Quantitative and functional characterisation of extracellular vesicles after passive loading with hydrophobic or cholesterol-tagged small molecules.

Extracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application potential as biological drug delivery vehicles. Much progress has been made for loading or decorating EVs with proteins, peptides or RNAs using genetically engineered donor cells, but post-isolation loading with synthetic drugs and using EVs from natural sources remains challenging. In particular, quantitative and unambiguous data assessing whether and how small molecules associate with EVs versus other components in the samples are still lacking.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field.

Facile Synthesis of a Next Generation Safety-Catch Acid-Labile Linker, SCAL-2, Suitable for Solid-Phase Synthesis, On-Support Display and for Post-Synthesis Tagging.

The SCAL linker, a safety catch linker, is amongst the most versatile linkers for solid phase synthesis. It was originally described in 1991 by Pátek and Lebl. Yet, its application has been hindered by the low yields of published synthetic routes.

A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking.

Identification and X-ray co-crystal structure of a small-molecule activator of LFA-1-ICAM-1 binding.

Stabilization of protein-protein interactions by small molecules is a concept with few examples reported to date. Herein we describe the identification and X-ray co-crystal structure determination of IBE-667, an ICAM-1 binding enhancer for LFA-1. IBE-667 was designed based on the SAR information obtained from an on-bead screen of tagged one-bead one-compound combinatorial libraries by confocal nanoscanning and bead picking (CONA).

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT).

A two-channel detection method for autofluorescence correction and efficient on-bead screening of one-bead one-compound combinatorial libraries using the COPAS fluorescence activated bead sorting system.

One-bead one-compound combinatorial library beads exhibit varying levels of autofluorescence after solid phase combinatorial synthesis. Very often this causes significant problems for automated on-bead screening using TentaGel beads and fluorescently labeled target proteins. Herein, we present a method to overcome this limitation when fluorescence activated bead sorting is used as the screening method.

Towards mimicking short linear peptide motifs: identification of new mixed α,β-peptidomimetic ligands for SLAM-Associated Protein (SAP) by confocal on-bead screening.

Unlabelled: An array of chemical modifications have recently emerged, designed to improve the stability of natural peptides that inherently suffer from short in vivo half-lives, thereby preventing their use as therapeutics. The resultant peptidomimetics resemble native peptides; however, they contain synthetic elements (e.g.

On-bead screens sample narrower affinity ranges of protein-ligand interactions compared to equivalent solution assays.

Conceptually, on-bead screening is one of the most efficient high-throughput screening (HTS) methods. One of its inherent advantages is that the solid support has a dual function: it serves as a synthesis platform and as a screening compartment. Compound purification, cleavage and storage and extensive liquid handling are not necessary in bead-based HTS.

Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits.

Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively.

Analysis of protein-small molecule interactions by microscale equilibrium dialysis and its application as a secondary confirmation method for on-bead screening.

On-bead screening of one-bead one compound (OBOC) libraries is an ultra fast surface based primary high-throughput screening (HTS) method. Typically the binding of a tagged target protein to bead immobilized compounds or its altered enzymatic activity are detected. For an efficient and reliable ligand discovery process secondary assays to confirm on-bead compound activity in homogeneous solution are key to exclude artifacts and weak binders.

Identification of a small molecule inhibitor of importin β mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries.

In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described.

Single bead labeling method for combining confocal fluorescence on-bead screening and solution validation of tagged one-bead one-compound libraries.

Screening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities.

Confocal nanoscanning, bead picking (CONA): PickoScreen microscopes for automated and quantitative screening of one-bead one-compound libraries.

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation.

A highly potent and cellularly active beta-peptidic inhibitor of the p53/hDM2 interaction.

New and improved: The incorporation of a 6-chlorotryptophan (6-Cl-Trp) into a beta-peptide (M)-3(14) helix leads to a high-affinity hDM2 inhibitor, as demonstrated by fluorescence fluctuation analysis at single molecule resolution. When conjugated to penetratin, the newly derived hDM2 binder specifically inhibits tumour cell growth in vitro.

Terminal adenosyl transferase activity of posttranscriptional regulator HuR revealed by confocal on-bead screening.

Posttranscriptional regulation and RNA metabolism have become central topics in the understanding of mammalian gene expression and cell signalling, with the 3' untranslated region emerging as the coordinating unit. The 3' untranslated region trans-acting factor Hu protein R (HuR) forms a central posttranscriptional pathway node bridging between AU-rich element-mediated processes and microRNA regulation. While (m)RNA control by HuR has been extensively characterized, the molecular mode of action still remains elusive.

Single-bead, single-molecule, single-cell fluorescence: technologies for drug screening and target validation.

According to many current reports, the pharmaceutical business will hit a wall over the next few years. The generic competition is expected to wipe out a double-digit billion-dollar amount from top companies' annual sales between 2007 and 2012 (Wall Street Journal, online, December 6, 2007). The industry's science engine has stalled, new blockbusters are lacking, and patent expirations are a big problem.

Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR.

Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.

In vivo detection of amyloid-beta deposits by near-infrared imaging using an oxazine-derivative probe.

As Alzheimer's disease pathogenesis is associated with the formation of insoluble aggregates of amyloid beta-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices.

The chemical hunt for the identification of drugable targets.

Chemical biology has emerged as a new scientific discipline to change the way scientists approach and study the interface between chemistry, biology, and physics. By integrating the knowledge base of the human genome with the power of diverse and flexible chemical technology platforms, the ultimate goal is to define the 'rules of engagement' for small molecules and their use in basic biology and in drug discovery. Herein, we highlight the current counterpoles of the chemical biology philosophy in the framework between conformational diversity and informational complexity.