Molecular recognition of Fc‐specific ligands binding onto the consensus binding site of IgG: insights from molecular simulation

Immunoglobulin G (IgG) plays an important role in clinical diagnosis and therapeutics. Meanwhile, the consensus binding site (CBS) on the Fc domain of IgG is responsible for ligand recognition, especially for Fc‐specific ligands. In this study, molecular simulation methods were used to investigate molecular interactions between the CBS of the Fc domain and seven natural Fc‐specific ligands. The analysis on the binding energy of the Fc–ligand complex indicated that hydrophobic interactions provide the main driving force for the Fc–ligand binding processes. The hot spots on the ligands and Fc were identified with the computational alanine scanning approach. It was found that the residues of tryptophan and tyrosine on the ligands have significant contributions for the Fc–ligand binding, while Met252, Ile253, Asn434, His435, and Tyr436 are the key residues of Fc. Moreover, two binding modes based on tryptophan or tyrosine were summarized and constructed according to the pairwise interaction analysis. Guidelines for the rational design of CBS‐specific ligands with high affinity and specificity were proposed. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine‐based affinity ligands

This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine‐based ligands immobilized onto agarose‐derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2‐ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine‐based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd.     

Structural studies on ligand–DNA systems: A robust approach in drug design

Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.     

Incorporating replacement free energy of binding‐site waters in molecular docking

Binding‐site water molecules play a crucial role in protein‐ligand recognition, either being displaced upon ligand binding or forming water bridges to stabilize the complex. However, rigorously treating explicit binding‐site waters is challenging in molecular docking, which requires to fully sample ensembles of waters and to consider the free energy cost of replacing waters. Here, we describe a method to incorporate structural and energetic properties of binding‐site waters into molecular docking. We first developed a solvent property analysis (SPA) program to compute the replacement free energies of binding‐site water molecules by post‐processing molecular dynamics trajectories obtained from ligand‐free protein structure simulation in explicit water. Next, we implemented a distance‐dependent scoring term into DOCK scoring function to take account of the water replacement free energy cost upon ligand binding. We assessed this approach in protein targets containing important binding‐site waters, and we demonstrated that our approach is reliable in reproducing the crystal binding geometries of protein‐ligand‐water complexes, as well as moderately improving the ligand docking enrichment performance. In addition, SPA program (free available to academic users upon request) may be applied in identifying hot‐spot binding‐site residues and structure‐based lead optimization. Proteins 2014; 82:1765–1776. © 2014 Wiley Periodicals, Inc.     

Investigation of ligand selectivity in CYP3A7 by molecular dynamics simulations

Cytochrome P450 (CYP) 3A7 plays a crucial role in the biotransformation of the metabolized endogenous and exogenous steroids. To compare the metabolic capabilities of CYP3A7–ligands complexes, three endogenous ligands were selected, namely dehydroepiandrosterone (DHEA), estrone, and estradiol. In this study, a three-dimensional model of CYP3A7 was constructed by homology modeling using the crystal structure of CYP3A4 as the template and refined by molecular dynamics simulation (MD). The docking method was adopted, combined with MD simulation and the molecular mechanics generalized born surface area method, to probe the ligand selectivity of CYP3A7. These results demonstrate that DHEA has the highest binding affinity, and the results of the binding free energy were in accordance with the experimental conclusion that estrone is better than estradiol. Moreover, several key residues responsible for substrate specificity were identified on the enzyme. Arg372 may be the most important residue due to the low interaction energies and the existence of hydrogen bond with DHEA throughout simulation. In addition, a cluster of Phe residues provides a hydrophobic environment to stabilize ligands. This study provides insights into the structural features of CYP3A7, which could contribute to further understanding of related protein structures and dynamics.     

Molecular simulations study of novel 1,4‐dihydropyridines derivatives with a high selectivity for Cav3.1 calcium channel

1,4‐Dihydropyridines (DHPs) have been developed to treat hypertension, angina, and nerve system disease. They are thought to mainly target the L‐type calcium channels, but low selectivity prompts them to block Cav1.2 and Cav3.1 channels simultaneously. Recently, some novel DHPs with different hydrophobic groups have been synthesized and among them M12 has a higher selectivity for Cav3.1. However, the structural information about Cav3.1‐DHPs complexes is not available in the experiment. Thus, we combined homology modeling, molecular docking, molecular dynamics simulations, and binding free energy calculations to quantitatively elucidate the inhibition mechanism of DHPs. The calculated results indicate that our model is in excellent agreement with experimental results. On the basis of conformational analysis, we identify the main interactions between DHPs and calcium channels and further elaborate on the different selectivity of ligands from the micro perspective. In conjunction with energy distribution, we propose that the binding sites of Cav3.1‐DHPs is characterized by several interspersed hydrophobic amino acid residues on the IIIS6 and IVS6 segments. We also speculate the favorable function groups on prospective DHPs. Besides, our model provides important information for further mutagenesis experiments.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Optimization of protein–protein docking for predicting Fc–protein interactions

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc–protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc‐binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc–protein complexes are available, numerous others have not yet been determined. Protein–protein docking could be used to investigate Fc–protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking‐based structural bioinformatics approach is developed for predicting the structures of Fc–protein complexes. Based on the available set of X‐ray structures of Fc–protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc–protein complexes, as well as in the design of peptides and proteins that target Fc.     

Design,synthesis and application of benzyl‐sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

The human anti‐human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity‐ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4‐aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS‐Trz‐4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS‐Trz‐4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two‐step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S‐Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS‐Trz‐4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and enzyme‐linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure‐guided screening of chemical database to identify NS3‐NS2B inhibitors for effective therapeutic application in dengue infection

Dengue infection is the most common arthropod‐borne disease caused by dengue viruses, predominantly affecting millions of human beings annually. To find out promising chemical entities for therapeutic application in Dengue, in the current research, a multi‐step virtual screening effort was conceived to screen out the entire “screening library” of the Asinex database. Initially, through “Lipinski rule of five” filtration criterion almost 0.6 million compounds were collected and docked with NS3‐NS2B protein. Thereby, the chemical space was reduced to about 3500 compounds through the analysis of binding affinity obtained from molecular docking study in AutoDock Vina. Further, the “Virtual Screening Workflow” (VSW) utility of Schrödinger suite was used, which follows a stepwise multiple docking programs such as ‐ high‐throughput virtual screening (HTVS), standard precision (SP), and extra precision (XP) docking, and in postprocessing analysis the MM‐GBSA based free binding energy calculation. Finally, five potent molecules were proposed as potential inhibitors for the dengue NS3‐NS2B protein based on the investigation of molecular interactions map and protein‐ligand fingerprint analyses. Different pharmacokinetics and drug‐likeness parameters were also checked, which favour the potentiality of selected molecules for being drug‐like candidates. The molecular dynamics (MD) simulation analyses of protein‐ligand complexes were explained that NS3‐NS2B bound with proposed molecules quite stable in dynamic states as observed from the root means square deviation (RMSD) and root means square fluctuation (RMSF) parameters. The binding free energy was calculated using MM‐GBSA method from the MD simulation trajectories revealed that all proposed molecules possess such a strong binding affinity towards the dengue NS3‐NS2B protein. Therefore, proposed molecules may be potential chemical components for effective inhibition of dengue NS3‐NS2B protein subjected to experimental validation.     

HIV-1跨膜蛋白gp41与N-取代吡咯衍生物的结合模式研究

采用分子对接,分子动力学(MD)模拟和分子力学/泊松-波尔兹曼溶剂可有面积方法与分子力学/广义伯恩溶剂可及面积方法(MM-PBSA/MM-GBSA),预测两种N-取代吡咯衍生物与HIV-1 跨膜蛋白gp41疏水口袋的结合模式与作用机理．分子对接采用多种受体构象,并从结果中选取几种可能的结合模式进行MD 模拟,然后通过MM-PBSA计算结合能的方法识别最优的结合模式. MM-PBSA计算结果表明,范德华相互作用是结合的主要驱动力,而极性相互作用决定了配体在结合过程中的取向．进一步的结合能分解显示,配体的羧基与gp41残基Arg579的静电相互作用对结合有重要贡献．上述工作为进一步优化N-取代吡咯衍生物类的HIV-1融合抑制剂建立了良好的理论基础．     

Comparative study of the interaction of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP) in its free base and Fe derivative form with oligo(dA.dT)15 and oligo(dG.dC)15

Interaction between a cationic porphyrin and its ferric derivative with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ was studied by UV–vis spectroscopy, resonance light scattering (RLS), and circular dichroism (CD) at different ionic strengths; molecular docking and molecular dynamics simulation were also used for completion. Followings are the observed changes in the spectral properties of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP), as a free-base porphyrin with no axial ligand, and its Fe derivative (FeTMAP) upon interaction with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅: (1) the substantial red shift and hypochromicity at the Soret maximum in the UV–vis spectra; (2) the increased RLS intensity by increasing the ionic strength; and (3) an intense bisignate excitonic CD signal. All of them are the reasons for TMAP and FeTMAP binding to oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ with the outside binding mode, accompanied by the self-stacking of the ligands along the oligonucleotide helix. The CD results demonstrated a drastic change from excitonic in monomeric behavior at higher ionic strengths, which indicates the groove binding of the ligands with oligonucleotides. Molecular docking also confirmed the groove binding mode of the ligands and estimated the binding constants and energies of the interactions. Their interaction trend was further confirmed by molecular dynamics technique and structure parameters obtained from simulation. It showed that TMAP reduced the number of intermolecular hydrogen bonds and increased the solvent accessible surface area in the oligonucleotide. The self-aggregation of ligands at lower concentrations was also confirmed.     

Selectivity of cytosolic phospholipase A2 type IV toward arachidonyl phospholipids

Cytosolic phospholipase A2 (cPLA₂) is an interesting protein involved in inflammatory processes and various diseases. Its catalytic mechanism as well as its substrate specificity for arachidonyl phospholipids is not typical for other phospolipases. Furthermore, a lid structure, which ensures a hydrophilic surface of the protein without any substrate bound and the movement of this flexible loop to make the hydrophobic active site accessible, is of high interest. Therefore, the focus of this work was to determine the binding mode of cPLA₂ with various substrates, such as arachidonic acid, a synthetic inhibitor, a saturated phospholipid, and most importantly an arachidonyl phospholipid. To understand the selectivity of the protein toward the arachidonyl phospholipid and the interaction in a protein–ligand complex, molecular dynamics simulations were performed using the GROMOS suite of simulation programs. The simulations provide insight into the protein and showed that selective binding of arachidonyl phospholipids is because of the shape of the sn‐2 tail. The amino acids Asn555 and Ala578 are involved in the strongest interactions observed in the protein–ligand complexes. Copyright © 2015 John Wiley & Sons, Ltd.     

Profiling the interaction of 1‐phenylbenzimidazoles to cyclooxygenases

In the design of 1‐phenylbenzimidazoles as model cyclooxygenase (COX) inhibitors, docking to a series of crystallographic COX structures was performed to evaluate their potential for high‐affinity binding and to reproduce the interaction profile of well‐known COX inhibitors. The effect of ligand‐specific induced fit on the calculations was also studied. To quantitatively compare the pattern of interactions of model compounds to the profile of several cocrystallized COX inhibitors, a geometric parameter, denominated ligand‐receptor contact distance (LRCD), was developed. The interaction profile of several model complexes showed similarity to the profile of COX complexes with inhibitors such as iodosuprofen, iodoindomethacin, diclofenac, and flurbiprofen. Shaping of high‐affinity binding sites upon ligand‐specific induced fit mostly determined both the affinity and the binding mode of the ligands in the docking calculations. The results suggest potential of 1‐phenylbenzimidazole derivatives as COX inhibitors on the basis of their predicted affinity and interaction profile to COX enzymes. The analyses also provided insights into the role of induced fit in COX enzymes. While inhibitors produce different local structural changes at the COX ligand binding site, induced fit allows inhibitors in diverse chemical classes to share characteristic interaction patterns that ensure key contacts to be achieved. Different interaction patterns may also be associated with different inhibitory mechanisms.     

Structure-based screening and molecular dynamics simulations offer novel natural compounds as potential inhibitors of Mycobacterium tuberculosis isocitrate lyase

Mycobacterium tuberculosis is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. M. tuberculosis isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M. tuberculosis, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with ?96.462, ?143.549, and ?122.526 kJ mol^?1 for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.     

Mechanisms of tetraethylammonium ion block in the KcsA potassium channel

We report results from automated docking and microscopic molecular dynamics simulations of the tetraethylammonium (TEA) complexes with KcsA. Binding modes and energies for TEA binding at the external and internal sides of the channel pore are examined utilising the linear interaction energy method. Effects of the channel ion occupancy (based on our previous results for the ion permeation mechanisms) on the binding energies are considered. Calculations show that TEA forms stable complexes at both the external and internal entrances of the selectivity filter. Furthermore, the effects of the Y82V mutation are evaluated and the results show, in agreement with experimental data, that the mutant has a significantly reduced binding affinity for TEA at the external binding site, which is attributed to stabilising hydrophobic interactions between the ligand and the tyrosines.     

Investigation into mechanism of orotidine 5′-monophosphate decarboxylase enzyme by MM-PBSA/MM-GBSA and molecular docking

In this work, we studied the binding affinity of orotidine 5′-monophosphate (OMP) and 6-hydroxy-UMP (BMP) for Saccharomyces cerevisiae orotidine 5′-monophosphate decarboxylase (OMPDC) enzyme by using Molecular Mechanics-Poisson–Boltzmann Surface Area (MM-PBSA) and the Molecular Mechanics-Generalised Born Surface Area (MM-GBSA) calculations. In all simulations, Asp91, which is an important residue in the enzyme active site, was considered in both anionic (present in the native form of the enzyme) and neutral states. A series of 10-ns molecular dynamics simulations were performed for the four OMPDC–ligand complexes, two ligand-free enzymes and two free ligands, followed by MM-PBSA/MM-GBSA calculations on the collected snapshots, and molecular docking calculations using the free enzymes and ligands. The results of MM-PBSA/MM-GBSA calculations indicate that all of the OMPDC–ligand complexes form favourable systems in water, which is in agreement with corresponding experimental data. The results of the MM-PBSA and molecular docking methods also showed that OMPDC–BMP complexes, transition state analogue and inhibitor of the OMPDC enzyme have the highest binding affinities. The fact that in the native anionic state BMP shows a higher binding affinity compared with the substrate suggests the contribution of a transition state stabilisation mechanism in the debatable catalytic mechanism of the OMPDC enzyme.