Identification of a disruptor of the MDM2-p53 protein–protein interaction facilitated by high-throughput in silico docking

NSC 333003 has been identified from the NCI Diversity Set as an inhibitor of the MDM2-p53 protein–protein interaction by in silico docking (virtual screening). Its potency and chemical characteristics render it well suited for lead optimization studies that can result in more potent analogs with improved drug-like properties. Its synthesis was achieved using an acid catalyzed condensation reaction from commercially available benzothiazole hydrazine and pyridyl phenyl ketone in refluxing methanol. Stereochemical implications for this compound are described.     

Rational design of the survivin/CDK4 complex by combining protein–protein docking and molecular dynamics simulations

Survivin, the smallest inhibitor of apoptosis protein (IAP), is a valid target for cancer research. It mediates both the apoptosis pathway and the cell cycle and has been proposed to form a complex with the cyclin-dependent kinase protein CDK4. The resulting complex transports CDK4 from the cytosol to the nucleus, where CDK4 participates in cell division. Survivin has been recognized as a node protein that interacts with several partners; disruption of the formed complexes can lead to new anticancer compounds. We propose a rational model of the survivin/CDK4 complex that fulfills the experimental evidence and that can be used for structure-based design of inhibitors modifying its interface recognition. In particular, the suggested complex involves the alpha helical domain of survivin and resembles the mode of binding of survivin in the survivin/borealin X-ray structure. The proposed model has been obtained by combining protein–protein docking, fractal-based shape complementarity, electrostatics studies and extensive molecular dynamics simulations.

3D-QSAR pharmacophore-based virtual screening,molecular docking and molecular dynamics simulation toward identifying lead compounds for NS2B–NS3 protease inhibitors

NS2B–NS3 protease has been identified to serve as lead drug design target due to its significant role in West Nile viral (WNV) and dengue virus (DENV) reproduction and replication. There are currently no approved chemotherapeutic drugs and effective vaccines to inhibit DENV and WNV infections. In this work, 3D-QSAR pharmacophore model has been developed to discover potential inhibitory candidates. Validation through Fischer’s model and decoy test indicate that the developed 3D pharmacophore model is highly predictive for DENV inhibitors, which was then employed to screen ZINC chemical library to obtain reasonable hits. Following ADMET filtering, 15 hits were subjected to further filter through molecular docking and CoMFA modeling. Finally, top three hits were identified as lead compounds or potential inhibitory candidates with IC₅₀ values of ～0.4637?µM and fitness of ～57.73. It is implied from CoMFA modeling that substituents at the side site of benzotriazole such as a p-nitro group (e.g. biphenyl head) and a carbonyl (e.g. carboxylate function) at the side site of furan or amino group may improve bioactivity of ZINC85645245, respectively. Molecular dynamics simulations (MDS) were performed to discover new interactions and reinforce the binding modes from docking for the hits also. The QSAR and MDS results obtained from this work should be useful in determining structural requirements for inhibitor development as well as in designing more potential inhibitors for NS2B–NS3 protease.     

A simple and widely applicable hit validation strategy for protein–protein interaction inhibitors based on a quantitative ligand displacement assay

Identification of inhibitors for protein–protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.     

Benzimidazole inhibitors of the protein kinase CHK2: Clarification of the binding mode by flexible side chain docking and protein–ligand crystallography

Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.     

Validation of a new restraint docking method for solution structure determinations of protein–ligand complexes

A new method is proposed for docking ligands into proteins in cases where an NMR-determined solution structure of a related complex is available. The method uses a set of experimentally determined values for protein–ligand, ligand–ligand, and protein–protein restraints for residues in or near to the binding site, combined with a set of protein–protein restraints involving all the other residues which is taken from the list of restraints previously used to generate the reference structure of a related complex. This approach differs from ordinary docking methods where the calculation uses fixed atomic coordinates from the reference structure rather than the restraints used to determine the reference structure. The binding site residues influenced by replacing the reference ligand by the new ligand were determined by monitoring differences in ¹H chemical shifts. The method has been validated by showing the excellent agreement between structures of L. casei dihydrofolate reductase.trimetrexate calculated by conventional methods using a full experimentally determined set of restraints and those using this new restraint docking method based on an L. casei dihydrofolate reductase.methotrexate reference structure.     

In silico–based combinatorial pharmacophore modelling and docking studies of GSK-3β and GK inhibitors of Hippophae

Type 2 diabetes is an inevitably progressive disease, with irreversible β cell failure. Glycogen synthase kinase and Glukokinase, two important enzymes with diverse biological actions in carbohydrate metabolism, are promising targets for developing novel antidiabetic drugs. A combinatorial structure-based molecular docking and pharmacophore modelling study was performed with the compounds of Hippophae salicifolia and H. rhamnoides as inhibitors. Docking with Discovery Studio 3.5 revealed that two compounds from H. salicifolia, viz Lutein D and an analogue of Zeaxanthin, and two compounds from H. rhamnoides, viz Isorhamnetin-3-rhamnoside and Isorhamnetin-7-glucoside, bind significantly to the GSK-3 β receptor and play a role in its inhibition; whereas in the case of Glucokinase, only one compound from both the plants, i.e. vitamin C, had good binding characteristics capable of activation. The results help to understand the type of interactions that occur between the ligands and the receptors. Toxicity predictions revealed that none of the compounds had hepatotoxic effects and had good absorption as well as solubility characteristics. The compounds did not possess plasma protein-binding, crossing blood–brain barrier ability. Further, in vivo and in vitro studies need to be performed to prove that these compounds can be used effectively as antidiabetic drugs.     

Modeling EphB4-EphrinB2 protein–protein interaction using flexible docking of a short linear motif

Background

Many protein–protein interactions are mediated by a short linear motif. Usually, amino acid sequences of those motifs are known or can be predicted. It is much harder to experimentally characterize or predict their structure in the bound form. In this work, we test a possibility of using flexible docking of a short linear motif to predict the interaction interface of the EphB4-EphrinB2 complex (a system extensively studied for its significance in tumor progression).

Methods

In the modeling, we only use knowledge about the motif sequence and experimental structures of EphB4-EphrinB2 complex partners. The proposed protocol enables efficient modeling of significant conformational changes in the short linear motif fragment during molecular docking simulation. For the docking simulations, we use the CABS-dock method for docking fully flexible peptides to flexible protein receptors (available as a server at http://biocomp.chem.uw.edu.pl/CABSdock/). Based on the docking result, the protein–protein complex is reconstructed and refined.

Results

Using this novel protocol, we obtained an accurate EphB4-EphrinB2 interaction model.

Conclusions

The results show that the CABS-dock method may be useful as the primary docking tool in specific protein–protein docking cases similar to EphB4-EphrinB2 complex—that is, where a short linear motif fragment can be identified.

     

Identification of potential PKC inhibitors through pharmacophore designing, 3D-QSAR and molecular dynamics simulations targeting Alzheimer’s disease

Protein kinases are ubiquitously expressed as Serine/Threonine kinases, and play a crucial role in cellular activities. Protein kinases have evolved through stringent regulation mechanisms. Protein kinases are also involved in tauopathy, thus are important targets for developing Anti-Alzheimer’s disease compounds. Structures with an indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors for human protein kinase C, here we report the generation of four point 3D geometric featured pharmacophore model. In order to identify novel and potent PKCθ inhibitors, the pharmacophore model was screened against 80,000,00 compounds from various chemical databases such as., ZINC, SPEC, ASINEX, which resulted in 127 compound hits, and were taken for molecular docking filters (HTVS, XP docking). After in-depth analysis of binding patterns, induced fit docking (flexible) was employed for six compounds along with the cocrystallized inhibitor. Molecular docking study reveals that compound 6F found to be tight binder at the active site of PKCθ as compared to the cocrystal and has occupancy of 90 percentile. MM-GBSA also confirmed the potency of the compound 6F as better than cocrystal. Molecular dynamics results suggest that compound 6F showed good binding stability of active sites residues similar to cocrystal 7G compound. Present study corroborates the pharmacophore-based virtual screening, and finds the compound 6F as a potent Inhibitor of PKC, having therapeutic potential for Alzheimer’s disease. Worldwide, 46.8 million people are believed to be living with Alzheimer’s disease. When elderly population increases rapidly and neurodegenerative burden also increases in parallel, we project the findings from this study will be useful for drug developing efforts targeting Alzheimer’s disease.     

High-throughput docking for the identification of new influenza A virus polymerase inhibitors targeting the PA–PB1 protein–protein interaction

A high-throughput molecular docking approach was successfully applied for the selection of potential inhibitors of the Influenza RNA-polymerase which act by targeting the PA–PB1 protein–protein interaction. Commercially available compounds were purchased and biologically evaluated in vitro using an ELISA-based assay. As a result, some compounds possessing a 3-cyano-4,6-diphenyl-pyridine nucleus emerged as effective inhibitors with the best ones showing IC₅₀ values in the micromolar range.     

Discovery of Klotho peptide antagonists against Wnt3 and Wnt3a target proteins using combination of protein engineering,protein–protein docking,peptide docking and molecular dynamics simulations

The Klotho is known as lifespan enhancing protein involved in antagonizing the effect of Wnt proteins. Wnt proteins are stem cell regulators, and uninterrupted exposure of Wnt proteins to the cell can cause stem and progenitor cell senescence, which may lead to aging. Keeping in mind the importance of Klotho in Wnt signaling, in silico approaches have been applied to study the important interactions between Klotho and Wnt3 and Wnt3a (wingless-type mouse mammary tumor virus (MMTV) integration site family members 3 and 3a). The main aim of the study is to identify important residues of the Klotho that help in designing peptides which can act as Wnt antagonists. For this aim, a protein engineering study is performed for Klotho, Wnt3 and Wnt3a. During the theoretical analysis of homology models, unexpected role of number of disulfide bonds and secondary structure elements has been witnessed in case of Wnt3 and Wnt3a proteins. Different in silico experiments were carried out to observe the effect of correct number of disulfide bonds on 3D protein models. For this aim, total of 10 molecular dynamics (MD) simulations were carried out for each system. Based on the protein–protein docking simulations of selected protein models of Klotho with Wnt3 and Wnt3a, different peptides derived from Klotho have been designed. Wnt3 and Wnt3a proteins have three important domains: Index finger, N-terminal domain and a patch of ～10 residues on the solvent exposed surface of palm domain. Protein–peptide docking of designed peptides of Klotho against three important domains of palmitoylated Wnt3 and Wnt3a yields encouraging results and leads better understanding of the Wnt protein inhibition by proposed Klotho peptides. Further in vitro studies can be carried out to verify effects of novel designed peptides as Wnt antagonists.     

Inhibition of protein–protein interaction of HER2–EGFR and HER2–HER3 by a rationally designed peptidomimetic

Protein–protein interactions (PPI) play a crucial role in many biological processes and modulation of PPI using small molecules to target hot spots has therapeutic value. As a model system we will use PPI of human epidermal growth factor receptors (EGFRs). Among the four EGFRs, EGFR–HER2 and HER2–HER3 are well known in cancer. We have designed a small molecule that is targeted to modulate HER2-mediated signaling. Our approach is novel because the small molecule designed disrupts dimerization not only of EGFR–HER2, but also of HER2–HER3. In the present study we have shown, using surface plasmon resonance analysis, that a peptidomimetic, compound 5, binds specifically to HER2 protein extracellular domain and disrupts the dimerization of EGFRs. To evaluate the effect of compound 5 on HER2 signaling in vitro, Western blot and PathHunter assays were used. Results indicated that compound 5 inhibits the phosphorylation of HER2 kinase domain and inhibits the heterodimerization in a dose-dependent manner. Molecular modeling methods were used to model the PPI of HER2–HER3 heterodimer.     

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K⁺ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K⁺ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.     

Atom-based 3D-QSAR,molecular docking and molecular dynamics simulation assessment of inhibitors for thyroid hormone receptor α and β

HCl, HNO₃ and H₂SO₄ are implicated in atmospheric processes in areas such as polar stratospheric clouds in the stratosphere. Ternary complexes of HCl, HNO₃ and H₂SO₄ were investigated by ab initio calculations at B3LYP level of theory with aug-cc-pVTZ and aug-cc-pVQZ basis sets, taking into account basis set superposition error (BSSE). The results were assessed in terms of structures (five hexagonal cyclic structures and two quasi-pentagonal cyclic structures), inter-monomeric parameters (all ternary complexes built three hydrogen bonds), energetics (seven minima obtained), infrared harmonic vibrational frequencies (red shifting of complexes from monomers), and relative stability of complexes, which were favorable when the temperature decreases under stratospheric conditions, from 298 K to 188 K, and in concrete, at 210 K, 195 K and 188 K.     

NLDB: a database for 3D protein–ligand interactions in enzymatic reactions

NLDB (Natural Ligand DataBase; URL: http://nldb.hgc.jp) is a database of automatically collected and predicted 3D protein–ligand interactions for the enzymatic reactions of metabolic pathways registered in KEGG. Structural information about these reactions is important for studying the molecular functions of enzymes, however a large number of the 3D interactions are still unknown. Therefore, in order to complement such missing information, we predicted protein–ligand complex structures, and constructed a database of the 3D interactions in reactions. NLDB provides three different types of data resources; the natural complexes are experimentally determined protein–ligand complex structures in PDB, the analog complexes are predicted based on known protein structures in a complex with a similar ligand, and the ab initio complexes are predicted by docking simulations. In addition, NLDB shows the known polymorphisms found in human genome on protein structures. The database has a flexible search function based on various types of keywords, and an enrichment analysis function based on a set of KEGG compound IDs. NLDB will be a valuable resource for experimental biologists studying protein–ligand interactions in specific reactions, and for theoretical researchers wishing to undertake more precise simulations of interactions.     

Identification of inhibitors against interaction between pro-inflammatory sPLA2-IIA protein and integrin αvβ3

Increased concentrations of secreted phospholipase A2 type IIA (sPLA2-IIA), have been found in the synovial fluid of patients with rheumatoid arthritis. It has been shown that sPLA2-IIA specifically binds to integrin αvβ3, and initiates a signaling pathway that leads to cell proliferation and inflammation. Therefore, the interaction between integrin and sPLA2-IIA could be a potential therapeutic target for the treatment of proliferation or inflammation-related diseases. Two one-bead-one-compound peptide libraries were constructed and screened, and seven target hits were identified. Herein we report the identification, synthesis, and biological testing of two pyrazolylthiazole-tethered peptide hits and their analogs. Biological assays showed that these compounds were able to suppress the sPLA2-IIA–integrin interaction and sPLA2-IIA-induced migration of monocytic cells and that the blockade of the sPLA2-IIA–integrin binding was specific to sPLA2-IIA and not to the integrin.     

Andrographolide,a new potential NF-κB inhibitor: docking simulation and evaluation of drug-likeness

Andrographolide is a potent anticancer and anti-inflammatory agent isolated from the plant Andrographis paniculata. It is found to be cytotoxic against various cancer cell lines (in vitro) and also reported to act as an anti-inflammatory agent by interfering with NF-κB protein. Andrographolide induced higher percentage of apoptosis in glutathione-depleted lymphoma cell lines. Andrographolide was also reported to form dehydrated adduct with reduced glutathione at 50^° C. On the basis of these observations, the docking analysis of andrographolide with its target protein (NF-κB/p50) and its proposed anti-target protein (glutathione S-transferase) was carried out. Docking analysis predicted the best putative pose of andrographolide molecule in the active site of NF-κB and glutathione S-transferase proteins. Here we report that the furan ring of andrographolide interacts with cysteine 59 of NF-κB/p50 and thereby inhibiting the protein action. Docking studies showed the andrographolide binding to the H-site of glutathione S-transferase enzyme which resembles the behaviour of susceptible xenobiotics inactivated by glutathione S-transferase enzyme. Andrographolide obeys Pfizer's rule but drug-likeness value for andrographolide is found to be negative as the molecule showed low drug score. Hence, inactivation by glutathione S-transferase and low drug score could possibly be the reasons to make andrographolide inactive in clinical trial.     

Novel P2–P4 macrocyclic inhibitors of HCV NS3/4A protease by P3 succinamide fragment depeptidization strategy

Hepatitis C represents a serious worldwide health-care problem. Recently, we have disclosed a novel class of P2–P4 macrocyclic inhibitors of NS3/4A protease containing a carbamate functionality as capping group at the P3 N-terminus. Herein we report our work aimed at further depeptidizing the P3 region by replacement of the urethane function with a succinamide motif. This peptidomimetic approach has led to the discovery of novel P2–P4 macrocyclic inhibitors of HCV NS3/4A protease with sub-nanomolar enzyme affinities. In addition to being potent inhibitors of HCV subgenomic replication, optimized analogues within this series have also presented attractive PK properties and showed promising liver levels in rat following oral administration.     

Protein–protein interaction networks studies and importance of 3D structure knowledge

Protein–protein interaction networks (PPINs) are a powerful tool to study biological processes in living cells. In this review, we present the progress of PPIN studies from abstract to more detailed representations. We will focus on 3D interactome networks, which offer detailed information at the atomic level. This information can be exploited in understanding not only the underlying cellular mechanisms, but also how human variants and disease-causing mutations affect protein functions and complexes’ stability. Recent studies have used structural information on PPINs to also understand the molecular mechanisms of binding partner selection. We will address the challenges in generating 3D PPINs due to the restricted number of solved protein structures. Finally, some of the current use of 3D PPINs will be discussed, highlighting their contribution to the studies in genotype–phenotype relationships and in the optimization of targeted studies to design novel chemical compounds for medical treatments.     

3D QSAR studies,pharmacophore modeling,and virtual screening of diarylpyrazole–benzenesulfonamide derivatives as a template to obtain new inhibitors,using human carbonic anhydrase II as a model protein

A 3D-QSAR modeling was performed on a series of diarylpyrazole-benzenesulfonamide derivatives acting as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The compounds were collected from two datasets with the same scaffold, and utilized as a template for a new pharmacophore model to screen the ZINC database of commercially available derivatives. The datasets were divided into training, test, and validation sets. As the first step, comparative molecular field analysis (CoMFA), CoMFA region focusing and comparative molecular similarity indices analysis (CoMSIA) in parallel with docking studies were applied to a set of 41 human (h) CA II inhibitors. The validity and the prediction capacity of the resulting models were evaluated by leave-one-out (LOO) cross-validation approach. The reliability of the model for the prediction of possibly new CA inhibitors was also tested.