Identification of terfenadine as an inhibitor of human CD81-receptor HCV-E2 interaction: synthesis and structure optimization

Terfenadine (4-[4-(hydroxydiphenylmethyl)-1-piperidyl]-1-(4-tert-butylphenyl)-butan-1-ol) was identified in a biological screening to be a moderate inhibitor (27% inhibition) of the CD81-LEL-HCV-E2 interaction. To increase the observed biological activity, 63 terfenadine derivates were synthesized via microwave assisted nucleophilic substitution. The prepared compounds were tested for their inhibitory potency by means ofa fluorescence labeled antibody assay using HUH7.5 cells. Distinct structure-activity relationships could be derived. Optimization was successful, leading to 3g, identified as the most potent compound (69 % inhibition). Experiments with viral particles revealed that there might be additional HCV infection reducing mechanisms.     

Water-mediated binding of agents that target the DNA minor groove

Small molecule complexes with DNA that incorporate linking water molecules are rare, and the DB921-DNA complex has provided a unique and well-defined system for analysis of water-mediated binding in the context of a DNA complex. DB921 has a benzimidazole-biphenyl system with terminal amidines that results in a linear conformation that does not possess the appropriate radius of curvature to match the minor groove shape and represents a new paradigm that does not fit the classical model of minor groove interactions. To better understand the role of the bound water molecule observed in the X-ray crystal structure of the DB921 complex, synthetic modifications have been made in the DB921 structure, and the interactions of the new compounds with DNA AT sites have been evaluated with an array of methods, including DNase I footprinting, biosensor-surface plasmon resonance, isothermal titration microcalorimetry, and circular dichroism. The interaction of a key compound, which has the amidine at the phenyl shifted from the para position in DB921 to the meta position, has also been examined by X-ray crystallography. The detailed structural, thermodynamic, and kinetic results provide valuable new information for incorporation of water molecules in the design of new lead scaffolds for targeting DNA in chemical biology and therapeutic applications.     

Mapping the binding site topology of amyloid protein aggregates using multivalent ligands

A key process in the development of neurodegenerative diseases such as Alzheimer''s and Parkinson''s diseases is the aggregation of proteins to produce fibrillary aggregates with a cross β-sheet structure, amyloid. The development of reagents that can bind these aggregates with high affinity and selectivity has potential for early disease diagnosis. By linking two benzothiazole aniline (BTA) head groups with different length polyethylene glycol (PEG) spacers, fluorescent probes that bind amyloid fibrils with low nanomolar affinity have been obtained. Dissociation constants measured for interaction with Aβ, α-synuclein and tau fibrils show that the length of the linker determines binding affinity and selectivity. These compounds were successfully used to image α-synuclein aggregates in vitro and in the post-mortem brain tissue of patients with Parkinson''s disease. The results demonstrate that multivalent ligands offer a powerful approach to obtain high affinity, selective reagents to bind the fibrillary aggregates that form in neurodegenerative disease.

Multivalent ligands offer a powerful approach to obtain high affinity reagents to bind the aggregates that form in neurodegenerative disease. Selectivity for different proteins was achieved by using different linkers to connect the head groups.     

Identification of novel ligands for the Z-DNA binding protein by structure-based virtual screening

We describe the first discovery of small molecules that bind to the Z-DNA binding domain of human ADAR1 (Adenosine Deaminase Acting on RNA 1) by structure-based virtual screening of chemical database. These molecules bind to Z-DNA binding domain to inhibit the interaction with the Z-DNA. Many viruses have Z-DNA binding proteins, which are structurally similar to Z-DNA binding domain of human ADAR1, and the ability of Z-DNA binding protein to bind the Z-DNA is essential for their pathogenicity. Therefore, the molecules identified in this study may serve as novel leads for the design of agents that inhibit biological functions of those pathogenic viruses.     

Optimization of tether length in nonglycosidically linked bivalent ligands that target sites 2 and 1 of a Shiga-like toxin

A series of bivalent ligands for a Shiga-like toxin have been synthesized, their experimentally determined inhibitory activities were compared with a simplified thermodynamic model, and computer simulations were used to predict the optimal tether length in bivalent ligands. The design of the inhibitors exploits the proximity of the C-2' hydroxyl groups of two P(k)-trisaccharides when bound to two different, neighboring carbohydrate recognizing binding sites located on the surface of Shiga-like toxin. NMR studies of the complex between the toxin and bivalent ligands show that site 2 and site 1 of a single B subunit are simultaneously occupied by a tethered P(k)-trisaccharide dimer. A simplified thermodynamic treatment provides the intrinsic affinities and binding energies for the intermolecular and intramolecular association events and permits the deconvolution of the contributions to the relative binding energies for the set of bivalent ligands. Conformational analysis based on MD simulations for bivalent galabioside dimers containing different tethers demonstrated that the calculated local concentrations of the pendant ligand at the second binding site correlate with the experimentally determined relative affinity values of the respective bivalent ligands, thereby providing a predictive method to optimize tether length.     

Thermodynamic analysis of a hydrophobic binding site: probing the PDZ domain with nonproteinogenic peptide ligands

[structure: see text] Isothermal titration calorimetry (ITC) is used to study the thermodynamic consequences of systematically modifying the hydrophobic character of a single residue in a series of protein-binding ligands. By substituting standard and nonproteinogenic aliphatic amino acids for the C-terminal valine of the hexapeptide KKETEV, binding to the third PDZ domain (PDZ3) of the PSD-95 protein is characterized by distinct changes in the Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) parameters. One notable observation is that peptide binding affinity can be improved with a nonstandard residue.     

Sphingosine kinase 1 (SK1) allosteric inhibitors that target the dimerization site

The sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) signaling pathway is a crucial target for numerous human diseases from cancer to cardiovascular diseases. However, available SK1 inhibitors that target the active site suffer from poor potency, selectivity and pharmacokinetic properties. The selectivity issue of the kinases, which share a highly-conserved ATP-pocket, can be overcome by targeting the less-conserved allosteric sites. SK1 is known to function minimally as a dimer; however, the crystal structure of the SK1 dimer has not been determined. In this study, a template-based algorithm implemented in PRISM was used to predict the SK1 dimer structure and then the possible allosteric sites at the dimer interface were determined via SiteMap. These sites were used in a virtual screening campaign that includes an integrated workflow of structure-based pharmacophore modeling, virtual screening, molecular docking, re-screening of common scaffolds to propose a series of compounds with different scaffolds as potential allosteric SK1 inhibitors. Finally, the stability of the SK1-ligand complexes was analyzed by molecular dynamics simulations. As a final outcome, ligand 7 having a 4,9-dihydro-1H-purine scaffold and ligand 12 having a 2,3,4,9-tetrahydro-1H-β-carboline scaffold were found to be potential selective inhibitors for SK1.     

Adamantyl-substituted ligands of colchicine binding site in tubulin: different effects on microtubule network in cancer cells

Structural Chemistry - Four novel conjugates of adamantane connected at a bridgehead position via four-bond linker to structurally different ligands interacting with colchicine binding site of...     

Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain.

BACKGROUND: Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) that promotes motility, adhesion, and proliferation in mammalian cells, as mediated by cell-surface HA receptors. We sought to identify non-carbohydrate ligands that would bind to and activate cell-surface HA receptors. Such analogs could have important therapeutic uses in the treatment of cancer, wound healing, and arthritis, since such ligands would be resistant to degradation by hyaluronidase (HAse). RESULTS: Peptide ligands that bind specifically to the recombinant HA binding domain (BD) of the receptor for hyaluronan-mediated motility (RHAMM) were obtained by screening two peptide libraries: (i) random 8-mers and (ii) biased 8-mers with alternating acidic side chains, i.e. XZXZXZXZ (X=all-L-amino acids except Cys, Lys, or Arg; Z=D-Asp, L-Asp, D-Glu, or L-Glu). Selectivity of the peptide ligands for the HABD was established by (i) detection of binding of biotin- or fluorescein-labeled peptides to immobilized proteins and (ii) fluorescence polarization of FITC-labeled peptides with the HABD in solution. HA competitively displaced binding of peptides to the HABD, while other GAGs were less effective competitors. The stereochemistry of four biased octapeptides was established by synthesis of the 16 stereoisomers of each peptide. Binding assays demonstrated a strong preference for alternating D and L configurations for the acidic residues, consistent with the calculated orientation of glucuronic acid moieties of HA. CONCLUSIONS: Two classes of HAse-resistant peptide mimetics of HA were identified with high affinity, HA-compatible binding to the RHAMM HABD. This demonstrated that non-HA ligands specific to a given HA binding protein could be engineered, permitting receptor-specific targeting.     

Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion measurements

It is important to characterize drug-albumin binding during drug discovery and lead optimization as strong binding may reduce bioavailability and/or increase the drug's in vivo half-life. Despite knowing about the location of human serum albumin (HSA) drug binding sites and the residues important for binding, less is understood about the binding dynamics between exogenous drugs and endogenous fatty acids. In contrast to highly specific antibody-antigen interactions, the conformational flexibility of albumin allows the protein to adopt multiple conformations of approximately equal energy in order to accommodate a variety of ligands. Nuclear magnetic resonance (NMR) diffusion measurements are a simple way to quantitatively describe ligand-protein interactions without prior knowledge of the number of binding sites or the binding stoichiometry. This method can also provide information about ligand orientation at the binding site due to buildup of exchange-transferred NOE (trNOE) on the diffusion time scale of the experiment. The results of NMR diffusion and NOE experiments reveal multiple binding interactions of HSA with dansylglycine, a drug site II probe, and caprylate, a medium-chain fatty acid that also has primary affinity for HSA's drug site II. Interligand NOE (ilNOE) detected in the diffusion analysis of a protein solution containing both ligands provides insight into the conformations adopted by these ligands while bound in common HSA binding pockets. The results demonstrate the ability of NMR diffusion experiments to identify ternary complex formation and show the potential of this method for characterizing other biologically important ternary structures, such as enzyme-cofactor-inhibitor complexes.     

Discrimination between enantioselective and non-selective binding sites on cellobiohydrolase-based stationary phases by site specific competing ligands.

A systematic study was performed to investigate the influence of cellobiose or lactose on the enantioselective retention behaviour of some beta-blockers in liquid chromatography using Cellobiohydrolase (CHB) I from Trichoderma reesei or Cellobiohydrolase 58 from Phanerochaete chrysosporium immobilized on silica as stationary phases. The results revealed that the retention could be described by the function [equation; see text] where the observed capacity factor corresponds to the sum of an enantioselective mode being influenced by a site specific competing ligand (competitor) and a non-selective mode unaffected by the competitor. A non-constrained non-linear least-square regression gave in all cases virtually identical nondisplacable capacity factors (k'ns) for both enantiomers of the same drug. The experimental capacity factors (k'(x,C)) of the enantiomers all show a close fit to the adapted function. The Kd values calculated for the competitor were also virtually identical for each pair of enantiomers and were in accordance with Ki data determined for the competitors in classical enzyme kinetics experiments, demonstrating that one unique site; namely, the catalytic site, was responsible for the enantioselective binding. Similar results were obtained with the resolution of rac-alprenolol and rac-metoprolol on CBH I phase.     

Identification of pyrazosulfuron-ethyl binding affinity and binding site subdomain IIA in human serum albumin by spectroscopic methods

Pyrazosulfuron-ethyl (PY) is a sulfonylurea herbicide developed by DuPont which has been widely used for weed control in cereals. The determination of PY binding affinity and binding site in human serum albumin (HSA) by spectroscopic methods is the subject of this work. From the fluorescence emission, circular dichroism and three-dimensional fluorescence results, the interaction of PY with HSA caused secondary structure changes in the protein. Fluorescence data demonstrated that the quenching of HSA fluorescence by PY was the result of the formation of HSA–PY complex at 1:1 molar ratio, a static mechanism was confirmed to lead to the fluorescence quenching. Hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results show that hydrophobic patches are the major sites for PY binding on HSA. The thermodynamic parameters ΔH° and ΔS° were calculated to be ?36.32 kJ mol^?1 and ?35.91 J mol^?1 K^?1, which illustrated van der Waals forces and hydrogen bonds interactions were the dominant intermolecular force in stabilizing the complex. Also, site marker competitive experiments showed that the binding of PY to HSA took place primarily in subdomain IIA (Sudlow's site I). What presented in this paper binding research enriches our knowledge of the interaction between sulfonylurea herbicides and the physiologically important protein HSA.     

Identification of critical residues in loop E in the 5-HT3ASR binding site

Background  

The serotonin type 3 receptor (5-HT₃R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.     

Evidence that bcl-2 is the target of three photosensitizers that induce a rapid apoptotic response

We originally proposed that the subcellular target for one class of photosensitizing agents was the mitochondrion. This classification was based on effects that occur within minutes of irradiation of photosensitized cells: rapid loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol and activation of caspase-3. These effects were followed by the appearance of an apoptotic morphology within 30-90 min. Fluorescence localization studies on three sensitizers initially classified as 'mitochondrial' revealed that these agents bind to a variety of intracellular membranes. The earliest detectable effect of photodamage is the selective loss of the antiapoptotic protein bcl-2 leaving the proapoptotic protein bax undamaged. Bcl-2 photodamage can be detected directly after irradiation of cells at 10 degrees C. Subsequent warming of cultures to 37 degrees C results in loss of delta psi m, release of cytochrome c and activation of caspase-3. The latter appears to amplify the other two effects. Based on results reported here we propose that the apoptotic response to these photosensitizers is derived from selective photodamage to the antiapoptotic protein bcl-2 while leaving the proapoptotic protein bax unaffected.     

O2 chemistry of dicopper complexes with alkyltriamine ligands. Comparing synergistic effects on O2 binding

Two dicopper(I) complexes containing tertiary N-methylated hexaaza ligands which impose different steric constraints to the Cu ions have been synthesized, and their reactivity toward O2 has been compared with a mononuclear related system, highlighting the importance of cooperative effects between the metal centers in O2 activation.     

Fast O2 binding at dicopper complexes containing Schiff-base dinucleating ligands

A new family of dicopper(I) complexes [CuI2RL](X)2 (R=H, 1X, R=tBu, 2X and R=NO2, 3X, X=CF3SO3, ClO4, SbF6, or BArF, BArF=[B{3,5-(CF3)2C6H3}4]-), where RL is a Schiff-base ligand containing two tridentate binding sites linked by a xylyl spacer, has been prepared and characterized, and its reaction with O2 has been studied. The complexes were designed with the aim of reproducing structural aspects of the active site of type 3 dicopper proteins; they contain two three-coordinate copper sites and a rather flexible podand ligand backbone. The solid-state structures of 1ClO4, 2CF3SO3, 2ClO4, and 3BArF.CH3CN have been established by single-crystal X-ray diffraction analysis. 1ClO4 adopts a polymeric structure in the solid state while 2CF3SO3, 2ClO4, and 3BArF.CH3CN are monomeric. The complexes have been studied in solution by means of 1H and 19F NMR spectroscopy, which put forward the presence of dynamic processes. 1-3BArF and 1-3CF3SO3 in acetone react rapidly with O2 to generate metaestable [CuIII2(mu-O)2(RL)]2+ 1-3(O2) and [CuIII2(mu-O)2(CF3SO3)(RL)]+ 1-3(O2)(CF3SO3) species, respectively, that have been characterized by UV-vis spectroscopy and resonance Raman analysis. Instead, reaction of 1-3BArF with O2 in CH2Cl2 results in intermolecular O2 binding. DFT methods have been used to study the chemical identities and structural parameters of the O2 adducts, and the relative stability of the CuIII2(mu-O)2 form with respect to the CuII2(mu-eta2:eta2-O2) isomer. The reaction of 1X, X = CF3SO3 and BArF, with O2 in acetone has been studied by stopped-flow UV-vis exhibiting an unexpected very fast reaction rate (k=3.82(4)x10(3) M-1 s-1, DeltaH=4.9+/-0.5 kJ.mol-1, DeltaS=-148+/-5 J.K-1.mol-1), nearly 3 orders of magnitude faster than in the parent [CuI2(m-XYLMeAN)]2+. Thermal decomposition of 1-3(O2) does not result in aromatic hydroxylation. The mechanism and kinetics of O2 binding to 1X (X=CF3SO3 and BArF) are discussed and compared with those associated with selected examples of reported models of O2-processing copper proteins. A synergistic role of the copper ions in O2 binding and activation is clearly established from this analysis.     

Investigation on the binding site in heparin by spectrophotometry

Heparin has a variety of biological activities, most of them due to heparin’s high sulfate groups. To gain insight into the mechanism of activation of the spectroscopic probe with sulfate groups of heparin in vitro, we have used a cationic dye by a spectrophotometric method. It is considered that the combination of heparin with methylene blue is due to noncovalent binding forces. Dye binding requires an organic chain structure form with sulfate groups. The solution equilibria of the reaction system are discussed. A new linear regression equation has been proposed, in which the maximum binding number N expresses the binding ability of methylene blue (MB) with sulfate groups of heparin. The linear regression equation can estimate this parameter.