1,721,002 research outputs found
Cysteine sulfinic acid and sulfinylated peptides
No full textCysteine sulfinic acid (CSA) is a stable post translational modification in nature. While long considered to be an irreversible by-product of accidental overoxidation of the cysteine sulfur, evidence in the last two decades has accumulated for its role in numerous and tightly regulated mechanisms. Proteomics studies in the last two decades have identified CSA in hundreds of cellular proteins, highlighting its omnipresence at the core of the cysteine redoxome. Elsewhere, structural studies have shed initial light on the molecular mechanisms underlying CSA reduction in vivo by the sulfiredoxin (Srx) enzyme. While peroxiredoxins have for a long time been the only known substrates to be turned over by Srx, recent studies have uncovered a plethora of potential new substrates of Srx, opening new avenues of investigation in fundamental biology, but also possibly opening new opportunities for developing novel medicines targeting the redoxome, especially in cancer and neurodegeneration. This review first summarises important knowledge surrounding the stereo-electronics and biochemical properties of CSA, including how it is reduced by Srx. In a second part, it highlights the chemical methods recently developed for CSA characterisation, with important examples of electrophilic probes for CSA covalent adduct formation. Crucially, in vitro biochemical studies of CSA and its peptides have historically proven difficult, in great part due to the limitations associated with the few existing synthetic methods available. Here, we also provide a summary of synthetic methods currently available for CSA incorporation into peptides, and their current limitations.</p
Current strategies for the design of PROTAC linkers: a critical review
No full textPROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands; an “anchor” to bind to an E3 ubiquitin ligase and a “warhead” to bind to a protein of interest, connected by a chemical linker. Targeted protein degradation by PROTACs has emerged as a new modality for the knock down of a range of proteins, with the first agents now reaching clinical evaluation. It has become increasingly clear that the length and composition of the linker play critical roles on the physicochemical properties and bioactivity of PROTACs. While linker design has historically received limited attention, the PROTAC field is evolving rapidly and currently undergoing an important shift from synthetically tractable alkyl and polyethylene glycol to more sophisticated functional linkers. This promises to unlock a wealth of novel PROTAC agents with enhanced bioactivity for therapeutic intervention. Here, the authors provide a timely overview of the diverse linker classes in the published literature, along with their underlying design principles and overall influence on the properties and bioactivity of the associated PROTACs. Finally, the authors provide a critical analysis of current strategies for PROTAC assembly. The authors highlight important limitations associated with the traditional “trial and error” approach around linker design and selection, and suggest potential future avenues to further inform rational linker design and accelerate the identification of optimised PROTACs. In particular, the authors believe that advances in computational and structural methods will play an essential role to gain a better understanding of the structure and dynamics of PROTAC ternary complexes, and will be essential to address the current gaps in knowledge associated with PROTAC design
Biophysical survey of small-molecule β-catenin inhibitors: a cautionary tale
No full textThe canonical Wingless-related integration site signaling pathway plays a critical role in human physiology, and its dysregulation can lead to an array of diseases. β-Catenin is a multifunctional protein within this pathway and an attractive yet challenging therapeutic target, most notably in oncology. This has stimulated the search for potent small-molecule inhibitors binding directly to the β-catenin surface to inhibit its protein-protein interactions and downstream signaling. Here, we provide an account of the claimed (and some putative) small-molecule ligands of β-catenin from the literature. Through in silico analysis, we show that most of these molecules contain promiscuous chemical substructures notorious for interfering with screening assays. Finally, and in line with this analysis, we demonstrate using orthogonal biophysical techniques that none of the examined small molecules bind at the surface of β-catenin. While shedding doubts on their reported mode of action, this study also reaffirms β-catenin as a prominent target in drug discovery
Kinetic method for the large-scale analysis of the binding mechanism of histone deacetylase inhibitors
No full textPerforming kinetic studies on protein ligand interactions provides important information on complex formation and dissociation. Beside kinetic parameters such as association rates and residence times, kinetic experiments also reveal insights into reaction mechanisms. Exploiting intrinsic tryptophan fluorescence a parallelized high-throughput Förster resonance energy transfer (FRET)-based reporter displacement assay with very low protein consumption was developed to enable the large-scale kinetic characterization of the binding of ligands to recombinant human histone deacetylases (HDACs) and a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes. For the binding of trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and two other SAHA derivatives to HDAH, two different modes of action, simple one-step binding and a two-step mechanism comprising initial binding and induced fit, were verified. In contrast to HDAH, all compounds bound to human HDAC1, HDAC6, and HDAC8 through a two-step mechanism. A quantitative view on the inhibitor-HDAC systems revealed two types of interaction, fast binding and slow dissociation. We provide arguments for the thesis that the relationship between quantitative kinetic and mechanistic information and chemical structures of compounds will serve as a valuable tool for drug optimization
Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes
Get PDFThermodynamic studies on ligand–protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n?=?4–7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of deltaH0/deltaG0. The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design
Luminescent lanthanide helicates self-assembled from ditopic ligands bearing phosphonic acid or phosphoester units
Get PDFA series of hexadentate ditopic receptors incorporating benzimidazole moieties have been designed, which are fitted with phosphonic acid or phosphoethylester coordinating units. In addition, poly(oxyethylene) pendants have been introduced on the benzimidazole backbone of two ligands to increase water solubility. The ligands self-assemble with lanthanide ions under stoichiometric conditions, yielding triple-stranded homobimetallic helicates, as ascertained by mass spectrometry and UV-visible titrations. The helicates display large thermodynamic stability, for example, log beta(23) approximately 21-24 for all the Eu(III) complexes. Photophysical measurements reveal sensitization of the metal-centered luminescence in the europium and terbium complexes, which is modulated by the nature of the ligand. Hydration numbers determined by the lifetime method are essentially zero. The Eu((5)D(0)) lifetimes are long and reach values up to 3.2 ms, while quantum yields as high as 25% are obtained in water at pH 7.4. Back transfer limits the sensitization efficiency for Tb(III) luminescence, and both lifetimes and quantum yields are much smaller. The properties of the helicates are discussed with respect to those self-assembled from ligands bearing carboxylate coordinating units
SOPS for preparation of solutions for testing NHS respirators
No full textThe fit of the respirators used by NHS workers in treating COVID-19 patients is tested to ensure they are properly protected. This is done by spraying a strong-tasting solution in the face of the person wearing a respirator. If they can taste the solution, the respirator does not fit properly or is otherwise faulty. Suitable solutions are sold commercially but there appears to be a shortage and some hospitals are struggling to secure the volumes needed to keep NHS staff protected. As solutions identical to the commercial solutions are easy to prepare and the materials required are cheap, the University of Southampton has successfully prepared stocks for use in Southampton General Hospital and neighbouring NHS trusts and services. The solutions conform to British Standard BS ISO 16975‑3:2017 and the US Occupational Safety and Health Administration (OSHA) in 29 CFR 1910.134 are rigorously quality checked before delivery.
In this document we have provided the QC form we used to validate our process and the procedures we used internally to ensure we produced solutions to the required specifications that were sterilised for storage and properly labelled for the end user. Please note these are not “instructions” – in undertaking this work you are to use your own expert knowledge and assess all risks and other requirements yourself.
The University of Southampton does not accept any legal liability. </span
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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