Luc Brunsveld

Modulators of protein-protein interactions.

Lech-Gustav Milroy,† Tom N. Grossmann,‡,§ Sven Hennig,‡ Luc Brunsveld,† and Christian Ottmann*,† †Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, Technische Universiteit Eindhoven, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands ‡Chemical Genomics Centre of the Max Planck Society, Otto-Hahn Straße 15, 44227 Dortmund, Germany Department of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

Hierarchical formation of helical supramolecular polymers via stacking of hydrogen-bonded pairs in water

Bifunctional ureido-s-triazines provided with penta(ethylene oxide) side chains are able to self assemble in water, leading to helical columns via cooperative stacking of the hydrogen-bonded pairs (DADA array). Monofunctional ureido-s-triazines do not form such helical architectures. The presence of a linker, covalently connecting the two ureido-s-triazine units, is essential as it generates a high local concentration of aromatic units, favorable for stacking interactions. This hydrophobic stacking of the aromatic units occurs at concentrations as low as 5⋅10−6 M and can be visualized by using fluorescence spectroscopy. The stacking generates a hydrophobic microenvironment that allows intermolecular hydrogen bonding to occur at higher concentrations because the hydrogen bonds are shielded from competitive hydrogen bonding with water. This hierarchical process results in the formation of a helical self-assembled polymer in water at concentrations above 10−4 M. Chiral side chains attached to the ureido-s-triazine units bias the helicity of these columns as concluded from CD spectroscopy and “Sergeants and Soldiers” experiments.

Pre- and Postfunctionalized Self-Assembled π-Conjugated Fluorescent Organic Nanoparticles for Dual Targeting

There is currently a high demand for novel approaches to engineer fluorescent nanoparticles with precise surface properties suitable for various applications, including imaging and sensing. To this end, we report a facile and highly reproducible one-step method for generating functionalized fluorescent organic nanoparticles via self-assembly of prefunctionalized π-conjugated oligomers. The engineered design of the nonionic amphiphilic oligomers enables the introduction of different ligands at the extremities of inert ethylene glycol side chains without interfering with the self-assembly process. The intrinsic fluorescence of the nanoparticles permits the measurement of their surface properties and binding to dye-labeled target molecules via Förster resonance energy transfer (FRET). Co-assembly of differently functionalized oligomers is also demonstrated, which enables the tuning of ligand composition and density. Furthermore, nanoparticle prefunctionalization has been combined with subsequent postmodification of azide-bearing oligomers via click chemistry. This allows for expanding ligand diversity at two independent stages in the nanoparticle fabrication process. The practicability of the different methods entails greater control over surface functionality. Through labeling with different ligands, selective binding of proteins, bacteria, and functionalized beads to the nanoparticles has been achieved. This, in combination with the absence of unspecific adsorption, clearly demonstrates the broad potential of these nanoparticles for selective targeting and sequestration. Therefore, controlled bifunctionalization of fluorescent π-conjugated oligomer nanoparticles represents a novel approach with high applicability to multitargeted imaging and sensing in biology and medicine.

Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants

Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed.

Supramolecular control of enzyme activity through cucurbit[8]uril-mediated dimerization.

At the double: Cucurbit[8]uril-mediated protein dimerization enables reversible control over strong enzyme activation of caspases. Simple addition of a short N-terminal FGG motif allows for a supramolecular-mediated 50-fold enhancement of caspase-9 catalytic activity.

Reversible and Oriented Immobilization of Ferrocene-Modified Proteins

Adopting supramolecular chemistry for immobilization of proteins is an attractive strategy that entails reversibility and responsiveness to stimuli. The reversible and oriented immobilization and micropatterning of ferrocene-tagged yellow fluorescent proteins (Fc-YFPs) onto β-cyclodextrin (βCD) molecular printboards was characterized using surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in combination with electrochemistry. The proteins were assembled on the surface through the specific supramolecular host-guest interaction between βCD and ferrocene. Application of a dynamic covalent disulfide lock between two YFP proteins resulted in a switch from monovalent to divalent ferrocene interactions with the βCD surface, yielding a more stable protein immobilization. The SPR titration data for the protein immobilization were fitted to a 1:1 Langmuir-type model, yielding K(LM) = 2.5 × 10(5) M(-1) and K(i,s) = 1.2 × 10(3) M(-1), which compares favorably to the intrinsic binding constant presented in the literature for the monovalent interaction of ferrocene with βCD self-assembled monolayers. In addition, the SPR binding experiments were qualitatively simulated, confirming the binding of Fc-YFP in both divalent and monovalent fashion to the βCD monolayers. The Fc-YFPs could be patterned on βCD surfaces in uniform monolayers, as revealed using fluorescence microscopy and atomic force microscopy measurements. Both fluorescence microscopy imaging and SPR measurements were carried out with the in situ capability to perform cyclic voltammetry and chronoamperometry. These studies emphasize the repetitive desorption and adsorption of the ferrocene-tagged proteins from the βCD surface upon electrochemical oxidation and reduction, respectively.

Interaction of 14-3-3 proteins with the Estrogen Receptor Alpha F domain provides a drug target interface

Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T594) for high-affinity interaction. We confirm that T594 is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T594–specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.

Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

Significance It has been shown that 14-3-3 proteins increase trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane by binding to its regulatory (R) domain. This paper contains a detailed characterization of the 14-3-3/CFTR interaction, showing that multiple phosphorylated binding sites in the CFTR R-domain are necessary for significant binding with 14-3-3. We find that one of these binding sites serves as an anchor, while surrounding weaker sites enhance the interaction. Furthermore, we show the druggability of this interaction using natural-product fusicoccin-A, which stabilizes the 14-3-3/CFTR interaction by selectively modifying a weaker binding site. This mechanism of action can serve as a model for the development of new trafficking corrector molecules to treat cystic fibrosis. Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics.

Directed Supramolecular Surface Assembly of SNAP‐tag Fusion Proteins

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized SNAP-fusion proteins on cyclodextrin-coated surfaces yielded stable monolayers. The binding of the fusion proteins is specific and occurs with an affinity in the order of 10(6) M(-1) as determined by surface plasmon resonance. Reversible micropatterns of the fusion proteins on micropatterned cyclodextrin surfaces were visualized by using fluorescence microscopy. Furthermore, the guest-functionalized proteins could be assembled out of solution specifically onto the surface of cyclodextrin vesicles. The SNAP-tag labeling of proteins thus allows for assembly of modified proteins through a host-guest interaction on different surfaces. This provides a new strategy in fabricating protein patterns on surfaces and takes advantage of the high labeling efficiency of the SNAP-tag with designed supramolecular elements.

Semiconductive, one-dimensional, self-assembled nanostructures based on oligopeptides with π-conjugated segments

p Stacking between the s sheets: A newly designed p-conjugated peptide can be assembled into a one-dimensional nanostructure with strong p–p intermolecular electronic communication. A nanoelectronic device based on the achieved individual nanostructure was used to measure conductivity