De Novo Design of Selective Quadruplex–Duplex Junction Ligands and Structural Characterisation of Their Binding Mode: Targeting the G4 Hot-Spot

Targeting the interface between DNA quadruplex and duplex regions by small molecules holds significant promise in both therapeutics and nanotechnology. Herein, a new pharmacophore is reported, which selectively binds with high affinity to quadruplex–duplex junctions, while presenting a poorer affinity for G-quadruplex or duplex DNA alone. Ligands complying with the reported pharmacophore exhibit a significant affinity and selectivity for quadruplex–duplex junctions, including the one observed in the HIV-1 LTR-III sequence. The structure of the complex between a quadruplex–duplex junction with a ligand of this family has been determined by NMR methods. According to these data, the remarkable selectivity of this structural motif for quadruplex–duplex junctions is achieved through an unprecedented interaction mode so far unexploited in medicinal and biological chemistry: the insertion of a benzylic ammonium moiety into the centre of the partially exposed G-tetrad at the interface with the duplex. Further decoration of the described scaffolds with additional fragments opens up the road to the development of selective ligands for G-quadruplex-forming regions of the genome.     

A High-Affinity “Synthavidin” Receptor for Squaraine Dyes

Strong-binding host–guest pairings in aqueous media have potential as “supramolecular glues” in biomedical techniques, complementing the widely-used (strept)avidin-biotin combination. We have previously found that squaraine dyes are bound very strongly by tetralactam macrocycles possessing anthracenyl units as cavity walls. Here we show that replacing the anthracenes with pentacyclic 5,7,12,14-tetrahydro-5,7,12,14-tetraoxapentacene (TOP) units generates receptors which bind squaraines with increased affinities (around K_a=10¹⁰ m ⁻¹) and improved selectivities. Binding can be followed through changes to squaraine fluorescence and absorbance. The TOP units are easy to prepare and potentially variable, while the TOP-based receptor shows improved photostability, both in itself and in complex with squaraines. The results suggest that this system could prove valuable in the further development of practical “synthavidin” chemistry.     

Binding Site Switch by Dispersion Interactions: Rotational Signatures of Fenchone–Phenol and Fenchone–Benzene Complexes

Non-covalent interactions between molecules determine molecular recognition and the outcome of chemical and biological processes. Characterising how non-covalent interactions influence binding preferences is of crucial importance in advancing our understanding of these events. Here, we analyse the interactions involved in smell and specifically the effect of changing the balance between hydrogen-bonding and dispersion interactions by examining the complexes of the common odorant fenchone with phenol and benzene, mimics of tyrosine and phenylalanine residues, respectively. Using rotational spectroscopy and quantum chemistry, two isomers of each complex have been identified. Our results show that the increased weight of dispersion interactions in these complexes changes the preferred binding site in fenchone and sets the basis for a better understanding of the effect of different residues in molecular recognition and binding events.     

Competition of Ligands and the 18-mer Binding Domain of the RHAU Helicase for G-Quadruplexes: Orthosteric or Allosteric Binding Mechanism?

Stabilizing the DNA and RNA structures known as G-quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G-quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G-quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also to free external G-quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu–ttpy (ttpy=4′-tolyl-2,2′:6′,2′′-terpyridine) induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug–target interactions, but also how the binding to other partners is affected.     

Ligand Migration in the Gaseous Insulin-CB7 Complex—A Cautionary Tale About the Use of ECD-MS for Ligand Binding Site Determination

Knowledge of the structure of protein?Cligand complexes can aid in understanding their roles within complex biological processes. Here we use electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer to investigate the noncovalent binding of the macrocycle cucurbit[7]uril (CB7) to bovine insulin. Recent condensed-phase experiments (Chinai et al., J. Am. Chem. Soc. 133:8810?C8813, 2011) indicate that CB7 binds selectively to the N-terminal phenylalanine of the insulin B-chain. Competition experiments employing ESI mass spectrometry to assess complex formation between CB7 and wild type insulin B-chain vs. a mutant B-chain, confirm that the N-terminal phenylalanine plays in important role in solution-phase binding. However, analysis of fragment ions produced by electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the insulin B-chain suggests a different picture. The apparent gas-phase binding site, as identified by the ECD, lies further along the insulin B-chain. Together, these studies thus indicate that the CB7 ligand migrates in the ESI mass spectrometry analysis. Migration is likely aided by the presence of additional interactions between CB7 and the insulin B-chain, which are not observed in the crystal structure. While this conformational difference may result simply from the removal of solvent and addition of excess protons by the ESI, we propose that the migration may be enhanced by charge reduction during the ECD process itself because ion-dipole interactions are key to CB7 binding. The results of this study caution against using ECD-MS as a stand-alone structural probe for the determination of solution-phase binding sites.     

Polyamine-Functionalized 2′-Amino-LNA in Oligonucleotides: Facile Synthesis of New Monomers and High-Affinity Binding towards ssDNA and dsDNA

Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2′-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2′-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson–Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3′-nucleolytic digestion relative to the non-functionalized controls.     

Synthesis of Chiral Sulfamide–Amine Alcohol Ligands for Enantioselective Alkylation of Aldehydes

A series of chiral sulfamide–amine alcohols (SAA) (1–6) has been easily synthesized from commercially available chiral amino alcohols. In the absence of Ti(O ⁱ Pr)₄, ligand 4 catalyzed the asymmetric addition of diethylzinc to aromatic aldehydes with moderate to good yields and enantioselectivities.     

Selection of Peptide Ligands Specific for Baculovirus DNA‐Binding Protein from the Flitrx™ Random Peptide Display Library

Abstract

Autographa californica nucleopolyhedrovirus (AcNPV) is a baculovirus that is widely employed as a vector for the expression of foreign genes and pest control. Although baculoviruses, including AcNPV, efficiently replicate in the nuclei of arthropod cells, the dynamics and mechanism of DNA replication within the infected cell are still poorly understood. It has been found that the DNA‐binding protein (DBP) is an early gene product and appears to be crucial for viral DNA replication.

Presented here is the selection of peptide ligands that specifically bind to DBP for AcNPV from the FliTrx? random peptide display library; this entails the amplification, cloning of the DNA‐binding protein (DBP) gene from AcNPV and the construction of the expression plasmid for DBP, and the expression and purification of the recombinant His.Tag AcNPV DBP that was used as a target molecule for the selection of the peptide ligands specific for AcNPV DBP. The affinity and efficiency of such peptide ligands were then measured by ELISA procedures.

The beneficial aspect of this research is the monospecificity quality of the peptide ligands specific for AcNPV DBP. They could be used for the study of the dynamics of the viral genome and its replication within the infected cell, for the development of a quantitative method for the determination of the presence of baculovirus in various samples, for the development of a peptide ligandbased assay for the determination of baculovirus titers; or they could be immobilized on a chromatographic support for an improved affinity purification of AcNPV DBP.     

Active Site Models for the Cu(A) Site of Peptidylglycine α-Hydroxylating Monooxygenase and Dopamine β-Monooxygenase

A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated product. The present results have indicated that a superoxo species having a four-coordinate distorted tetrahedral geometry could be reactive enough to induce the direct C-H bond activation of aliphatic substrates in the enzymatic systems.     

Hexafluoropropylene Oxide–Alcohol: A Convenient System for Silica Dissolution

Abstract

A new method of silica dissolution is described. It involves the formation of a stable SiF₄ · n ROH complex (1, 1a) just from SiO₂ and anhydrous alcoholic HF generated in situ from commercially available hexafluoropropene oxide. Alcoholic SiF₄ complexes can be easily converted to different organosilicon compounds of the type SiF₄L₂ and (LH)₂SiF₆ [L = 1,10-phenantroline (2a), 2,2′-dipyridyl (2b), Me₂SO (2c), pyridine (2d), triethanolamine (3a)]. Different silica-containing compounds can be used in this strategy—silicagel, sand, alumosilicates, and even rice husk.

GRAPHICAL ABSTRACT     

Binding affinity prediction for binary drug–target interactions using semi-supervised transfer learning

Journal of Computer-Aided Molecular Design - In the field of drug–target interactions prediction, the majority of approaches formulated the problem as a simple binary classification task....     

Resolving Multiple Protein–Peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease

Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE–FL) can resolve multiple MHC–peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE–FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.     

Bismuth Chloride–Sodium Nitrite: A Novel Reagent for Chemoselective N-Nitrosation

Bismuth(III) chloride–sodium nitrite was used as a mild and efficient reagent for N-nitrosation of various tetrazoles, secondary amines, and amides under ambient conditions. Nitrosation took place chemoselectively at the nitrogen atom, giving corresponding N-nitroso derivatives in good to excellent yield.     

Chemo-enzymatic Synthesis and RAFT Polymerization of 6-O-Methacryloyl Mannose: A Suitable Glycopolymer for Binding to the Tetrameric Lectin Concanavalin A?

Summary: The chemo-enzymatic synthesis of 6-O-methacryloyl mannose (MaM) glycomonomer was successfully performed for the first time. Subsequent aqueous RAFT polymerization of the monomer yielded well-defined, linear poly(6-O-methacryloyl mannose) (PMaM) glycopolymers without the need for protecting and deprotecting group chemistry. As well as investigating the RAFT polymerization kinetics of this monomer using various initial monomer to chain transfer agent concentration ratios, the protein binding ability of the generated glycopolymer was tested using concanavalin A, a known mannose-residue binding lectin.     

Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures

Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein–ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein–ligand binding interactions. The protocol utilizes an H₂¹⁶O₂ and H₂¹⁸O₂ labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein–ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the ¹⁸O/¹⁶O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique’s ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein–protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).