A flexible-protein molecular docking study of the binding of ruthenium complex compounds to PIM1, GSK-3β, and CDK2/Cyclin A protein kinases

We employ ensemble docking simulations to characterize the interactions of two enantiomeric forms of a Ru-complex compound (1-R and 1-S) with three protein kinases, namely PIM1, GSK-3β, and CDK2/cyclin A. We show that our ensemble docking computational protocol adequately models the structural features of these interactions and discriminates between competing conformational clusters of ligand-bound protein structures. Using the determined X-ray crystal structure of PIM1 complexed to the compound 1-R as a control, we discuss the importance of including the protein flexibility inherent in the ensemble docking protocol, for the accuracy of the structure prediction of the bound state. A comparison of our ensemble docking results suggests that PIM1 and GSK-3β bind the two enantiomers in similar fashion, through two primary binding modes: conformation I, which is very similar to the conformation presented in the existing PIM1/compound 1-R crystal structure; conformation II, which represents a 180° flip about an axis through the NH group of the pyridocarbazole moiety, relative to conformation I. In contrast, the binding of the enantiomers to CDK2 is found to have a different structural profile including a suggested bound conformation, which lacks the conserved hydrogen bond between the kinase and the ligand (i.e., ATP, staurosporine, Ru-complex compound). The top scoring conformation of the inhibitor bound to CDK2 is not present among the top-scoring conformations of the inhibitor bound to either PIM1 or GSK-3β and vice-versa. Collectively, our results help provide atomic-level insights into inhibitor selectivity among the three kinases.

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.

DFT study of the Lewis acid mediated synthesis of 3-acyltetramic acids

The recent emergence and re-emergence of alphaviruses, in particular the chikungunya virus (CHIKV), in numerous countries has invoked a worldwide threat to human health, while simultaneously generating an economic burden on affected countries. There are currently no vaccines or effective drugs available for the treatment of the CHIKV, and with few lead compounds reported, the vital medicinal chemistry is significantly more challenging. This study reports on the discovery of potential inhibitors for the nsP3 macro domain of CHIKV using molecular docking, virtual screening, and molecular dynamics simulations, as well as work done to evaluate and confirm the active site of nsP3. Virtual screening was carried out based on blind docking as well as focused docking, using the database of 1541 compounds from NCI Diversity Set II, to identify hit compounds for nsP3. The top hit compounds were further subjected to molecular dynamic simulations, yielding a greater understanding of the dynamic behavior of nsP3 and its complexes with various ligands, concurrently confirming the outcomes of docking, and establishing in silico lead compounds which target the CHIKV nsP3 enzyme.

In-silico screening of cancer associated mutation on PLK1 protein and its structural consequences

The Polo-like kinases (Plks) are a conserved subfamily of serine-threonine protein kinases that have significant roles in cell proliferation. The serine/threonine protein kinases or polo-like kinase 1 (PLK1) exist in centrosome during interphase and is an important regulatory enzyme in cell cycle progression during M phase. Mutations in mammalian PLK1 were found to be over expressed in various human cancers and it is disrupting the binding ability of polo box domain with target peptide. In this analysis we implemented a computational approach to filter the most deleterious and cancer associated mutation on PLK1 protein. We found W414F as the most deleterious and cancer associated by Polyphen 2.0, SIFT, I-mutant 3.0, PANTHER, PhD-SNP, SNP&GO, Mutpred and Dr Cancer tools. Molecular docking and molecular dynamics simulation (MDS) approach was used to investigate the structural and functional behavior of PLK1 protein upon mutation. MDS and docking results showed stability loss in mutant PLK1 protein. Due to mutation, PLK1 protein became more flexible and alters the dynamic property of protein which might affect the interaction with target peptide and leads to cell proliferation. Our study provided a well designed computational methodology to examine the cancer associated nsSNPs and their molecular mechanism. It further helps scientists to develop a drug therapy against PLK1 cancer-associated diseases.

MiR-424 and miR-155 deregulated expression in cytogenetically normal acute myeloid leukaemia: correlation with NPM1 and FLT3 mutation status

Background

Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML). This has made FLT3 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to generate a sensitive substrate for assays of the FLT3 enzymatic activity.

Methods

We expressed in Escherichia coli cells a glutathione S-transferase (GST) fusion protein designated GST-FLT3S, which contains a peptide sequence derived from an autophosphorylation site of FLT3. The protein was used to analyze tyrosine kinase activity of baculovirus-expressed FLT3 and crude cell extracts of bone marrow cells from AML patients. It was also employed to perform FLT3 kinase assays for FLT3 inhibitor screening.

Results

GST-FLT3S in solution or on beads was strongly phosphorylated by recombinant proteins carrying the catalytic domain of wild type FLT3 and FLT3D835 mutants, with the latter exhibiting much higher activity and efficiency. GST-FLT3S was also able to detect elevated tyrosine kinase activity in bone marrow cell extracts from AML patients. A small-scale inhibitor screening led to identification of several potent inhibitors of wild type and mutant forms of FLT3.

Conclusions

GST-FLT3S is a sensitive protein substrate for FLT3 assays. It may find applications in diagnosis of diseases related to abnormal FLT3 activity and in inhibitor screening for drug development.     

The CH3PH2 and CH3PH isomers: isomerization,hydrogen release,thermodynamic, and spectroscopy properties

Curcumin has been reported to be therapeutically active but has poor bioavailability, half life, and high rate of metabolic detoxifcation. Most of the hydrophobic and acidic drugs get transported through human serum albumin (HSA). Binding of drugs to serum protein increases their half-life. The present study is focused to analyze interaction of curcumin with HSA by NMR and docking studies. In order to investigate the binding affinity of curcumin with HSA, NMR based diffusion techniques and docking study have been carried out. We report that curcumin has shown comparable binding affinity value vis-a-vis standard, the accessible surface area (ASA) of human serum albumin (uncomplexed) and its docked complex with curcumin at both binding sites was calculated and found to be close to that of warfarin and diazepam respectively. Conclusion drawn from our study demonstrates that curcumin interacts with HSA strongly thereby its poor half life is due to high rate of its metabolic detoxification as reported in literature.

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu⁵⁴³-Asp⁵⁴⁴ in this linker or Leu⁶¹¹, Arg⁶⁸³, or Phe⁶⁹⁴ in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.The Janus family of tyrosine kinases (JAKs)² are key regulators of cytokine receptor signaling in hematopoiesis and immune responses (1). Of the four mammalian JAK kinases, JAK2 transmits signals for a variety of cytokine receptors, including the erythropoietin receptor (EpoR) that is essential for red blood cell production (2). Upon Epo stimulation, JAK2 activates downstream signaling, such as STAT5, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/AKT pathways (2). Mice deficient in Epo, EpoR, or JAK2 die embryonically due to the absence of definitive erythropoiesis (3–5).In addition to regulation by phosphatases and suppressors of cytokine signaling (6, 7), JAK2 kinase activity is critically controlled by an autoinhibitory mechanism. Like other JAK members, JAK2 contains an N-terminal segment followed by a pseudokinase domain and a C-terminal tyrosine kinase domain. The N-terminal segment, consisting of a FERM (protein 4.1, ezrin, moezin, radixin homologous) domain and an atypical SH2 domain (1), mediates association with the membrane-proximal region of the cytokine receptors (8). Binding of JAK2 through its N-terminal segment to the EpoR is essential for EpoR surface expression (9). The pseudokinase domain is predicted to adopt a kinase fold but lacks residues essential for catalysis (10). Deletion of the pseudokinase domain leads to a marked increase in JAK2 kinase activity and loss of response to cytokine stimulation (11–13). Therefore, this domain is essential for JAK2 autoinhibition and is essential for JAK2 activation upon cytokine stimulation. Consistent with this notion, a point mutation in the JAK2 pseudokinase domain was identified in the majority of myeloproliferative disorder patients, including 90% of polycythemia vera (PV) patients (14–18). This mutation, V617F, in the presence of a dimerized receptor scaffold, such as the EpoR, resulted in the constitutive activation of JAK2 and downstream signaling effectors (19, 20) and caused erythrocytosis in a murine bone marrow transplant model (14, 21–23). Recently, mutations immediately adjacent to the JAK2 pseudokinase domain in the SH2-pseudokinase domain linker were identified in PV patients and shown to cause constitutive activation of JAK2 and a PV-like phenotype in mice (24–26). The molecular mechanisms underlying the control of JAK2 activity (i.e. the swift augmentation of its activity upon receptor activation) are poorly understood. The residues involved in the autoinhibition in JAK2 are unknown.In this work, we sought to characterize the regulatory mechanisms controlling JAK2 kinase activity. Using a functional screen for activating JAK2 mutations that signal constitutively, we discovered 13 mutations in the pseudokinase domain and in the SH2-pseudokinase domain linker. These mutations identified specific residues that are important for the inhibition of basal JAK2 kinase activity and for cytokine-induced JAK2 activation. In addition, we showed that the SH2-pseudokinase domain linker is essential for interaction with the EpoR, autoinhibitory regulation, and Epo-inducible JAK2 activation and may act as a switch in relaying cytokine receptor engagement to JAK2 activation by flexing the pseudokinase domain hinge.     

Discovery of novel low-molecular-weight HIV-1 inhibitors interacting with cyclophilin A using in silico screening and biological evaluations

Cyclophilin A has attracted attention recently as a new target of anti-human immunodeficiency virus type 1 (HIV-1) drugs. However, so far no drug against HIV-1 infection exhibiting this mechanism of action has been approved. To identify new potent candidates for inhibitors, we performed in silico screening of a commercial database of more than 1,300 drug-like compounds by using receptor-based docking studies. The candidates selected from docking studies were subsequently tested using biological assays to assess anti-HIV activities. As a result, two compounds were identified as the most active. Specifically, both exhibited anti-HIV activity against viral replication at a low concentration and relatively low cytotoxicity at the effective concentration inhibiting viral growth by 50 %. Further modification of these molecules may lead to the elucidation of potent inhibitors of HIV-1.

Interaction Between Umami Peptide and Taste Receptor T1R1/T1R3

The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. The homology models of the ligand binding domains of the human umami receptor have been constructed based on crystallographic structures of the taste receptor of the central nervous system. Furthermore, the molecular simulations of the ligand binding domain show that the likely conformation was that T1R1 protein exists in the closed conformation, and T1R3 in the open conformation in the heterodimer. The molecular docking study of T1R1 and T1R3 in complex with four peptides, including Lys–Gly–Asp–Glu–Ser–Leu–Leu–Ala, Ser–Glu–Glu, G1u–Ser, and Asp–Glu–Ser, displayed that the amino acid residue of SER146 and Glu277 in T1R3 may play great roles in the synergism of umami taste. This docking result further validated the robustness of the model. In the paper, binding of umami peptide and the T1R1/T1R3 receptor was first described and the interaction is the base of umami activity theory.     

Cytotoxic effect and molecular docking of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide—a novel topoisomerase II inhibitor

The preliminary cytotoxic effect of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide hydrochloride (1)—a potent topoisomerase II inhibitor—was measured using a MTT assay. It was found that the compound decreased the number of viable cells in both estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231breast cancer cells, with IC₅₀ values of 146?±?2 and 132?±?2 μM, respectively. To clarify the molecular basis of the inhibitory action of 1, molecular docking studies were carried out. The results suggest that 1 targets the ATP binding pocket.

Discovery of potent inhibitors for interleukin-2-inducible T-cell kinase: structure-based virtual screening and molecular dynamics simulation approaches

In our study, a structure-based virtual screening study was conducted to identify potent ITK inhibitors, as ITK is considered to play an important role in the treatment of inflammatory diseases. We developed a structure-based pharmacophore model using the crystal structure (PDB ID: 3MJ2) of ITK complexed with BMS-50944. The most predictive model, SB-Hypo1, consisted of six features: three hydrogen-bond acceptors (HBA), one hydrogen-bond donor (HBD), one ring aromatic (RA), and one hydrophobic (HY). The statistical significance of SB-Hypo1 was validated using wide range of test set molecules and a decoy set. The resulting well-validated model could then be confidently used as a 3D query to screen for drug-like molecules in a database, in order to retrieve new chemical scaffolds that may be potent ITK inhibitors. The hits retrieved from this search were filtered based on the maximum fit value, drug-likeness, and ADMET properties, and the hits that were retained were used in a molecular docking study to find the binding mode and molecular interactions with crucial residues at the active site of the protein. These hits were then fed into a molecular dynamics simulation to study the flexibility of the activation loop of ITK upon ligand binding. This combination of methodologies is a valuable tool for identifying structurally diverse molecules with desired biological activities, and for designing new classes of selective ITK inhibitors.

Retrospective molecular docking study of WY-25105 ligand to β-secretase and bias of the three-dimensional structure flexibility

β-Secretase (BACE) is a very promising target in the search for a treatment for Alzheimer’s disease using a protein–ligand inhibition approach. Given the many published X-ray structures of BACE protein, structure-based drug design has been used extensively to support new inhibitor discovery programs. Due to the high flexibility and large catalytic site of this protein, sampling of the huge conformational space of the binding site is the big challenge to overcome and is the main limitation of the most widely used docking programs. Incorrect treatment of these pitfalls can introduce bias into ligand docking and could affect the results. This is especially the case with the WY-25105 compound reported by the Wyeth Corporation as a BACE ligand that did not fit into any of the known crystal structures. In the present retrospective study, a set of available X-ray enzyme structures was selected and molecular dynamics simulations were conducted to generate more diverse representative BACE protein conformations. These conformations were then used for a docking study of the WY-25105 compound. The results confirmed the need to use an ensemble of structures in protein–ligand docking for identification of new binding modes in structure-based drug design of BACE inhibitors.

Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages

The mechanisms by which lipopolysaccharide (LPS) is recognized, and how such recognition leads to innate immune responses, are poorly understood. Stimulation with LPS induces the activation of a variety of proteins, including mitogen-activated protein kinases (MAPKs) and NF-B. Activation of protein tyrosine kinases (PTKs) is also necessary for a number of biological responses to LPS. We used a murine macrophage-like cell line, RAW264.7, to demonstrate that Janus kinase (JAK)2 is tyrosine phosphorylated immediately after LPS stimulation. Anti-Toll-like receptor (TLR)4 neutralization antibody inhibits the phosphorylation of JAK2 and the c-Jun NH₂-terminal protein kinase (JNK). Both the JAK inhibitor AG490 and the kinase-deficient JAK2 protein reduce the phosphorylation of JNK and phosphatidylinositol 3-kinase (PI3K) via LPS stimulation. Pharmacological inhibition of the kinase activity of PI3K with LY-294002 decreases the phosphorylation of JNK. Finally, we show that JAK2 is involved in the production of IL-1 and IL-6. PI3K and JNK are also important for the production of IL-1. These results suggest that LPS induces tyrosine phosphorylation of JAK2 via TLR4 and that JAK2 regulates phosphorylation of JNK mainly through activation of PI3K. Phosphorylation of JAK2 via LPS stimulation is important for the production of IL-1 via the PI3K/JNK cascade. Thus JAK2 plays a pivotal role in LPS-induced signaling in macrophages. cytokine; toll-like receptor-4; c-Jun NH₂-terminal kinase     

Entropy versus aromaticity in the conformational dynamics of aromatic rings

Comparison of the results of Car-Parrinello molecular dynamics simulations of isolated benzene, pyrimidine and 1,2,4-triazine molecules reveals that the unusually low population of planar geometry of the benzene ring is caused by entropy effects despite its high aromaticity. The decrease in symmetry of the molecule results in smaller changes in entropy and Gibbs free energy due to out-of-plane deformations of the ring, leading to an increase in the population of planar geometry of the ring. This leads to differences in the topology of potential energy and Gibbs free energy surfaces.

Plasmonic Heating-Assisted Transformation of SiO2/Au Core/Shell Nanospheres (Au Nanoshells): Caveats and Opportunities for SERS and Direct Laser Writing

The selective modification of silica/gold nanospheres (gold nanoshells) driven by plasmonic heating is demonstrated. Direct laser writing and reshaping of nanoshell assemblies can be easily controlled and exploited for nanofabrication purposes. The modified nanoshells exhibit improved surface enhanced Raman scattering, allowing to settle most of the issues related to nanoshell stability under working conditions.

Density functional investigation of CO adsorption on Ni-doped single-walled armchair (5,5) boron nitride nanotubes

The adsorption of CO onto Ni-doped boron nitride nanotubes (BNNTs) was investigated using density functional theory at the B3LYP/LanL2DZ level of theory. The structures of the Ni-doped BNNTs and their CO-adsorbed configurations were obtained. It was found that the strength of adsorption of CO onto Ni-doped perfect BNNTs is higher than that on defective BNNTs. The electronic properties of all of the adsorption configurations of CO on Ni-doped BNNTs are reported.