Structural insight into the ligand-receptor interaction between glycyrrhetinic acid (GA) and the high-mobility group protein B1 (HMGB1)-DNA complex

Structural analysis of the high-mobility group protein B1 (HMGB1)-DNA complex and a docking simulation between glycyrrhetinic acid (GA) and the HMGB1-DNA complex were performed with a software package the Molecular Operating Environment (MOE). An HMGB1-DNA (PDB code: 2GZK) was selected for the 3D structure modeling of the HMGB1-DNA complex. The Site Finder module of the MOE identified 16 possible ligand-binding sites in the modeled HMGB1-DNA complex. The docking simulation revealed that GA possibly inhibits functions of HMGB1 interfering with Lys₉₀, Arg₉₁, Ser₁₀₁, Tyr₁₄₉, C₂₃₀ and C₂₃₁ in the HMGB1-DNA complex. To the best of our knowledge, this is the first report of an HMGB1-DNA complex with GA, and our data verify that the GA-HMGB1-DNA model can be utilized for application to target HMGB1 for the development of antitumor drugs.

Abbreviations

ASE-Dock - alpha sphere and excluded volume-based ligand-protein docking, CNS - central nervous system, GA - glycyrrhetinic acid, GL - glycyrrhizin, HMGB1 - high-mobility group protein B1, LBS - ligand-biding site, MOE - Molecular Operating Environment, SRY - sex-determining region on the Y chromosome.     

Dopamine transporter comparative molecular modeling and binding site prediction using the LeuT(Aa) leucine transporter as a template

Pharmacological and behavioral studies indicate that binding of cocaine and the amphetamines by the dopamine transporter (DAT) protein is principally responsible for initiating the euphoria and addiction associated with these drugs. The lack of an X-ray crystal structure for the DAT or any other member of the neurotransmitter:sodium symporter (NSS) family has hindered understanding of psychostimulant recognition at the atomic level; structural information has been obtained largely from mutagenesis and biophysical studies. The recent publication of a crystal structure for the bacterial leucine transporter LeuT(Aa), a distantly related NSS family homolog, provides for the first time a template for three-dimensional comparative modeling of NSS proteins. A novel computational modeling approach using the capabilities of the Molecular Operating Environment program MOE 2005.06 in conjunction with other comparative modeling servers generated the LeuT(Aa)-directed DAT model. Probable dopamine and amphetamine binding sites were identified within the DAT model using multiple docking approaches. Binding sites for the substrate ligands (dopamine and amphetamine) overlapped substantially with the analogous region of the LeuT(Aa) crystal structure for the substrate leucine. The docking predictions implicated DAT side chains known to be critical for high affinity ligand binding and suggest novel mutagenesis targets in elucidating discrete substrate and inhibitor binding sites. The DAT model may guide DAT ligand QSAR studies, and rational design of novel DAT-binding therapeutics.     

A homology model for <Emphasis Type="Italic">Clostridium difficile</Emphasis> methionyl tRNA synthetase: active site analysis and docking interactions

Treatment of C. difficile infection is one of the most difficult biomedical challenges. To develop novel antibacterials, researchers have been targeting bacterial molecular functions that are essential for its growth. The methionyl tRNA synthetase (MetRS) is strictly required for protein biosynthesis and success was reported in developing antibacterials to inhibit this enzyme. The present study was aimed at building and analyzing a homology model for C. difficile MetRS in the context of drug design. A homology model of C. difficile MetRS was constructed using Molecular Operating Environment (MOE) software. A. aeolicus MetRS was the main template while the query zinc binding domain was modeled using T. thermophilus MetRS. The model has been assessed and compared to its main template (Ramachandran, ERRAT and ProSA). The active site of the query protein has been predicted from its sequence using a detailed conservation analysis (ClustalW2). Using MOE software, suitable ligands were docked in the constructed model, including a C. difficile MetRS inhibitor REP3123 and the enzyme natural substrate, and the key active site residues and interactions were identified. These docking studies have validated the active site conformation in the constructed model and identified binding interactions.     

In silico identification and study of potential anti-mosquito juvenile hormone binding protein (MJHBP) compounds as candidates for dengue virus - Vector insecticides

Dengue has become a huge global health burden. It is currently recognized as the most rapidly spreading mosquito-borne viral disease. Yet, there are currently no licensed vaccines or specific therapeutics to manage the virus, thus, scaling up vector control approaches is important in controlling this viral spread. This study aimed to identify and study in silico, potential anti-mosquito compounds targeting Juvenile hormone (JH) mediated pathways via the Mosquito Juvenile Hormone Binding Protein (MJHBP). The study was implemented using series of computational methods. The query compounds included pyrethroids and those derived from ZINC and ANPDB databases using a simple pharmacophore model in Molecular Operating Environment (MOE). Molecular docking of selected compounds’ library was implemented in MOE. The resultant high-score compounds were further validated by molecular dynamics simulation via Maestro 12.3 module and the respective Prime/Molecular Mechanics Generalized Born Surface Area (Prime/MM-GBSA) binding energies computed. The study identified compounds-pyrethroids, natural and synthetic - with high docking energy scores (ranging from 10.91–12.34 kcal/mol). On further analysis of the high-ranking (in terms of docking scores) compounds using MD simulation, the compounds - Ekeberin D4, Maesanin, Silafluofen and ZINC16919139- revealed very low binding energies (?122.99, ?72.91 -104.50 and,-74.94 kcal/mol respectively), fairly stable complex and interesting interaction with JH-binding site amino acid residues on MJHBP. Further studies can explore these compounds in vitro/in vivo in the search for more efficient mosquito vector control.     

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family.     

A leishmaniasis study: Structure-based screening and molecular dynamics mechanistic analysis for discovering potent inhibitors of spermidine synthase

Protozoa Leishmania donovani (Ld) is the main cause of the endemic disease leishmaniasis. Spermidine synthase (SS), an important enzyme in the synthetic pathway of polyamines in Ld, is an essential element for the survival of this protozoan. Targeting SS may provide an important aid for the development of drugs against Ld. However, absence of tertiary structure of spermidine synthase of Leishmania donovani (LSS) limits the possibilities of structure based drug designing. Presence of the same enzyme in the host itself further challenges the drug development process. We modeled the tertiary structure of LSS using homology modeling approach making use of homologous X-ray crystallographic structure of spermidine synthase of Trypanosoma cruzi (TSS) (2.5? resolution). The modeled structure was stabilized using Molecular Dynamics simulations. Based on active site structural differences between LSS and human spermidine synthase (HSS), we screened a large dataset of compounds against modeled protein using Glide virtual screen docking and selected two best inhibitors based on their docking scores (-10.04 and -13.11 respectively) with LSS and having least/no binding with the human enzyme. Finally Molecular Dynamics simulations were used to assess the dynamic stability of the ligand bound structures and to elaborate on the binding modes. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.     

Novel benzothiazolyl urea and thiourea derivatives with potential cytotoxic and antimicrobial activities

A novel series of benzothiazole urea and thiourea derivatives was synthesized and evaluated for its in vitro cytotoxicity against MCF-7 breast cancer cells. The N1-(benzothiazol-2-yl)-N3-morpholinourea 3 displayed the highest cytotoxic activity in this series. A docked pose of 3 was obtained bound to G-quadruplex of human telomere DNA active site using the Molecular Operating Environment (MOE) module. Moreover, the synthesized compounds were screened for their antimicrobial activity against Mycobacterium tuberculosis H37Rv, E. coli, S. aureus and C. albicans. Again, 3 showed the best activity against M. tuberculosis H37Rv while other compounds were equipotent with ampicillin against S. aureus and E. coli.     

Molecular docking and structural analysis of cofactor-protein interaction between NAD⁺ and 11β-hydroxysteroid dehydrogenase type 2

Molecular docking and structural analysis of the cofactor-protein interaction between NAD⁺ and human (h) or mouse (m) 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) were performed with the molecular operating environment (MOE). 11βHSD1 (PDB code: 3HFG) was selected as a template for the 3D structure modeling of 11βHSD2. The MOE docking (MOE-dock) and the alpha sphere and excluded volume-based ligand-protein docking (ASE-dock) showed that both NAD⁺-h11βHSD2 and NAD⁺-m11βHSD2 models have a similar binding orientation to the template cofactor-protein model. Our present study also revealed that Asp91, Phe94, Tyr232 and Thr267 could be of importance in the interaction between NAD⁺ and 11βHSD2. NADP⁺ was incapable of entering into the cofactor-binding site of the 11βHSD2 models. The present study proposes the latest models for 11βHSD2 and its cofactor NAD⁺, and to the best of our knowledge, this is the first report of a m11βHSD2 model with NAD⁺.     

Predicting P-glycoprotein-mediated drug transport based on support vector machine and three-dimensional crystal structure of P-glycoprotein

Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening.     

A structural model for the open conformation of the mdr1 P-glycoprotein based on the MsbA crystal structure

The validity of the structure of the Escherichia coli MsbA lipid transporter as a model from the mdr1 P-glycoprotein has been evaluated. Comparative sequence analyses, motif search and secondary structure prediction indicated that each of the two P-glycoprotein halves is structurally similar to the MsbA monomer and also suggested that the open dimer structure is valid for P-glycoprotein. Homology modeling was used to predict the structure of P-glycoprotein using MsbA as a template. The resulting modeled structure allowed a detailed study of the interactions between the intracellular domain and the nucleotide binding domain and suggested that these contacts are involved in mediating the coupling between nucleotide binding domain conformational changes and transmembrane helices reorientation during transport. In P-glycoprotein, the internal chamber open to the inner leaflet and the inner medium is significantly different in size and charge than in MsbA. These differences can be related to those of the transported substrates. Moreover an ensemble of 20 conserved aromatic residues appears to border the periphery of each side of the chamber in P-glycoprotein. These may be important for size selection and proper positioning of drugs for transport. The relevance of the modeled conformation to P-gp function is discussed.     

Predicting binding to p-glycoprotein by flexible receptor docking

P-glycoprotein (P-gp) is an ATP-dependent transport protein that is selectively expressed at entry points of xenobiotics where, acting as an efflux pump, it prevents their entering sensitive organs. The protein also plays a key role in the absorption and blood-brain barrier penetration of many drugs, while its overexpression in cancer cells has been linked to multidrug resistance in tumors. The recent publication of the mouse P-gp crystal structure revealed a large and hydrophobic binding cavity with no clearly defined sub-sites that supports an "induced-fit" ligand binding model. We employed flexible receptor docking to develop a new prediction algorithm for P-gp binding specificity. We tested the ability of this method to differentiate between binders and nonbinders of P-gp using consistently measured experimental data from P-gp efflux and calcein-inhibition assays. We also subjected the model to a blind test on a series of peptidic cysteine protease inhibitors, confirming the ability to predict compounds more likely to be P-gp substrates. Finally, we used the method to predict cellular metabolites that may be P-gp substrates. Overall, our results suggest that many P-gp substrates bind deeper in the cavity than the cyclic peptide in the crystal structure and that specificity in P-gp is better understood in terms of physicochemical properties of the ligands (and the binding site), rather than being defined by specific sub-sites.     

Structure modeling of novel DNA glycosylase enzyme from oral pathogen Streptococcus sanguinis

The novel 3-methyladenine DNA glycosylase enzyme from oral pathogen Streptococcus sanguinisin involves in DNA repair mechanisms and participates in base excision repair. Its 3D structure is still unknown which may be a potential drug target, therefore here we proposed its putative 3D structure by homology modeling approach. EsyPred3d software produced more precise modeled structure as compare to Swiss model software. The modeled structure was further verified by PROCHECK analysis and subjected to functional site prediction servers for active site residues prediction. The functional site was further validated by molecular docking approach with ligand EDA (3- [2- Deoxyribofuranosyl] - 3H- 1, 3, 4, 5A, 8-Pentaaza- Asindacene-5- monophosphate) from 1F4R. The EDR docked at the cavity of modeled structure of 3-methyladenine DNA glycosylase enzyme with highest Patchdock score of 3966 and lowest Autodock 4 docking energy of -10.30 Kcal/mol. The YA51, LA105, RA107 residues are surrounding the EDA and matching with ligand binding residues predicted by PROFUNC server.     

A discovery of novel Mycobacterium tuberculosis pantothenate synthetase inhibitors based on the molecular mechanism of actinomycin D inhibition

Mycobacterium tuberculosis pantothenate synthetase is a potential anti-tuberculosis target, and a high-throughput screening system was previously developed to identify its inhibitors. Using a similar system, we screened a small library of compounds and identified actinomycin D (ActD) as a weak inhibitor of pantothenate synthetase. A new method was established to discover more effective inhibitors by determining the molecular mechanism of ActD inhibition followed by structure-based virtual screening. The molecular interaction of inhibition was determined by circular dichroism and tryptophan fluorescence quenching. The structure-based search and virtual screening were performed using the Molecular Operating Environment (MOE) program and SYBYL 7.5, respectively. Two inhibitors were identified with an IC₅₀ for pantothenate synthetase that was at least ten times better than that of ActD.     

Molecular dynamics simulation and molecular docking studies of 1,4-Dihydropyridines as P-glycoprotein’s allosteric inhibitors

P-glycoprotein (P-gp) is a main factor contributing to multidrug resistance. The effect of this transporter protein on limiting the effectiveness of chemotherapy has been shown by various studies. In a previous report, we synthesized some 14-dihydropyridine (DHP) derivatives as inhibitors of human P-gp. In the present study, a computational approach has been exploited to reveal the main interactions between DHPs and P-gp. In order to do this, homology modeling was performed to obtain a model of the protein. Then, molecular dynamics simulation was used to refine the constructed model of P-gp in the presence of the lipids bilayer. Model validation was performed with several tools. Finally, molecular docking followed by MD simulation of ligand–protein complex was employed to elucidate the binding mode and the dynamical changes of protein with/without DHPs bound. The results emphasized that interaction of the residues Gln912, Ser909, Arg905, Ser474, Val472 with DHPs play a crucial role in the inhibitory of these ligands and this was in a relatively good accordance with the results reported in the experimental studies.     

Modeling of angiogenin - 3-NMP complex

Angiogenin belongs to the Ribonuclease superfamily and has a weak enzymatic activity that is crucial for its biological function of stimulating blood vessel growth. Structural studies on ligand bound Angiogenin will go a long way in understanding the mechanism of the protein as well as help in designing drugs against it. In this study we present the first available structure of nucleotide ligand bound Angiogenin obtained by computer modeling. The importance of this study in itself notwithstanding, is a precursor to modeling a full dinucleotide substrate onto Angiogenin. Bovine Angiogenin, the structure of which has been solved at a high resolution, was earlier subjected to Molecular Dynamics simulations for a nanosecond. The MD structures offer better starting points for docking as they offer lesser obstruction than the crystal structure to ligand binding. The MD structure with the least serious short contacts was modeled to obtain a steric free Angiogenin - 3' mononucleotide complex structure. The structures were energetically minimized and subjected to a brief spell of Molecular Dynamics. The results of the simulation show that all the ligand-Angiogenin interactions and hydrogen bonds are retained, redeeming the structure and docking procedure. Further, following ligand - protein interactions in the case of the ligands 3'-CMP and 3'-UMP we were able to speculate on how Angiogenin, a predominantly prymidine specific ribonuclease prefers Cytosine to Uracil in the first base position.     

Homology modeling,molecular docking and spectra assay studies of sterol 14α-demethylase from <Emphasis Type="Italic">Penicillium digitatum</Emphasis>

Sterol 14α-demethylase from Penicillium digitatum (PdCYP51) is a prime target of antifungal drugs for citrus disease in plants. To design novel antifungal compounds, a homology model of PdCYP51 was constructed using the recently reported crystal structure of human CYP51 as the template. Molecular docking was performed to investigate the interaction of four commercial fungicides with the modeled enzyme. The side chain of these compounds interplayed with PdCYP51 mainly through hydrophobic and van der Waals interactions. Biochemical spectra analysis of inhibitors combined with PdCYP51 are also compatible with the docking results. This is the first molecular modeling for PdCYP51 based on the eukaryotic crystal structure of CYP51. The structural information and binding site mapping of PdCYP51 for different inhibitors obtained from this study could aid in screening and designing new antifungal compounds targeting this enzyme.     

Molecular epidemiology of antibiotic-resistant genes and potent inhibitors against TEM,CTX-M-14, CTX-M-15, and SHV-1 proteins of Escherichia coli in district Peshawar,Pakistan

The Extended Spectrum Beta-Lactamases (ESBLs) producing bacteria is an issue of concern for clinicians resulting in minimize the treatment options. To overcome resistance mechanisms, novel inhibitors with good Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties must inhibit the ESBLs resistant genes. The current study aimed to identify the antibiotic resistance genes of ESBLs producing E. coli and a single inhibitor was designed to inhibit all the resistant proteins. The results showed that 42.9% ESBL producers had CTX-M (69.9%), TEM (63.4%), SHV (34.5%) and CTX-M-14 (17.5%) genes. The ESBLs producing isolates were resistant to cephalosporins, quinolones, and sulfonamide with Minimum Inhibitory Concentration (MICs) ranging from 64 to >256 μg/ml. To design multi inhibitory ligands, RECAP synthesis was used for the de-novo discovery of 1000 inhibitors database. Protein crystal structures were retrieved from Protein Data Base (PDB). Lipinski’s rules of five were applied to the novel inhibitors database to improve the ADMET properties. The novel inhibitors database was selected for docking simulations. Placement of the ligand was used by the London dG algorithm implemented in Molecular Operating Environment (MOE), while GBVI/WSA dG algorithm was used for final refinement. Based on docking score, visual inspection of ligands interaction with key residues, binding affinity, and binding energy of ligands with proteins, ten compounds were selected for ESBLs proteins with best ADMET properties, binding energy, and binding affinity the reported ones. These hits compounds have unique scaffolds and are predicted to be a starting point for developing potent inhibitors against antibiotic-resistant proteins.