was accounted. In this model, hydrogen bonding of the carbonyl and carboxyl group of the norfloxacin rings with the DNA bases was present, and norfloxacin binds to the amine group of the guanine base which protrudes toward the minor groove of B-DNA.     

In silico characterization and molecular dynamics simulation of Pfcyc-1, a cyclin homolog of Plasmodium falciparum

Malaria is still one of the deadly diseases resulting in deaths of millions of people worldwide and situation has become worse due to alarming rise in anti-malarial drug resistance. Genome sequence availability of Plasmodium falciparum, the main causal organism of severe malaria in humans, has enabled identification of various parasite cell cycle regulators like several cyclins and cyclin dependent kinases or CDKs which are promising novel drug targets for Malaria. Here, we present in silico characterization of tertiary structure of Pfcyc-1, a P. falciparum cyclin homolog, which enables identification of key structural elements that contribute to its tertiary structure and function. We have investigated the structure and dynamics of Pfcyc-1 structural model by performing 10?ns molecular dynamics (MD) simulation. Our study indicates that despite poor sequence similarities with cyclin H and A, the characteristic structural cyclin domains are conserved in Pfcyc-1 too. The Pfcyc-1 model reveals a cyclin box, consisting of two tandemly repeating five-helix bundles separated by a linker hinge peptide. Furthermore, the amino acid residues in other known cyclins mediating cyclin-CDK interactions are conserved in Pfcyc-1. The model and its MD simulation offer a first ever structural annotation of any plasmodium cyclin, which along with sequence comparisons, helps in identification of important functional residues mediating the Pfcyc-1-CDK like interactions.     

Characterization of adenosine receptor in its native environment: insights from molecular dynamics simulations of palmitoylated/glycosylated, membrane-integrated human A2B adenosine receptor

Selective A(2B) receptor antagonists and agonists may play a role in important pathologies such as gastrointestinal, neurological (i.e., Alzheimer disease and dementia) and hypersensitive disorders (i.e., asthma), diabetes, atherosclerosis, restenosis and cancer. Hence, it is regarded as a good target for the development of clinically useful agents. In this study, the effects of lipid bilayer, N-acetylglucosamine and S-palmitoyl on the dynamic behavior of A(2B)AR model is explored. Homology modeling, molecular docking and molecular dynamics simulations were performed to explore structural features of A(2B)AR in the presence of lipid bilayer. Twenty ns MD simulation was performed on the constructed model inserted in a hydrated lipid bilayer to examine stability of the best model. OSIP339391 as the most potent antagonist was docked in the active site of the model. Another MD simulation was performed on the ligand-protein complex to explore effects of the bilayer on this complex. A similar procedure was performed for the modified protein with N-acetylglucosamine and S-palmitoyl moieties in its structure. Phe173 and Glu174 located in EL2 were determined to be involved in ligand-receptor interactions through π-π stacking and hydrogen bonding. Asn254 was crucial to form hydrogen-bonding. The reliability of the model was assessed through docking using both commercial and synthetic antagonists and an r(2) of 0.70 was achieved. Our results show that molecular dynamics simulations of palmitoylated/glycosylated, membrane-integrated human A(2B)AR in its native environment is a possible approach and this model can be used for designing potent and selective A(2B)AR antagonists.     

Modeling and prediction of the mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monoliths

n-Octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monoliths were prepared for the rapid screening and determination of puerarin content of a crude extract Radix puerariae. The mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified monoliths were investigated using a variety of solvent systems, chromatographic evaluation and molecular dynamics (MDs) modeling. The equilibrated conformations between cross-linked polymers and target molecules were obtained from MD modeling. Both the polymer skeleton and functional groups played important roles in the recognition process. The cross-linker formed a structural network skeleton, in which recognition cavities were formed surrounded by functional groups. The polymer network structures provided good interaction access for isoflavones. The active groups recognized isoflavones by both intermolecular hydrogen bonding and hydrophobic interaction. The interaction energies and retention factors between polymers and target molecules were also evaluated and compared. A higher value of interaction energy corresponded to a higher value of retention factor. The potential of using modeling technology for predicting the chromatographic performances of target molecules was explored.     

Insight into molecular interactions between two PB1 domains

Specific protein-protein interactions play crucial roles in the regulation of any biological process. Recently, a new protein-protein interaction domain termed PB1 (Phox and Bem1) was identified, which is conserved throughout evolution and present in diverse proteins functioning in signal transduction, cell polarity and survival. Here, we investigated the structure and molecular interactions of the PB1 heterodimer complex composed of the PB1 domains of the yeast proteins Bem1 and Cdc24. A structural model of the Cdc24 PB1 was built by homology modeling and molecular dynamics simulations, and experimentally validated by 15N nuclear Overhauser effect spectroscopy (NOESY)-heteronuclear single quantum coherence (HSQC) analysis. Residues at the interface of the complex for both proteins were identified by NMR titration experiments. A model of the heterodimer was obtained by docking of the two PB1 domains with HADDOCK, which applies ambiguous interaction restraints on residues at the interface to drive the docking procedure. The refined model was validated by site-directed mutagenesis on both Bem1 and Cdc24. Finally, the docking was repeated from the newly published NMR structure of Cdc24, allowing us to assess the performance of homology-based docking. Our results provide insight into the molecular structure of the Bem1-Cdc24 PB1-mediated heterodimer, which allowed identification of critical residues at the binding interface.     

Multiscale simulation of the interaction of calreticulin-thrombospondin-1 complex with a model membrane microdomain

Cell surface calreticulin (CRT) binding to thrombospondin-1 (TSP1), regulates cell adhesion, migration, anoikis resistance, and collagen production. Due to the essential role of membrane microdomains in CRT-mediated focal adhesion disassembly, we previously studied the effect of raft-like bilayers on TSP1–CRT interactions with all-atom molecular dynamics (AAMD) simulations. However, the simulated systems of protein on the surface of the bilayer(s) in the explicit solvent are too large for long timescale AAMD simulations due to computational expense. In this study, we adopted a multiscale modeling approach of combining AAMD, coarse-grained molecule dynamics (CGMD), and reversed AAMD (REV AAMD) simulations to investigate the interactions of single CRT or of the TSP1–CRT complex with a membrane microdomain at microsecond timescale. Results showed that CRT conformational stabilization by binding of TSP1 in AAMD simulation was undetectable in CGMD simulation, but it was recovered in REV AAMD simulation. Similarly, interactions of the CRT N-domain and TSP1 with the membrane microdomain were lost in CGMD simulations but they were re-gained in the REV AAMD simulations. There was the higher coordination of the CRT P-domain in the TSP1–CRT complex with the lipid components of membrane microdomain compared to that of single CRT, which could directly affect the conformation of CRT and further mediate CRT recruitment of LDL receptor-related protein for signaling events. This study provides structural and molecular insights into TSP1–CRT interactions in a membrane microdomain environment and demonstrates the feasibility of using multiscale simulations to investigate the interactions between protein and membrane microdomains at a long timescale.     

Multiscale modeling of lipids and lipid bilayers

A multiscale modeling approach is applied for simulations of lipids and lipid assemblies on mesoscale. First, molecular dynamics simulation of initially disordered system of lipid molecules in water within all-atomic model was carried out. On the next stage, structural data obtained from the molecular dynamics (MD) simulation were used to build a coarse-grained (ten sites) lipid model, with effective interaction potentials computed by the inverse Monte Carlo method. Finally, several simulations of the coarse-grained model on longer length- and time-scale were performed, both within Monte Carlo and molecular dynamics simulations: a periodical sample of lipid molecules ordered in bilayer, a free sheet of such bilayer without periodic boundary conditions, formation of vesicle from a plain membrane, process of self-assembly of lipids randomly dispersed in volume. It was shown that the coarse-grained model, developed exclusively from all-atomic simulation data, reproduces well all the basic features of lipids in water solution.     

Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (μ-TRTX-Hh2a)

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.