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. Here we describe the systematic and quantitative characterisation of passive EV loading with small molecules based on hydrophobic interactions - either through direct adsorption of hydrophobic compounds, or by membrane anchoring of hydrophilic ligands via cholesterol tags. As revealed by single vesicle imaging, both ligand types bind to CD63 positive EVs (exosomes), however also non-specifically to other vesicles, particles, and serum proteins. The hydrophobic compounds Curcumin and Terbinafine aggregate on EVs with no apparent saturation up to 106-107 molecules per vesicle as quantified by liquid chromatography - high resolution mass spectrometry (LC-HRMS). For both compounds, high density EV loading resulted in the formation of a population of large, electron-dense vesicles as detected by quantitative cryo-transmission electron microscopy (TEM), a reduced EV cell uptake and a toxic gain of function for Curcumin-EVs. In contrast, cholesterol tagging of a hydrophilic mdm2-targeted cyclic peptide saturated at densities of ca 104-105 molecules per vesicle, with lipidomics showing addition to, rather than replacement of endogenous cholesterol. Cholesterol anchored ligands did not change the EVs' size or morphology, and such EVs retained their cell uptake activity without inducing cell toxicity. However, the cholesterol-anchored ligands were rapidly shed from the vesicles in presence of serum. Based on these data, we conclude that (1) both methods allow loading of EVs with small molecules but are prone to unspecific compound binding or redistribution to other components if present in the sample, (2) cholesterol anchoring needs substantial optimization of formulation stability for in vivo applications, whereas (3) careful titration of loading densities is warranted when relying on hydrophobic interactions of EVs with hydrophobic compounds to mitigate changes in physicochemical properties, loss of EV function and potential cell toxicity.

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. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

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. Over time, the exceptional versatility of this linker has been demonstrated in several applications of advanced solid phase synthesis of peptides and peptidomimetics. Recently, an updated synthesis of the original linker has also been presented at the 22nd American Peptide Symposium, comprising 10 steps. Herein, the design and synthesis of a next generation SCAL linker, SCAL-2, is reported. SCAL-2 features a simplified molecular architecture, which allows for a more efficient synthesis in 8 steps with superior yields. Both linkers, SCAL and SCAL-2 are compared in terms of their cleavage properties adding valuable information on how to best utilize the versatility of these linkers for solid phase synthesis.

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. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.

Dynamics and interaction of interleukin-4 receptor subunits in living cells.

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (K(d,2D)) of 180 and 480 receptors per µm(2) for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a K(d,2D) of ∼1000 receptors per µm(2). The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

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). Cellular assays demonstrate the activity of IBE-667 in promoting the binding of LFA-1 on activated immune cells to ICAM-1.

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). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.

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. We have equipped the COPAS bead sorting instrument with a high-speed profiling unit and developed a spectral autofluorescence correction method. The correction method is based on a simple algebraic operation using the fluorescence data from two detection channels and is applied on-the-fly in order to reliably identify hit beads by COPAS bead sorting. Our method provides a practical tool for the fast and efficient isolation of hit beads from one-bead one-compound library screens using either fluorescently labeled target proteins or biotinylated target proteins. This method makes hit bead identification easier and more reliable. It reduces false positives and eliminates the need for time-consuming pre-sorting of library beads in order to remove autofluorescent beads.

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. Since the establishment of one-bead one-compound library synthesis, the properties of polymer beads in chemical reactions have been thoroughly investigated. However, the characterization of the kinetics and thermodynamics of protein-ligand interactions on the beads used for screening has received much less attention. Consequently, the majority of reported on-bead screens are based on empirically derived procedures, independent of measured equilibrium constants and rate constants of protein binding to ligands on beads. More often than not, on-bead screens reveal apparent high affinity binders through strong protein complexation on the matrix of the solid support. After decoding, resynthesis, and solution testing the primary hits turn out to be unexpectedly weak binders, or may even fall out of the detection limit of the solution assay. Only a quantitative comparison of on-bead binding and solution binding events will allow systematically investigating affinity differences as function of protein and small molecule properties. This will open up routes for optimized bead materials, blocking conditions and other improved assay procedures. By making use of the unique features of our previously introduced confocal nanoscanning (CONA) method, we investigated the kinetic and thermodynamic properties of protein-ligand interactions on TentaGel beads, a popular solid support for on-bead screening. The data obtained from these experiments allowed us to determine dissociation constants for the interaction of bead-immobilized ligands with soluble proteins. Our results therefore provide, for the first time, a comparison of on-bead versus solution binding thermodynamics. Our data indicate that affinity ranges found in on-bead screening are indeed narrower compared to equivalent interactions in homogeneous solution. A thorough physico-chemical understanding of the molecular recognition between proteins and surface bound ligands will further strengthen the role of on-bead screening as an ultimately cost-effective method in hit and lead finding.

Towards mimicking short linear peptide motifs: identification of new mixed α,ß-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. non-coded amino acids) which confer improved biophysical properties. An elegant approach towards the identification of peptidomimetics is through screening of large combinatorial chemical libraries incorporating both coded and non-coded amino acids (e.g. ß amino acids). We apply here our recently developed Integrated Chemical Biophysics (ICB) platform, which combines microscale one-bead one-compound screening with fluorescence tagging of retrieved hit beads and subsequent affinity determination of hit compounds in homogenous solution, to the task of identifying novel mixed α, ß peptidomimetic binders for the adaptor protein SLAM-associated protein (SAP), which acts as an intracellular adapter that transduces T and NK cell activation. An enhancement to the ICB process is introduced which enables ranking hit compounds from single-point measurements even if the library compound is <95% pure and without HPLC purification of single-bead-derived substance. Finally, a novel computational protocol enabling binding mode and SAR rationalisation of hit compounds is also described which we now utilise to inform future library design. Application of the full ICB process has allowed identification of a highly interesting motif, Ac-ß(3)-Pro-α-pTyr, as a mimic for the -1 and -2 positions of the natural binding motif and provides a promising starting point for further optimization towards higher-affinity SAP inhibitors with enhanced metabolic stability. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-011-0071-9) contains supplementary material, which is available to authorized users.

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. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.

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. Ideally they should allow to flag hit compounds with undesirable biophysical properties such as aggregation, unspecific binding, or insufficient solubility and the like. Here we demonstrate that miniaturized and parallelized equilibrium dialysis is an excellent and generic secondary confirmation method for hit compounds identified by on-bead screening. We further show that microscale dialysis can be reliably performed prior to decoding and resynthesis even with hit-compounds cleaved from the single beads. Down-scaling of the method takes advantage of the fluorescent tag, AIDA, which is integrated as permanent tracer in our library design. Our results suggest that microscale equilibrium dialysis followed by high performance liquid chromatography (HPLC) analysis is a generic, cheap, and meaningful confirmation method for identifying the most promising candidates within a series hit compounds derived from fluorescently tagged one-bead one-compound libraries.

Identification of a small molecule inhibitor of importin ß 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. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin ß with high nanomolar affinity and specifically inhibits importin α/ß mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding experiments suggest that karyostatin 1A acts by disrupting the interaction between importin ß and the GTPase Ran. As a selective inhibitor of the importin α/ß import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.

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. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.

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. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.

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. Here we describe the identification of the first RRM3 (RNA recognition motif 3) targeted low molecular weight HuR inhibitors from a one-bead-one-compound library screen using confocal nanoscanning. A further compound characterization revealed the presence of an ATP-binding pocket within HuR RRM3, associated with enzymatic activity. Centered around a metal-ion-coordinating DxD motif, the catalytic site mediates 3'-terminal adenosyl modification of non-polyadenylated RNA substrates by HuR. These findings suggest that HuR actively contributes to RNA modification and maturation and thereby shed an entirely new light on the role of HuR in RNA metabolism.

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. Also, the U.S. Food and Drug Administration is pulling back on approvals, requesting larger safety studies. Among the different approaches taken throughout the industry to improve productivity and to reduce the attrition rate of compounds in the drug discovery process, an extended application of quantitative biology and biophysical methods is ranked very high. Fluorescence spectroscopy and imaging represented the main detection technologies for assays and screening methods in recent years. Today, label-free detection methods, such as isothermal titration calorimetry, differential scanning calorimetry, tandem mass spectrometry (MS(n)), light scattering, or interferometry, start to provide viable alternative readouts for physicochemical characterization of leads and hit list triaging. However, the multidimensional nature of fluorescence along with its high sensitivity and single-molecule resolution remains an unparalleled source of molecular parameters to extract all different kinds of information on molecules and ligand-protein complexes in solution. Although fluorescence-based methods are currently applied throughout the different stages of the industrial drug discovery process, they are usually applied in an unconnected way. We have developed a fully integrated hit and lead discovery process combining bead-based synthesis and screening methods with confocal fluorescence microspectroscopy. The primary on-bead screening process provides fluorescent ligands that after a multistep characterization process ultimately leads to fully mechanistically characterized cellularly validated binders and inhibitors of target protein interactions. The unlabeled small-molecular inhibitors represent chemical starting points in drug discovery and target validation.

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.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.