Amino Acid Residues In Binding Site Research Articles

Pyrimidine-morpholine hybrids as potent druggable therapeutics for Alzheimer’s disease: Synthesis, biochemical and in silico analyses

The identification of effective and druggable cholinesterase inhibitors to treat progressive neurodegenerative Alzheimer’s disorder remains a continuous drug discovery hunt. In this perspective, the present study investigates the design and discovery of pyrimidine-morpholine hybrids (5a-l) as potent cholinesterase inhibitors. Palladium-catalyzed Suzuki-Miyaura cross-coupling reaction was employed to introduce the structural diversity on the pyrimidine heterocyclic core. A range of commercially available boronic acids was successfully coupled showing a high functional group tolerance. In vitro cholinesterase inhibitory potential using Ellman’s method revealed significantly strong potency. Compound 5h bearing a meta-tolyl substituent at 2-position of pyrimidine ring emerged as a lead candidate against AChE with an inhibitory potency of 0.43 ± 0.42 µM, ∼38-fold stronger value than neostigmine (IC50 = 16.3 ± 1.12 µM). Compound 5h also showed the lead inhibition against BuChE with an IC50 value of 2.5 ± 0.04 µM. The kinetics analysis of 5h revealed the non-competitive mode of inhibition against AChE whereas computational modelling results of potent leads depicted diverse contacts with the binding site amino acid residues. Molecular dynamics simulations revealed the stability of biomolecular system, while, ADME analysis demonstrated druglikeness behaviour of potent compounds. Overall, the investigated pyrimidine-morpholine scaffold presented a remarkable potential to be developed as efficacious anti-Alzheimer’s drugs.

Boronic modified quercetin molecules: Synthesis and biological investigations with molecular docking verification

Quercetin is an essential compound belonging to the flavanol group, which is a derivative of bioflavonoids found in fruits and vegetables. Quercetin has various pharmacological properties such as anticancer, antioxidant, anti-inflammatory, antiviral, antihistamine, and antitumor. Based on these important properties, the quercetin compound was modified with various boronic acids and new types of quercetin-based boronic (QB) compounds were synthesized. In the current study, we have synthesized quercetin-based boronic compounds to obtain more effective molecules by doping the biological activity properties of quercetin naturally in its structure. In this purpose, 6‑methoxy naphthalene boronic acid, 1,4-phenyl diboronic acid, 4‑methoxy phenylboronic acid, 6‑methoxy-3-pyridinylboronic acid, 3-formyl-4-methoxyphenyl boronic acid compounds were used. The synthesized compounds were structurally enlightened by mass, 13C NMR, 1H NMR, and FTIR spectroscopy. Then, the antioxidant, anticholinesterase, antiurease, antityrosinase and toxic-cytotoxic effects of these molecules were tested. QB compounds synthesized and examined in vitro in the study were also analyzed by molecular docking. Molecular docking, one of the structure-based drug design methods, has been applied to support experimental data. It was determined that QB compounds showed more significant efficacy than the reference substance employed in antioxidant and enzyme activity studies. As a result of molecular docking analysis, the binding energy parameter values and active binding site amino acid residues of the compounds were determined. Obtained novel boronic compounds might be associated as drug candidate due to consistent results both in experimental and molecular docking results.

Green Route Synthesis and Molecular Docking of Azines Using Cellulose Sulfuric Acid under Microwave Irradiation

A novel catalytic approach for the synthesis of symmetric and asymmetric azines has been developed. The environmentally benign protocol was achieved via condensation of 1-[(2-thienyl)ethylidene]hydrazine (1) with different aromatic aldehydes 2a–h and acetyl heterocyclic compounds (4, 6, 8, 10, and 12) in the presence of cellulose sulfuric acid (CSA) as the green catalyst. These procedures offer an interesting method for the large-scale industrial manufacture of azines due to their high percentage yield, mild reaction conditions, broad substrate range, and utilization of an economical and environmentally acceptable catalyst. Additionally, the molecular docking of the products to the monoamine oxidase (MAO-A) target protein was achieved to highlight the possible binding interaction with the amino acid residues Arg51, Glu43, Gly22, Gly49, Gly443, Ala272, Ile335, and Tyr407 at the point of binding. The binding interaction energy was discovered to be (− 6.48 kcal/mol) for the protein MAO-A (PDB ID: 2Z5X). The most effective azine derivatives 7 and 13 revealed some major conserved interactions between the MAO-A protein’s binding site amino acid residues and the PDB co-crystal ligand 2Z5X. Moreover, azine derivatives 3a and 3f showed the lowest binding activity with the target MAO-A.

BabyBoom: 3-Dimensional Structure-Based Ligand and Protein Interaction Prediction by Molecular Docking.

Baby Boom (BBM) is a key transcription factor that triggers embryogenesis, enhances transformation and regeneration efficiencies, and regulates developmental pathways in plants. Triggering or activating BBM in non-model crops could overcome the bottlenecks in plant breeding. Understanding BBM's structure is critical for functional characterization and determination of interacting partners and/or ligands. The current in silico study aimed to study BBM's sequence and conservation across all plant proteomes, predict protein-protein and protein-ligand interactions, and perform molecular docking and molecular dynamics (MD) simulation to specifically determine the binding site amino acid residues. In addition, peptide sequences that interact with BBM have also been predicted, which provide avenues for altered functional interactions and the design of peptide mimetics that can be experimentally validated for their role in tissue culture or transformation media. This novel data could pave the way for the exploitation of BBM's potential as the master regulator of specialized plant processes such as apomixes, haploid embryogenesis, and CRISPR/Cas9 transgenic development.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection.

Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it's missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

Investigation on the binding specificity of Agrocybe cylindracea galectin towards α(2,6)-linked sialyllactose by molecular modeling and molecular dynamics simulations

Galectins are β-galactoside binding proteins that act as potential cancer biomarkers. Agrocybe cylindracea galectin (ACG) is a fungal galectin which recognizes specifically α(2,3)-linked sialyllactose and play a pivotal role in biological recognition processes. This manuscript has presented the theoretical investigation on enhancing the binding specificity of ACG towards α(2,6)-linked sialyllactose. In silico single point and double point mutations were carried out at the binding site amino acid residues of ACG to enhance its binding to α(2,6)-linked sialyllactose, and the mutated protein-carbohydrate complexes were simulated for 30 ns durations. Analysis of the interaction patterns of the carbohydrate at the binding site of wild type and mutated lectins revealed that sialic acid contributed more to the binding when compared with other sugar units in the trisaccharide and enhanced the binding specificity of ACG towards α(2,6)-linked sialyllactose.

Identification of novel autoinducer-2 receptors in Clostridia reveals plasticity in the binding site of the LsrB receptor family

Autoinducer-2 (AI-2) is unique among quorum-sensing signaling molecules, as it is produced and recognized by a wide variety of bacteria and thus facilitates interspecies communication. To date, two classes of AI-2 receptors have been identified: the LuxP-type, present in the Vibrionales, and the LsrB-type, found in a number of phylogenetically distinct bacterial families. Recently, AI-2 was shown to affect the colonization levels of a variety of bacteria in the microbiome of the mouse gut, including members of the genus Clostridium, but no AI-2 receptor had been identified in this genus. Here, we identify a noncanonical, functional LsrB-type receptor in Clostridium saccharobutylicum. This novel LsrB-like receptor is the first one reported with variations in the binding-site amino acid residues that interact with AI-2. The crystal structure of the C. saccharobutylicum receptor determined at 1.35 Å resolution revealed that it binds the same form of AI-2 as the other known LsrB-type receptors, and isothermal titration calorimetry (ITC) assays showed that binding of AI-2 occurs at a submicromolar concentration. Using phylogenetic analysis, we inferred that the newly identified noncanonical LsrB receptor shares a common ancestor with known LsrB receptors and that noncanonical receptors are present in bacteria from different phyla. This led us to identify putative AI-2 receptors in bacterial species in which no receptors were known, as in bacteria belonging to the Spirochaetes and Actinobacteria phyla. Thus, this work represents a significant step toward understanding how AI-2-mediated quorum sensing influences bacterial interactions in complex biological niches.

Targeting isoprenoid biosynthesis pathway in Staphylococcus lugdunensis: Comparative docking and simulation studies of conventional and allosteric sites

The current study establishes the use of in silico subtractive genomics approach, molecular docking and molecular dynamic (MD) simulation studies for identifying and inhibiting a potential therapeutic target protein involved in vital metabolic pathways and is therefore crucial for the survival of the human pathogen Staphylococcus lugdunensis. Filtering all the druggable target proteins based on cellular localization and functional annotation has led to the selection of farnesyl diphosphate synthase (Fds). Fds is the key enzyme involved in isoprenoid biosynthesis pathway. Homology model of the protein was generated via MODELLER and other web-based servers. Most reliable protein model selected on the basis of comparative analysis was used as an input for molecular docking studies performed using a total library of 374 compounds. Both conventional and allosteric binding sites were probed to assess the inhibitor binding potential. Among the studied compounds, non-bisphosphonate bisamidines, compound 160 (GOLD Score 79.8) and compound 193 (GOLD Score 87.84) were the top scoring compounds against the normal and allosteric binding pockets, respectively. MD simulations were applied further to explore the dynamic behaviour of both complexes in order to get an insight into structural and conformational changes induced upon ligand binding. MD simulation results revealed that the allosteric binding pocket was slightly more compact and stable when compared to the conventional binding pocket. Significant conformational changes including considerable side chain movements were observed in the binding site amino acid residues accompanied by rearrangement of the hydrogen bonding pattern in conventional and allosteric binding pockets.

In Silico drug designing studies on Dengue Virus NS1 Protein

The key proteins involved in causing dengue are seven major proteins, which are considered as major therapeutic targets for dengue drug development. Recent studies have reported positively for NS1 protein in dysregulation of causing dengue process in humans. Dragon fruit seed phytochemicals are reported to have antioxidant and antiviral properties. In the present study we studied binding efficiency of 11 compounds that are present in the dragon fruit seeds with NS1 Protein through Insilico methods. By our virtual screening and docking result, we found that the Compound A, Compound B and Compound C have highest binding affinity with the NS1 Protein and also we predicted the binding site amino acid residues and the nature of hydrogen bonding. However more invivo experimental validation of our results with animal models will enlighten the development of more potent drugs from these compounds for treatment of dengue.

Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins

A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (−89° to −60°, −59° to −30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

Role of Noncovalent Interactions in Designing Inhibitors for H+,K+‐ATPase: Combined QM and MD Based Investigations

AbstractReversible acid suppressants represent a new therapeutic option for the treatment of acid‐related diseases. The forces responsible for the interaction of reversible inhibitors with gastric pump H+,K+‐ATPase is less explored. Quantum chemical calculations reveals that the drug molecule is mainly stabilized by non‐covalent interactions, such as π‐π, cation‐π, and NH‐π, with the active amino acid residues of the H+,K+‐ATPase enzyme. The M062X/6‐311++G(d,p) level of theory calculated interaction energy of SCH28080 is 149.52 kJ/mol and 148.26 kJ/mol for K+ ion with the inhibitor binding site amino acid residues. The results suggest that SCH28080 can block the acid secretion activity of gastric proton pump in the parietal cells. The designed Inhibitor‐3 structurally similar to the SCH28080 enjoys better non‐covalent interactions. We have also performed well‐tempered metadynamics (WTMtD) simulations of the protein‐drug complexes which corroborate the findings from DFT calculations. Our computational study demonstrated that noncovalent interactions are responsible for the inhibitor binding at the inhibitor binding site of H+,K+‐ATPase.

Usefulness of Molecular Modeling in Characterizing the Ligand-Binding Sites of Proteins: Experience with Human PDI, PDIp and COX

In this paper, we discussed our recent experience with the use of computational modeling tools in studying the binding interaction of small molecular weight ligands with their protein targets. Specific examples discussed here include the interaction of estrogens with human protein disulfide isomerase (PDI) and its pancreas-specific homolog (PDIp), and the interaction of dietary flavonoids with human cyclooxygenase (COX) I and II. Using human PDIp as an example, biochemical analysis revealed that the estrogen-binding activity is only associated with PDIp's b-b´ domain combination but not associated with the single b or b´ domain or any other domains. Homology modeling was then used to build a threedimensional structure of the human PDIp's b-b´ fragment. Docking analyses predicted that a hydrogen bond, formed between the 3-hydroxyl group of estradiol and His278 of PDIp's E2-binding site, is critical for the binding interaction. This binding model was then experimentally confirmed by a series of experiments, such as selective mutations of the predicted binding site amino acid residues and the selective modifications of the functional groups of the ligands. Similar combinatorial approaches were used successfully to identify the binding site structure of human PDI for estradiol and the binding site structures of human COX I and II for their phenolic co-substrates. The success with these combinatorial approaches provides the basis for using computational modeling-guided approaches in characterizing the ligand binding site structures of complex proteins whose structures are difficult to decipher with crystallographic studies.

Optimization of Benzoisothiazole dioxide inhibitory activity of the NS5B polymerase of HCV genotype 4 using ligand-steered homological modeling, reaction-driven scaffold-hopping and Enovo workflow.

Infection caused by hepatitis C virus (HCV) is a significant world health problem for which novel therapies are in urgent demand. The virus is highly prevalent in the Middle East and Africa particularly Egypt with more than 90% of infections due to genotype 4. Nonstructural (NS5B) viral proteins have emerged as an attractive target for HCV antivirals discovery. A potent class of inhibitors having benzisothiazole dioxide scaffold has been identified on this target, however they were mainly active on genotype 1 while exhibiting much lowered activity on other genotypes due to the high degree of mutation of its binding site. Based on this fact, we employed a novel strategy to optimize this class on genotype 4. This strategy depends on using a refined ligand-steered homological model of this genotype to study the mutation binding energies of the binding site amino acid residues, the essential features for interaction and provide a structure-based pharmacophore model that can aid optimization. This model was applied on a focused library which was generated using a reaction-driven scaffold-hopping strategy. The hits retrieved were subjected to Enovo pipeline pilot optimization workflow that employs R-group enumeration, core-constrained protein docking using modified CDOCKER and finally ranking of poses using an accurate molecular mechanics generalized Born with surface area method.

Characterization of the Estradiol-Binding Site Structure of Human Pancreas-Specific Protein Disulfide Isomerase: Indispensable Role of the Hydrogen Bond between His278 and the Estradiol 3-Hydroxyl Group

Estradiol (E(2)), a female sex hormone, has important biological functions. Human pancreas-specific protein disulfide isomerase (PDIp), a protein folding catalyst, was recently found to be able to bind E(2). Here we report the characterization of its E(2)-binding site by using biochemical methods coupled with molecular modeling tools. Analysis of various truncated PDIp proteins showed that the b-b' fragment contains an intact E(2)-binding site that has the same binding affinity as the full-length PDIp protein, with apparent K(d) values of approximately 170 nM. Computational modeling and docking analyses revealed that the E(2)-binding site in the b-b' fragment is located in a hydrophobic pocket composed mainly of the b' domain and partially of the b domain. The hydrogen bond, formed between the 3-hydroxyl group of E(2) (donor) and PDIp's His278 (acceptor), is indispensable for its binding. By contrast, the 17β-hydroxyl group of E(2) is of negligible importance for E(2) binding. This binding model was jointly confirmed by a series of experiments, such as selective mutation of the binding site amino acid residues and selective modification of the ligand structures.

A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.

Azidowarfarin photoaffinity probes of purified rat liver cytochrome P4501A1

Substrate specificity differences between various forms of cytochrome P450 (P450) are governed by substrate binding site amino acid residue differences. To determine the identities of these residues, four analogs of warfarin, a thoroughly investigated anticoagulant drug which is regio- and stereoselectively metabolized by many P450s, have been synthesized as photoaffinity probes. The probes 4′-, 6-, 7-, and 8-azidowarfarin were readily photolyzed in neutral solution by 254-nm light, with half-lives of less than 15 s. When the azidowarfarins were photolyzed in the presence of β-naphthoflavone-inducible P4501A1 (2.5 μ m) at −196 °C and the P450 was subsequently reconstituted for warfarin metabolism, 50% inactivation was achieved with 160 μ m 4′-azidowarfarin, 64 μ m 6-azidowarfarin, 127 μ m 7-azidowarfarin, and 29 μ m 8-azidowarfarin. This inactivation is irreversible. When these concentrations of the azidowarfarins were photolyzed prior to addition to P4501A1, less inhibition of P450 activity was detected and the inhibition was reversible. The CO-ferrous P450 spectrum of P4501A1 at 448 nm was diminished when photoactivated azidowarfarins bound to and inactivated the enzyme, with essentially no formation of P420 except in the case of 4′-azidowarfarin. The inactivation of P4501A1 by photoactivated 4′-azidowarfarin was prevented by 50% by 1.2 m m R-warfarin or 0.3 m m 4′-nitrowarfarin, consistent with the latter being a better P4501A1 substrate than R-warfarin. The photoinactivation of P4501A1 by each of the azidowarfarins was prevented to variable extents by R-warfarin or by 4′-, 6-, 7-, or 8-nitrowarfarin. Taken together these results demonstrate that all four azidowarfarins are potentially useful photoaffinity probes of the substrate binding site amino acid residues of P450s.

Physicochemical ProPerties and binding-site amino acid residues of galactoside-binding Protein of human Placenta

The galactose-binding lectin of human Placenta has been Purified to homogeneity by affinity chromatograPhy on asialo-fetuin column. The Protein, extractable from the tissue only with lactose is aPParently membrane-bound. Molecular weight determination of native Protein and subunit indicated a dimer of l3.4 kDa subunits. Inhibition of haemagglutination with various saccharides indicate that thiodigalactoside is the best inhibitor followed by lactose. However,P-nitroPhenyl-and 1-O-methyl derivatives of galactose showed that α-anomers inhibited slightly better than β-anomer. Modification of amino acid residues indicated involvement of arginine, lysine and histidine residues at the saccharidebinding site. Cysteine residue modificatioin also abolished haemagglutinating activity. Amino acid comPosition of the lectin is also Presented.

Binding site amino acid residues of jack fruit (artocarpus integrifolia) seed lectin: chemical modification and protein difference spectral studies

The effect of chemical modification of amino acid residues essential for sugar binding in the α-D-galactoside specific jack fruit (Artocarpus integrifolia) seed lectin and the protection of the residues by specific sugar from modification were studied. Citraconylation or maleylation of 75 % of its lysyl residues or acetylation of 70 % of the tyrosyl residues completely abolished sugar binding and agglutination without dissociation of subunits. 1-O- methyl α-D-galactoside could protect its essential lysyl and tyrosyl groups from modification. Tryptophan could not be detected in the protein. Difference absorption spectra on binding of the above sugar confirmed the role of tyrosine residues and showed an association constant K = 0·4 × 10 3 M -1 . Data suggests that the lectin could be immobilized without any loss of sugar binding activity.