Prof. Dr. Thomas Schrader
Institute of Organic Chemistry
University of Duisburg-Essen
- Binding Methylarginines and Methyllysines as Free Amino Acids: A Comparative Study of Multiple Host Classes**
Warmerdam, Z. and Kamba, B.E. and Le, M.-H. and Schrader, T. and Isaacs, L. and Bayer, P. and Hof, F.
ChemBioChem 23 (2022)Methylated free amino acids are an important class of targets for host-guest chemistry that have recognition properties distinct from those of methylated peptides and proteins. We present comparative binding studies for three different host classes that are each studied with multiple methylated arginines and lysines to determine fundamental structure-function relationships. The hosts studied are all anionic and include three calixarenes, two acyclic cucurbiturils, and two other cleft-like hosts, a clip and a tweezer. We determined the binding association constants for a panel of methylated amino acids using indicator displacement assays. The acyclic cucurbiturils display stronger binding to the methylated amino acids, and some unique patterns of selectivity. The two other cleft-like hosts follow two different trends, shallow host (clip) following similar trends to the calixarenes, and the other more closed host (tweezer) binding certain less-methylated amino acids stronger than their methylated counterparts. Molecular modelling sheds some light on the different preferences of the various hosts. The results identify hosts with new selectivities and with affinities in a range that could be useful for biomedical applications. The overall selectivity patterns are explained by a common framework that considers the geometry, depth of binding pockets, and functional group participation across all host classes. © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH
view abstract 10.1002/cbic.202100502
- Molecular tweezers - a new class of potent broad-spectrum antivirals against enveloped viruses
Le, M.-H. and Taghuo K., E.S. and Schrader, T.
Chemical Communications 58 (2022)A new supramolecular approach to broad spectrum antivirals utilizes host guest chemistry between molecular tweezers and lysine/arginine as well as choline. Basic amino acids in amyloid-forming SEVI peptides (semen-derived enhancers of viral infection) are included inside the tweezer cavity leading to disaggregation and neutralization of the fibrils, which lose their ability to enhance HIV-1/HIV-2 infection. Lipid head groups contain the trimethylammonium cation of choline; this is likewise bound by molecular tweezers, which dock onto viral membranes and thus greatly enhance their surface tension. Disruption of the envelope in turn leads to total loss of infectiosity (ZIKA, Ebola, Influenza). This complexation event also seems to be the structural basis for an effective inihibition of cell-to-cell spread in Herpes viruses. The article describes the discovery of novel molecular recognition motifs and the development of powerful antiviral agents based on these host guest systems. It explains the general underlying mechanisms of antiviral action and points to future optimization and application as therapeutic agents. This journal is © The Royal Society of Chemistry
view abstract 10.1039/d1cc06737k
- CHAPTER 6: Molecular Tweezers and Clips that Modify Protein Function
Alavijeh, N.S. and Kirupakaran, A. and Klärner, F.-G. and Schrader, T.
Monographs in Supramolecular Chemistry 2021-January (2021)Protein recognition by designed protein ligands is highly challenging, but bears great opportunities. Supramolecular chemists have recently been able to synthesize tailored ligands with remarkable protein recognition properties which are absent in the natural binding partners, and which lead to synergistic effects, positive cooperativity and exquisite selectivity. Thus, the combination of powerful charged interactions with hydrophobic forces has recently led to new prototypes of protein surface binders. This review summarizes the development of molecular tweezers (part 1) and clips (part 2) as unique tools for protein recognition. The parts begin with molecular tweezers for basic amino acid inclusion and the discovery of diphosphate clips for efficient cofactor binding, respectively. Gratifyingly, both host molecules complement each other due to their different molecular shapes. Molecular tweezers will be presented first in their interaction with amino acids and small disordered peptides, where they generally complex each Lys and Arg; then the review will proceed to tweezer complexation with protein surfaces, elucidating the preference for well-accessible basic residues and various examples of protein targeting and interference with protein-protein interactions. Finally, we discuss the advantages of additional recognition elements on the tweezer skeleton, which opens the door to numerous advanced applications in chemical biology and drug discovery. For the clips, we describe in detail the inclusion of two important cationic cofactors, followed by applications on cofactor-mediated enzymatic processes. © The Royal Society of Chemistry 2021.
view abstract 10.1039/9781788019798-00161
- Inhibition of Staphylococcus aureus biofilm-forming functional amyloid by molecular tweezers
Malishev, R. and Salinas, N. and Gibson, J. and Eden, A.B. and Mieres-Perez, J. and Ruiz-Blanco, Y.B. and Malka, O. and Kolusheva, S. and Klärner, F.-G. and Schrader, T. and Sanchez-Garcia, E. and Wang, C. and Landau, M. and Bitan, G. and Jelinek, R.
Cell Chemical Biology 28 (2021)Biofilms are rigid and largely impenetrable three-dimensional matrices constituting virulence determinants of various pathogenic bacteria. Here, we demonstrate that molecular tweezers, unique supramolecular artificial receptors, modulate biofilm formation of Staphylococcus aureus. In particular, the tweezers affect the structural and assembly properties of phenol-soluble modulin α1 (PSMα1), a biofilm-scaffolding functional amyloid peptide secreted by S. aureus. The data reveal that CLR01, a diphosphate tweezer, exhibits significant S. aureus biofilm inhibition and disrupts PSMα1 self-assembly and fibrillation, likely through inclusion of lysine side chains of the peptide. In comparison, different peptide binding occurs in the case of CLR05, a tweezer containing methylenecarboxylate units, which exhibits lower affinity for the lysine residues yet disrupts S. aureus biofilm more strongly than CLR01. Our study points to a possible role for molecular tweezers as potent biofilm inhibitors and antibacterial agents, particularly against untreatable biofilm-forming and PSM-producing bacteria, such as methicillin-resistant S. aureus. © 2021 Elsevier Ltd
view abstract 10.1016/j.chembiol.2021.03.013
- Lysine-selective molecular tweezers are cell penetrant and concentrate in lysosomes
Li, Z. and Siddique, I. and Hadrović, I. and Kirupakaran, A. and Li, J. and Zhang, Y. and Klärner, F.-G. and Schrader, T. and Bitan, G.
Communications Biology 4 (2021)Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm. Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. The main impediment to answering these questions has been the low intrinsic fluorescence of the main compound tested to date, called CLR01. Here, we address these questions using new fluorescently labeled molecular tweezers derivatives. We show that these compounds are internalized in neurons and astrocytes, at least partially through dynamin-dependent endocytosis. In addition, we demonstrate that the molecular tweezers concentrate rapidly in acidic compartments, primarily lysosomes. Accumulation of molecular tweezers in lysosomes may occur both through the endosomal-lysosomal pathway and via the autophagy-lysosome pathway. Moreover, by visualizing colocalization of molecular tweezers, lysosomes, and tau aggregates we show that lysosomes likely are the main site for the intracellular anti-amyloid activity of molecular tweezers. These findings have important implications for the mechanism of action of molecular tweezers in vivo, explaining how administration of low doses of the compounds achieves high effective concentrations where they are needed, and supporting the development of these compounds as drugs for currently cureless proteinopathies. © 2021, The Author(s).
view abstract 10.1038/s42003-021-02603-2
- New tools to probe the protein surface: Ultrasmall gold nanoparticles carry amino acid binders
van der Meer, S.B. and Hadrovic, I. and Meiners, A. and Loza, K. and Heggen, M. and Knauer, S.K. and Bayer, P. and Schrader, T. and Beuck, C. and Epple, M.
Journal of Physical Chemistry B (2021)A strategy toward epitope-selective functionalized nanoparticles is introduced in the following: ultrasmall gold nanoparticles (diameter of the metallic core about 2 nm) were functionalized with molecular tweezers that selectively attach lysine and arginine residues on protein surfaces. Between 11 and 30 tweezer molecules were covalently attached to the surface of each nanoparticle by copper-catalyzed azide alkyne cycloaddition (CuAAC), giving multiavid agents to target proteins. The nanoparticles were characterized by high-resolution transmission electron microscopy, differential centrifugal sedimentation, and 1H NMR spectroscopy (diffusion-ordered spectroscopy, DOSY, and surface composition). The interaction of these nanoparticles with the model proteins hPin1 (WW domain; hPin1-WW) and Survivin was probed by NMR titration and by isothermal titration calorimetry (ITC). The binding to the WW domain of hPin1 occurred with a KD of 41 ± 2 μM, as shown by ITC. The nanoparticle-conjugated tweezers targeted cationic amino acids on the surface of hPin1-WW in the following order: N-terminus (G) ≈ R17 > R14 ≈ R21 > K13 > R36 > K6, as shown by NMR spectroscopy. Nanoparticle recognition of the larger protein Survivin was even more efficient and occurred with a KD of 8 ± 1 μM, as shown by ITC. We conclude that ultrasmall nanoparticles can act as versatile carriers for artificial protein ligands and strengthen their interaction with the complementary patches on the protein surface. © XXXX American Chemical Society
view abstract 10.1021/acs.jpcb.0c09846
- Prospects of ultraviolet resonance Raman spectroscopy in supramolecular chemistry on proteins
Kumar, V. and Holtum, T. and Voskuhl, J. and Giese, M. and Schrader, T. and Schlücker, S.
Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 254 (2021)Ultraviolet resonance Raman scattering (UVRR) has been frequently used for studying peptide and protein structure and dynamics, while applications in supramolecular chemistry are quite rare. Since UVRR offers the additional advantages of chromophore selectivity and high sensitivity compared with conventional non-resonant Raman scattering, it is ideally suited for label-free probing of relatively small artificial/supramolecular ligands exhibiting electronic resonances in the UV. In this perspective article, we first summarize results of UVRR spectroscopy in supramolecular chemistry in the context of peptide/protein recognition. We focus on selected artificial ligands which were rationally designed as selective carboxylate binders (guanidiniocarbonyl pyrrole, GCP, and guanidiniocarbonyl indole, GCI) and selective lysine binder (molecular tweezer, CLR01), respectively, via a combination of non-covalent interactions involving electrostatics, hydrogen bonding, and hydrophobic effects/van der Waals forces. Current limitations of applying UVRR as a universally applicable method for label-free and site-specific probing of molecular recognition between supramolecular ligands and proteins are highlighted. We then propose solutions to overcome these limitations for transforming UVRR spectroscopy into a generic tool in supramolecular chemistry on proteins, with an emphasis on mono- and multivalent GCP- and GCI-based ligands. Finally, we outline specific cases of supramolecular ligands such as molecular tweezers where alternative approaches such as laser-based mid-IR spectroscopy are required since UVRR can intrinsically not provide the required molecular information. © 2021 The Authors
view abstract 10.1016/j.saa.2021.119622
- Specific inhibition of the Survivin–CRM1 interaction by peptide-modified molecular tweezers
Meiners, A. and Bäcker, S. and Hadrović, I. and Heid, C. and Beuck, C. and Ruiz-Blanco, Y.B. and Mieres-Perez, J. and Pörschke, M. and Grad, J.-N. and Vallet, C. and Hoffmann, D. and Bayer, P. and Sánchez-García, E. and Schrader, T. and Knauer, S.K.
Nature Communications 12 (2021)Survivin’s dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein–protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin’s nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine. © 2021, The Author(s).
view abstract 10.1038/s41467-021-21753-9
- Supramolecular Enhancement of a Natural 14-3-3 Protein Ligand
Guillory, X. and Hadrović, I. and De Vink, P.J. and Sowislok, A. and Brunsveld, L. and Schrader, T. and Ottmann, C.
Journal of the American Chemical Society 143 (2021)Rational design of protein-protein interaction (PPI) inhibitors is challenging. Connecting a general supramolecular protein binder with a specific peptidic ligand provides a novel conceptual approach. Thus, lysine-specific molecular tweezers were conjugated to a peptide-based 14-3-3 ligand and produced a strong PPI inhibitor with 100-fold elevated protein affinity. X-ray crystal structure elucidation of this supramolecular directed assembly provides unique molecular insight into the binding mode and fully aligns with Molecular Dynamics (MD) simulations. This new supramolecular chemical biology concept opens the path to novel chemical tools for studying PPIs. © 2021 American Chemical Society.
view abstract 10.1021/jacs.1c07095
- The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301S-tau transgenic mice
Di, J. and Siddique, I. and Li, Z. and Malki, G. and Hornung, S. and Dutta, S. and Hurst, I. and Ishaaya, E. and Wang, A. and Tu, S. and Boghos, A. and Ericsson, I. and Klärner, F.-G. and Schrader, T. and Bitan, G.
Alzheimer's Research and Therapy 13 (2021)Background: Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein aggregation. A lead MT, called CLR01, has been demonstrated to inhibit the aggregation and toxicity of multiple amyloidogenic proteins in vitro and in vivo. Previously, we evaluated the effect of CLR01 in the 3 × Tg mouse model of Alzheimer’s disease, which overexpresses mutant human presenilin 1, amyloid β-protein precursor, and tau and found that subcutaneous administration of the compound for 1 month led to a robust reduction of amyloid plaques, neurofibrillary tangles, and microgliosis. CLR01 also has been demonstrated to inhibit tau aggregation in vitro and tau seeding in cell culture, yet because in Alzheimer’s disease (AD) and in the 3 × Tg model, tau hyperphosphorylation and aggregation are thought to be downstream of Aβ insults, the study in this model left open the question whether CLR01 affected tau in vivo directly or indirectly. Methods: To determine if CLR01 could ameliorate tau pathology directly in vivo, we tested the compound similarly using the P301S-tau (line PS19) mouse model. Mice were administered 0.3 or 1.0 mg/kg per day CLR01 and tested for muscle strength and behavioral deficits, including anxiety- and disinhibition-like behavior. Their brains then were analyzed by immunohistochemical and biochemical assays for pathological forms of tau, neurodegeneration, and glial pathology. Results: CLR01 treatment ameliorated muscle-strength deterioration, anxiety-, and disinhibition-like behavior. Improved phenotype was associated with decreased levels of pathologic tau forms, suggesting that CLR01 exerts a direct effect on tau in vivo. Limitations of the study included a relatively short treatment period of the mice at an age in which full pathology is not yet developed. In addition, high variability in this model lowered the statistical significance of the findings of some outcome measures. Conclusions: The findings suggest that CLR01 is a particularly attractive candidate for the treatment of AD because it targets simultaneously the two major pathogenic proteins instigating and propagating the disease, amyloid β-protein (Aβ), and tau, respectively. In addition, our study suggests that CLR01 can be used for the treatment of other tauopathies in the absence of amyloid pathology. © 2021, The Author(s).
view abstract 10.1186/s13195-020-00743-x
- The molecular tweezer clr01 inhibits antibody-resistant cell-to-cell spread of human cytomegalovirus
Brenner, S. and Braun, B. and Read, C. and Weil, T. and Walther, P. and Schrader, T. and Münch, J. and von Einem, J.
Viruses 13 (2021)Human cytomegalovirus (HCMV) uses two major ways for virus dissemination: infection by cell-free virus and direct cell-to-cell spread. Neutralizing antibodies can efficiently inhibit infection by cell-free virus but mostly fail to prevent cell-to-cell transmission. Here, we show that the ‘molecular tweezer’ CLR01, a broad-spectrum antiviral agent, is not only highly active against infection with cell-free virus but most remarkably inhibits antibody-resistant direct cell-to-cell spread of HCMV. The inhibition of cell-to-cell spread by CLR01 was not limited to HCMV but was also shown for the alphaherpesviruses herpes simplex viruses 1 and 2 (HSV-1,-2). CLR01 is a rapid acting small molecule that inhibits HCMV entry at the attachment and penetration steps. Electron microscopy of extracellular virus particles indicated damage of the viral envelope by CLR01, which likely impairs the infectivity of virus particles. The rapid inactivation of viral particles by CLR01, the viral envelope as the main target, and the inhibition of virus entry at different stages are presumably the key to inhibition of cell-free virus infection and cell-to-cell spread by CLR01. Importance: While cell-free spread enables the human cytomegalovirus (HCMV) and other herpesviruses to transmit between hosts, direct cell-to-cell spread is thought to be more relevant for in vivo dissemination within infected tissues. Cell-to-cell spread is resistant to neutralizing antibodies, thus contributing to the maintenance of virus infection and virus dissemination in the presence of an intact immune system. Therefore, it would be therapeutically interesting to target this mode of spread in order to treat severe HCMV infections and to prevent dissemination of virus within the infected host. The molecular tweezer CLR01 exhibits broad-spectrum antiviral activity against a number of enveloped viruses and efficiently blocks antibody-resistant cell-to-cell spread of HCMV, thus representing a novel class of small molecules with promising antiviral activity. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
view abstract 10.3390/v13091685
- Artificial Signal Transduction
Bekus, R. and Schrader, T.
ChemistryOpen 9 (2020)Communication between and inside cells as well as their response to external stimuli relies on elaborated systems of signal transduction. They all require a directional transmission across membranes, often realized by primary messenger docking onto external receptor units and subsequent internalization of the signal in form of a released second messenger. This in turn starts a cascade of events which ultimately control all functions of the living cell. Although signal transduction is a fundamental biological process realized by supramolecular recognition and multiplication events with small molecules, chemists have just begun to invent artificial models which allow to study the underlying rules, and one day perhaps to rescue damaged transduction systems in nature. This review summarizes the exciting pioneering efforts of chemists to create simple models for the basic principles of signal transduction across a membrane. It starts with first attempts to establish molecular recognition events on liposomes with embedded receptor amphiphiles and moves on to simple transmembrane signaling across lipid bilayers. More elaborated systems step by step incorporate more elements of cell signaling, such as primary and secondary messenger or a useful cellular response such as cargo release. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
view abstract 10.1002/open.201900367
- Chirality sensing of terpenes, steroids, amino acids, peptides and drugs with acyclic cucurbit[: N] urils and molecular tweezers
Prabodh, A. and Bauer, D. and Kubik, S. and Rebmann, P. and Klärner, F.G. and Schrader, T. and Delarue Bizzini, L. and Mayor, M. and Biedermann, F.
Chemical Communications 56 (2020)Achiral chromophoric hosts, i.e. acyclic cucurbit[n]urils and molecular tweezers, were found to respond with characteristic Circular Dichroism (CD) spectra to the presence of micromolar concentrations of chiral hydrocarbons, terpenes, steroids, amino acids and their derivates, and drugs in water. In favourable cases, this allows for analyte identification or for reaction monitoring. This journal is © The Royal Society of Chemistry.
view abstract 10.1039/d0cc00707b
- The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease
Monaco, A. and Maffia, V. and Sorrentino, N.C. and Sambri, I. and Ezhova, Y. and Giuliano, T. and Cacace, V. and Nusco, E. and De Risi, M. and De Leonibus, E. and Schrader, T. and Klärner, F.-G. and Bitan, G. and Fraldi, A.
Molecular Therapy 28 (2020)Fraldi and colleagues demonstrated that multiple amyloid proteins progressively aggregate in neurons of a severe lysosomal storage disease, impairing autophagy degradation and triggering neurodegeneration. They also showed that inhibiting amyloid deposition protects against neurodegeneration, thus providing evidence that amyloid aggregation is a new attractive target for the treatment of LSDs. © 2020 The Author(s) Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid β progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a “molecular tweezer” that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs. © 2020 The Author(s)
view abstract 10.1016/j.ymthe.2020.02.005
- Bisphosphonate ligand mediated ultrasensitive capacitive protein sensor: complementary match of supramolecular and dynamic chemistry
Ertürk, G. and Akhoundian, M. and Lueg-Althoff, K. and Shinde, S. and Yeung, S.Y. and Hedström, M. and Schrader, T. and Mattiasson, B. and Sellergren, B.
New Journal of Chemistry 43 (2019)Modern healthcare demands rapid and accurate detection of proteins/enzymes at the ultratrace level. Herein we present a molecularly imprinted capacitive sensor for trypsin, developed by microcontact imprinting. High affinity and selectivity was achieved by doping the prepolymerization mixture with a stoichiometric amount of methacrylamide-based bisphosphonate (BP) monomer. Taking advantage of the specific interaction between bisphosphonate binding monomers and lysine/arginine residues on the surface of trypsin, we have constructed a powerful polymeric sensor. The BP based sensor has the ability to recognize trypsin over other arginine-rich proteins, even in high ionic strength buffers with a sub-picomolar detection limit (pM). We believe that the combination of supramolecular chemistry, molecular imprinting and advanced instrumentation has a potential for future drug development and diagnostics that extends beyond biomolecular recognition. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.
view abstract 10.1039/c8nj05238g
- Dissociation of the Signaling Protein K-Ras4B from Lipid Membranes Induced by a Molecular Tweezer
Li, L. and Erwin, N. and Möbitz, S. and Niemeyer, F. and Schrader, T. and Winter, R.H.A.
Chemistry - A European Journal 25 (2019)Oncogenic Ras mutations occur in more than 30 % of human cancers. K-Ras4B is the most frequently mutated isoform of Ras proteins. Development of effective K-Ras4B inhibitors has been challenging, hence new approaches to inhibit this oncogenic protein are urgently required. The polybasic domain of K-Ras4B with its stretch of lysine residues is essential for its plasma membrane targeting and localization. Employing CD and fluorescence spectroscopy, confocal fluorescence, and atomic force microscopy we show that the molecular tweezer CLR01 is able to efficiently bind to the lysine stretch in the polybasic domain of K-Ras4B, resulting in dissociation of the K-Ras4B protein from the lipid membrane and disintegration of K-Ras4B nanoclusters in the lipid bilayer. These results suggest that targeting of the polybasic domain of K-Ras4B by properly designed tweezers might represent an effective strategy for inactivation of K-Ras4B signaling. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
view abstract 10.1002/chem.201901861
- Major Differences between the Self-Assembly and Seeding Behavior of Heparin-Induced and in Vitro Phosphorylated Tau and Their Modulation by Potential Inhibitors
Despres, C. and Di, J. and Cantrelle, F.-X. and Li, Z. and Huvent, I. and Chambraud, B. and Zhao, J. and Chen, J. and Chen, S. and Lippens, G. and Zhang, F. and Linhardt, R. and Wang, C. and Klärner, F.-G. and Schrader, T. and Landrieu, I. and Bitan, G. and Smet-Nocca, C.
ACS Chemical Biology 14 (2019)Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer's disease and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that, unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications (PTM), aggregation inducers, or preformed seeds. The most common induction method is addition of polyanions, such as heparin; yet, this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in vitro phosphorylation of tau to produce aggregation-competent forms recently have been introduced (Despres et al. (2017) Proc. Natl. Acad. Sci. U.S.A., 114, 9080-9085). However, the oligomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat brain extract. Seeding activity was measured using a fluorescence-resonance energy transfer-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure-activity relationship study demonstrates that heparin-induced tau behaves differently from in vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resemble brain-derived tau strains suggesting that heparin-induced tau and in vitro phosphorylated tau have different conformations, properties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in vitro phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau. © 2019 American Chemical Society.
view abstract 10.1021/acschembio.9b00325
- Molecular Lysine Tweezers Counteract Aberrant Protein Aggregation
Hadrovic, I. and Rebmann, P. and Klärner, F.-G. and Bitan, G. and Schrader, T.
Frontiers in Chemistry 7 (2019)Molecular tweezers (MTs) are supramolecular host molecules equipped with two aromatic pincers linked together by a spacer (Gakh, 2018). They are endowed with fascinating properties originating from their ability to hold guests between their aromatic pincers (Chen and Whitlock, 1978; Zimmerman, 1991; Harmata, 2004). MTs are finding an increasing number of medicinal applications, e.g., as bis-intercalators for DNA such as the anticancer drug Ditercalinium (Gao et al., 1991), drug activity reverters such as the bisglycoluril tweezers Calabadion 1 (Ma et al., 2012) as well as radioimmuno detectors such as Venus flytrap clusters (Paxton et al., 1991). We recently embarked on a program to create water-soluble tweezers which selectively bind the side chains of lysine and arginine inside their cavity. This unique recognition mode is enabled by a torus-shaped, polycyclic framework, which is equipped with two hydrophilic phosphate groups. Cationic amino acid residues are bound by the synergistic effect of disperse, hydrophobic, and electrostatic interactions in a kinetically fast reversible process. Interactions of the same kind play a key role in numerous protein-protein interactions, as well as in pathologic protein aggregation. Therefore, these particular MTs show a high potential to disrupt such events, and indeed inhibit misfolding and self-assembly of amyloidogenic polypeptides without toxic side effects. The mini-review provides insight into the unique binding mode of MTs both toward peptides and aggregating proteins. It presents the synthesis of the lead compound CLR01 and its control, CLR03. Different biophysical experiments are explained which elucidate and help to better understand their mechanism of action. Specifically, we show how toxic aggregates of oligomeric and fibrillar protein species are dissolved and redirected to form amorphous, benign assemblies. Importantly, these new chemical tools are shown to be essentially non-toxic in vivo. Due to their reversible moderately tight binding, these agents are not protein-, but rather process-specific, which suggests a broad range of applications in protein misfolding events. Thus, MTs are highly promising candidates for disease-modifying therapy in early stages of neurodegenerative diseases. This is an outstanding example in the evolution of supramolecular concepts toward biological application. © Copyright © 2019 Hadrovic, Rebmann, Klärner, Bitan and Schrader.
view abstract 10.3389/fchem.2019.00657
- Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator
Nshanian, M. and Lantz, C. and Wongkongkathep, P. and Schrader, T. and Klärner, F.-G. and Blümke, A. and Despres, C. and Ehrmann, M. and Smet-Nocca, C. and Bitan, G. and Loo, J.A.
Journal of the American Society for Mass Spectrometry 30 (2019)Native top-down mass spectrometry (MS) and ion mobility spectrometry (IMS) were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer’s disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc.) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from non-denaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool used to determine not only the sites of post-translational modifications but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. The ESI-MS study of intact tau proteins indicates that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragment in the presence of CLR01 and phosphorylated tau reveal a shift towards a more compact structure. The mass spectrometry study suggests a picture for the molecular mechanism of the modulation of protein-protein interactions in tau by CLR01. [Figure not available: see fulltext.]. © 2018, American Society for Mass Spectrometry.
view abstract 10.1007/s13361-018-2027-6
- The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS
Malik, R. and Meng, H. and Wongkongkathep, P. and Corrales, C.I. and Sepanj, N. and Atlasi, R.S. and Klärner, F.-G. and Schrader, T. and Spencer, M.J. and Loo, J.A. and Wiedau, M. and Bitan, G.
Journal of Biological Chemistry 294 (2019)Mutations in superoxide dismutase 1 (SOD1) cause 15–20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1’s protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01–a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins–as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro. Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach. © 2019 Malik et al.
view abstract 10.1074/jbc.RA118.005940
- The molecular tweezer CLR01 reduces aggregated, pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy
Herrera-Vaquero, M. and Bouquio, D. and Kallab, M. and Biggs, K. and Nair, G. and Ochoa, J. and Heras-Garvin, A. and Heid, C. and Hadrovic, I. and Poewe, W. and Wenning, G.K. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Stefanova, N.
Biochimica et Biophysica Acta - Molecular Basis of Disease 1865 (2019)Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development. © 2019 Elsevier B.V.
view abstract 10.1016/j.bbadis.2019.07.007
- Cell entry of a host-targeting protein of oomycetes requires gp96
Trusch, F. and Loebach, L. and Wawra, S. and Durward, E. and Wuensch, A. and Iberahim, N.A. and De Bruijn, I. and MacKenzie, K. and Willems, A. and Toloczko, A. and Diéguez-Uribeondo, J. and Rasmussen, T. and Schrader, T. and Bayer, P. and Secombes, C.J. and Van West, P.
Nature Communications 9 (2018)The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants. © 2018 The Author(s).
view abstract 10.1038/s41467-018-04796-3
- Maßgeschneiderte Liganden für Protein-Protein-Interaktionen
Heid, C. and Kujawski, K. and Schrader, T.
BioSpektrum 24 (2018)The de novo design of artificial modulators for protein protein interactions remains a premier challenge in chemical biology. This article summarizes latest developments on the area of small supramolecular binders for hot spots as well as large polymer materials suitable to address extended protein interfaces. In recent years novel chemical tools have evolved which allow to block or activate essential protein contacts at will. © 2018, Springer-Verlag GmbH Deutschland, ein Teil von Springer Nature.
view abstract 10.1007/s12268-018-0934-5
- The molecular tweezer CLR01 inhibits Ebola and Zika virus infection
Röcker, A.E. and Müller, J.A. and Dietzel, E. and Harms, M. and Krüger, F. and Heid, C. and Sowislok, A. and Riber, C.F. and Kupke, A. and Lippold, S. and von Einem, J. and Beer, J. and Knöll, B. and Becker, S. and Schmidt-Chanasit, J. and Otto, M. and Vapalahti, O. and Zelikin, A.N. and Bitan, G. and Schrader, T. and Münch, J.
Antiviral Research 152 (2018)Ebola (EBOV) and Zika viruses (ZIKV) are responsible for recent global health threats. As no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer CLR01 may inhibit EBOV and ZIKV infection. This small molecule has previously been shown to inactivate HIV-1 and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. We found that CLR01 indeed blocked infection of EBOV and ZIKV in a dose-dependent manner. The tweezer inhibited infection of epidemic ZIKV strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. Our findings show that CLR01 is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent. © 2018 Elsevier B.V.
view abstract 10.1016/j.antiviral.2018.02.003
- Using molecular tweezers to remodel abnormal protein self-assembly and inhibit the toxicity of amyloidogenic proteins
Malik, R. and Di, J. and Nair, G. and Attar, A. and Taylor, K. and Teng, E. and Klärner, F.-G. and Schrader, T. and Bitan, G.
Methods in Molecular Biology 1777 (2018)Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly, which act by binding selectively to lysine and arginine residues. Through this unique mechanism of action, MTs inhibit formation of toxic oligomers and aggregates. Their efficacy and safety have been demonstrated in vitro, in cell culture, and in animal models. Here, we discuss the application of MTs in diverse in vitro and in vivo systems, the experimental details, the scope of their use, and the limitations of the approach. We also consider methods for administration of MTs in animal models to measure efficacy, pharmacokinetic, and pharmacodynamic parameters in proteinopathies. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.
view abstract 10.1007/978-1-4939-7811-3_24
- A Binary Bivalent Supramolecular Assembly Platform Based on Cucurbituril and Dimeric Adapter Protein 14-3-3
de Vink, P.J. and Briels, J.M. and Schrader, T. and Milroy, L.-G. and Brunsveld, L. and Ottmann, C.
Angewandte Chemie - International Edition 56 (2017)Interactions between proteins frequently involve recognition sequences based on multivalent binding events. Dimeric 14-3-3 adapter proteins are a prominent example and typically bind partner proteins in a phosphorylation-dependent mono- or bivalent manner. Herein we describe the development of a cucurbituril (Q8)-based supramolecular system, which in conjunction with the 14-3-3 protein dimer acts as a binary and bivalent protein assembly platform. We fused the phenylalanine–glycine–glycine (FGG) tripeptide motif to the N-terminus of the 14-3-3-binding epitope of the estrogen receptor α (ERα) for selective binding to Q8. Q8-induced dimerization of the ERα epitope augmented its affinity towards 14-3-3 through a binary bivalent binding mode. The crystal structure of the Q8-induced ternary complex revealed molecular insight into the multiple supramolecular interactions between the protein, the peptide, and Q8. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
view abstract 10.1002/anie.201701807
- A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein
Richter, F. and Subramaniam, S.R. and Magen, I. and Lee, P. and Hayes, J. and Attar, A. and Zhu, C. and Franich, N.R. and Bove, N. and de la Rosa, K. and Kwong, J. and Klärner, F.-G. and Schrader, T. and Chesselet, M.-F. and Bitan, G.
Neurotherapeutics (2017)Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The “molecular tweezer” CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28 days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies. © 2017 The American Society for Experimental NeuroTherapeutics, Inc.
view abstract 10.1007/s13311-017-0544-9
- An NMR Method To Pinpoint Supramolecular Ligand Binding to Basic Residues on Proteins
Hogeweg, A. and Sowislok, A. and Schrader, T. and Beuck, C.
Angewandte Chemie - International Edition 56 (2017)Targeting protein surfaces involved in protein–protein interactions by using supramolecular chemistry is a rapidly growing field. NMR spectroscopy is the method of choice to map ligand-binding sites with single-residue resolution by amide chemical shift perturbation and line broadening. However, large aromatic ligands affect NMR signals over a greater distance, and the binding site cannot be determined unambiguously by relying on backbone signals only. We herein employed Lys- and Arg-specific H2(C)N NMR experiments to directly observe the side-chain atoms in close contact with the ligand, for which the largest changes in the NMR signals are expected. The binding of Lys- and Arg-specific supramolecular tweezers and a calixarene to two model proteins was studied. The H2(C)N spectra track the terminal CH2 groups of all Lys and Arg residues, revealing significant differences in their binding kinetics and chemical shift perturbation, and can be used to clearly pinpoint the order of ligand binding. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/anie.201707950
- High-Affinity Copolymers Inhibit Digestive Enzymes by Surface Recognition
Gilles, P. and Wenck, K. and Stratmann, I. and Kirsch, M. and Smolin, D.A. and Schaller, T. and De Groot, H. and Kraft, A. and Schrader, T.
Biomacromolecules 18 (2017)This account presents a general method for the construction of polymeric surface binders for digestion enzymes. Two prominent parts, namely, the modification of the copolymer composition and the screening assay for the most powerful inhibitors are both amenable to parallelization. The concept hinges on the appropriate selection of amino-acid-selective comonomers, their free radical copolymerization, and subsequent screening of the resulting copolymer library for efficient enzyme inhibition. A microscale synthetic procedure for the copolymerization process was developed, which produces water-soluble affinity polymers that can be stored for years at room temperature. Initial parallel screening was conducted in standard enzyme assays to identify polymeric inhibitors, which were subsequently subjected to determination of IC50 values for their target enzyme. For all digestion enzymes, except elastase, a number of polymer inhibitors were found, some of which were selective toward one or two protein targets. Since the key monomers of the best inhibitors bind to amino acid residues in the direct vicinity of the active site, we conclude that efficient coverage of the immediate environment by the copolymers is critical. Strong interference with enzymatic activity is brought about by blocking the substrate access and product exit to and from the active site. © 2017 American Chemical Society.
view abstract 10.1021/acs.biomac.7b00162
- Inhibition of Huntingtin Exon-1 Aggregation by the Molecular Tweezer CLR01
Vöpel, T. and Bravo-Rodriguez, K. and Mittal, S. and Vachharajani, S. and Gnutt, D. and Sharma, A. and Steinhof, A. and Fatoba, O. and Ellrichmann, G. and Nshanian, M. and Heid, C. and Loo, J.A. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Wanker, E.E. and Ebbinghaus, S. and Sanchez-Garcia, E.
Journal of the American Chemical Society 139 (2017)Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity. © 2017 American Chemical Society.
view abstract 10.1021/jacs.6b11039
- Inhibition of Mutant αB crystallin-induced protein aggregation by a molecular tweezer
Xu, N. and Bitan, G. and Schrader, T. and Klärner, F.-G. and Osinska, H. and Robbins, J.
Journal of the American Heart Association 6 (2017)Background--Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. The development of therapeutics directed at proteotoxicity-based pathology in heart disease is just beginning. The molecular tweezer CLR01 is a broad-spectrum inhibitor of abnormal self-assembly of amyloidogenic proteins, including amyloid β-protein, tau, and α-synuclein. This small molecule interferes with aggregation by binding selectively to lysine side chains, changing the charge distribution of aggregation-prone proteins and thereby disrupting aggregate formation. However, the effects of CLR01 in cardiomyocytes undergoing proteotoxic stress have not been explored. Here we assess whether CLR01 can decrease cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated αB-crystallin (CryABR120G). Methods and Results--A proteotoxic model of desmin-related cardiomyopathy caused by cardiomyocyte-specific expression of CryABR120G was used to test the efficacy of CLR01 therapy in the heart. Neonatal rat cardiomyocytes were infected with adenovirus expressing either wild-type CryAB or CryABR120G. Subsequently, the cells were treated with different doses of CLR01 or a closely related but inactive derivative, CLR03. CLR01 decreased aggregate accumulation and attenuated cytotoxicity caused by CryABR120G expression in a dose-dependent manner, whereas CLR03 had no effect. Ubiquitin-proteasome system function was analyzed using a ubiquitin-proteasome system reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein. CLR01 improved proteasomal function in CryABR120G cardiomyocytes but did not alter autophagic flux. In vivo, CLR01 administration also resulted in reduced protein aggregates in CryABR120G transgenic mice. Conclusions--CLR01 can inhibit CryABR120G aggregate formation and decrease cytotoxicity in cardiomyocytes undergoing proteotoxic stress, presumably through clearance of the misfolded protein via increased proteasomal function. CLR01 or related compounds may be therapeutically useful in treating the pathogenic sequelae resulting from proteotoxic heart disease. © 2017 The Authors and Medtronic.
view abstract 10.1161/JAHA.117.006182
- Molecular Tweezers Inhibit PARP-1 by a New Mechanism
Wilch, C. and Talbiersky, P. and Berchner-Pfannschmidt, U. and Schaller, T. and Kirsch, M. and Klärner, F.-G. and Schrader, T.
European Journal of Organic Chemistry 2017 (2017)The inhibition of PARP-1 (poly[ADP-ribose]polymerase 1), a key enzyme for DNA quality control, has been achieved with synthetic molecular tweezers through a noncompetitive mechanism with an IC50 value of 3 µm. Displacement as well as electrophoretic mobility shift assays and molecular dynamics experiments point to a simultaneous inclusion of lysine side-chains in the cavity of the tweezers and the coordination of one phosphate arm to the central Zn2+ ion of the zinc finger; thereby, lesioned DNA is displaced. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
view abstract 10.1002/ejoc.201601596
- The Molecular Tweezer CLR01 Stabilizes a Disordered Protein-Protein Interface
Bier, D. and Mittal, S. and Bravo-Rodriguez, K. and Sowislok, A. and Guillory, X. and Briels, J. and Heid, C. and Bartel, M. and Wettig, B. and Brunsveld, L. and Sanchez-Garcia, E. and Schrader, T. and Ottmann, C.
Journal of the American Chemical Society 139 (2017)Protein regions that are involved in protein-protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein-protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular "Janus" ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein-protein interface, "freezes" one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein. © 2017 American Chemical Society.
view abstract 10.1021/jacs.7b07939
- Characterizing the Effect of Multivalent Conjugates Composed of Aβ-Specific Ligands and Metal Nanoparticles on Neurotoxic Fibrillar Aggregation
Streich, C. and Akkari, L. and Decker, C. and Bormann, J. and Rehbock, C. and Müller-Schiffmann, A. and Niemeyer, F.C. and Nagel-Steger, L. and Willbold, D. and Saccà, B. and Korth, C. and Schrader, T. and Barcikowski, S.
ACS Nano 10 (2016)Therapeutically active small molecules represent promising nonimmunogenic alternatives to antibodies for specifically targeting disease-relevant receptors. However, a potential drawback compared to antibody-antigen interactions may be the lower affinity of small molecules toward receptors. Here, we overcome this low-affinity problem by coating the surface of nanoparticles (NPs) with multiple ligands. Specifically, we explored the use of gold and platinum nanoparticles to increase the binding affinity of Aβ-specific small molecules to inhibit Aβ peptide aggregation into fibrils in vitro. The interactions of bare NPs, free ligands, and NP-bound ligands with Aβ are comprehensively studied via physicochemical methods (spectroscopy, microscopy, immunologic tests) and cell assays. Reduction of thioflavin T fluorescence, as an indicator for β-sheet content, and inhibition of cellular Aβ excretion are even more effective with NP-bound ligands than with the free ligands. The results from this study may have implications in the development of therapeutics for treating Alzheimer's disease. © 2016 American Chemical Society.
view abstract 10.1021/acsnano.6b02627
- Metabolic shift toward oxidative phosphorylation in docetaxel resistant prostate cancer cells
Ippolito, L. and Marini, A. and Cavallini, L. and Morandi, A. and Pietrovito, L. and Pintus, G. and Giannoni, E. and Schrader, T. and Puhr, M. and Chiarugi, P. and Taddei, M.L.
Oncotarget 7 (2016)Drug resistance of cancer cells is recognized as the primary cause of failure of chemotherapeutic treatment in most human cancers. Growing evidences support the idea that deregulated cellular metabolism is linked to such resistance. Indeed, both components of the glycolytic and mitochondrial pathways are involved in altered metabolism linked to chemoresistance of several cancers. Here we investigated the drug-induced metabolic adaptations able to confer advantages to docetaxel resistant prostate cancer (PCa) cells. We found that docetaxel-resistant PC3 cells (PC3-DR) acquire a pro-invasive behavior undergoing epithelial-to-mesenchymal-transition (EMT) and a decrease of both intracellular ROS and cell growth. Metabolic analyses revealed that PC3-DR cells have a more efficient respiratory phenotype than sensitive cells, involving utilization of glucose, glutamine and lactate by the mitochondrial oxidative phosphorylation (OXPHOS). Consequently, targeting mitochondrial complex I by metformin administration, impairs proliferation and invasiveness of PC3-DR cells without effects on parental cells. Furthermore, stromal fibroblasts, which cause a "reverse Warburg" phenotype in PCa cells, reduce docetaxel toxicity in both sensitive and resistant PCa cells. However, re-expression of miR-205, a microRNA strongly down-regulated in EMT and associated to docetaxel resistance, is able to shift OXPHOS to a Warburg metabolism, thereby resulting in an elevated docetaxel toxicity in PCa cells. Taken together, these findings suggest that resistance to docetaxel induces a shift from Warburg to OXPHOS, mandatory for conferring a survival advantage to resistant cells, suggesting that impairing such metabolic reprogramming could be a successful therapeutic approach.
view abstract 10.18632/oncotarget.11301
- Molecular tweezers for lysine and arginine-powerful inhibitors of pathologic protein aggregation
Schrader, T. and Bitan, G. and Klärner, F.-G.
Chemical Communications 52 (2016)Molecular tweezers represent the first class of artificial receptor molecules that have made the way from a supramolecular host to a drug candidate with promising results in animal tests. Due to their unique structure, only lysine and arginine are well complexed with exquisite selectivity by a threading mechanism, which unites electrostatic, hydrophobic and dispersive attraction. However, tweezer design must avoid self-dimerization, self-inclusion and external guest binding. Moderate affinities of molecular tweezers towards sterically well accessible basic amino acids with fast on and off rates protect normal proteins from potential interference with their biological function. However, the early stages of abnormal Aβ, α-synuclein, and TTR assembly are redirected upon tweezer binding towards the generation of amorphous non-toxic materials that can be degraded by the intracellular and extracellular clearance mechanisms. Thus, specific host-guest chemistry between aggregation-prone proteins and lysine/arginine binders rescues cell viability and restores animal health in models of AD, PD, and TTR amyloidosis. © The Royal Society of Chemistry 2016.
view abstract 10.1039/c6cc04640a
- Molecular tweezers target a protein-protein interface and thereby modulate complex formation
Trusch, F. and Kowski, K. and Bravo-Rodriguez, K. and Beuck, C. and Sowislok, A. and Wettig, B. and Matena, A. and Sanchez-Garcia, E. and Meyer, H. and Schrader, T. and Bayer, P.
Chemical Communications 52 (2016)Molecular tweezers for lysine and arginine select a few residues on a protein surface and by their unique complexation mode disrupt a critical protein-protein interaction. Detailed structural information was gained by NMR experiments, strongly supported by QM/MM calculations and further substantiated by ITC, fluorescence anisotropy, ELISA and bio-layer-interference studies. © 2016 The Royal Society of Chemistry.
view abstract 10.1039/c6cc08039a
- Neurotoxicity of the parkinson disease-associated pesticide ziram is synuclein-dependent in zebrafish embryos
Lulla, A. and Barnhill, L. and Bitan, G. and Ivanova, M.I. and Nguyen, B. and O’Donnell, K. and Stahl, M.C. and Yamashiro, C. and Klärner, F.-G. and Schrader, T. and Sagasti, A. and Bronstein, J.M.
Environmental Health Perspectives 124 (2016)Background: Exposure to the commonly used dithiocarbamate (DTC) pesticides is associated with an increased risk of developing Parkinson disease (PD), although the mechanisms by which they exert their toxicity are not completely understood. Objective: We studied the mechanisms of ziram’s (a DTC fungicide) neurotoxicity in vivo. Methods: Zebrafish (ZF) embryos were utilized to determine ziram’s effects on behavior, neuronal toxicity, and the role of synuclein in its toxicity. Results: Nanomolar-range concentrations of ziram caused selective loss of dopaminergic (DA) neurons and impaired swimming behavior. Because ziram increases α-synuclein (α-syn) concentrations in rat primary neuronal cultures, we investigated the effect of ziram on ZF γ-synuclein 1 (γ1). ZF express 3 synuclein isoforms, and ZF γ1 appears to be the closest functional homologue to α-syn. We found that recombinant ZF γ1 formed fibrils in vitro, and overexpression of ZF γ1 in ZF embryos led to the formation of neuronal aggregates and neurotoxicity in a manner similar to that of α-syn. Importantly, knockdown of ZF γ1 with morpholinos and disruption of oligomers with the molecular tweezer CLR01 prevented ziram’s DA toxicity. Conclusions: These data show that ziram is selectively toxic to DA neurons in vivo, and this toxicity is synuclein-dependent. These findings have important implications for understanding the mechanisms by which pesticides may cause PD. © 2016, Public Health Services, US Dept of Health and Human Services. All rights reserved.
view abstract 10.1289/EHP141
- Protein Camouflage: Supramolecular Anion Recognition by Ubiquitin
Mallon, M. and Dutt, S. and Schrader, T. and Crowley, P. B.
Chembiochem 17 (2016)Progress in the field of bio-supramolecular chemistry, the bottom-up assembly of protein-ligand systems, relies on a detailed knowledge of molecular recognition. To address this issue, we have characterised complex formation between human ubiquitin (HUb) and four supramolecular anions. The ligands were: pyrenetetrasulfonic acid (4PSA), p-sulfonato-calixarene (SCLX4), bisphosphate tweezers (CLR01) and meso-tetrakis (4-sulfonatophenyl)porphyrin (TPPS), which vary in net charge, size, shape and hydrophobicity. All four ligands induced significant changes in the HSQC spectrum of HUb. Chemical shift perturbations and line-broadening effects were used to identify binding sites and to quantify affinities. Supporting data were obtained from docking simulations. It was found that these weakly interacting ligands bind to extensive surface patches on HUb. A comparison of the data suggests some general indicators for the protein-binding specificity of supramolecular anions. Differences in binding were observed between the cavity-containing and planar ligands. The former had a preference for the arginine-rich, flexible Cterminus of HUb.
view abstract 10.1002/cbic.201500477
- Reducing synuclein accumulation improves neuronal survival after spinal cord injury
Fogerson, S.M. and van Brummen, A.J. and Busch, D.J. and Allen, S.R. and Roychaudhuri, R. and Banks, S.M.L. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Morgan, J.R.
Experimental Neurology 278 (2016)Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the "molecular tweezer" inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury. © 2016 Elsevier Inc.
view abstract 10.1016/j.expneurol.2016.02.004
- A molecular tweezer antagonizes seminal amyloids and HIV infection
Lump, E. and Castellano, L.M. and Meier, C. and Seeliger, J. and Erwin, N. and Sperlich, B. and Stürzel, C.M. and Usmani, S. and Hammond, R.M. and Von Einem, J. and Gerold, G. and Kreppel, F. and Bravo-Rodriguez, K. and Pietschmann, T. and Holmes, V.M. and Palesch, D. and Zirafi, O. and Weissman, D. and Sowislok, A. and Wettig, B. and Heid, C. and Kirchhoff, F. and Weil, T. and Klärner, F.G. and Schrader, T. and Bitan, G. and Sanchez-Garcia, E. and Winter, R. and Shorter, J. and Münch, J.
eLife 4 (2015)Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a ‘molecular tweezer’ specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion–amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses.We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses. © 2015, Lump et al.
view abstract 10.7554/eLife.05397
- Amyloid β-Protein Assembly: The Effect of Molecular Tweezers CLR01 and CLR03
Zheng, X. and Liu, D. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Bowers, M.T.
Journal of Physical Chemistry B 119 (2015)The early oligomerization of amyloid β-protein (Aβ) has been shown to be an important event in the pathology of Alzheimer's disease (AD). Designing small molecule inhibitors targeting Aβ oligomerization is one attractive and promising strategy for AD treatment. Here we used ion mobility spectrometry coupled to mass spectrometry (IMS-MS) to study the different effects of the molecular tweezers CLR01 and CLR03 on Aβ self-assembly. CLR01 was found to bind to Aβ directly and disrupt its early oligomerization. Moreover, CLR01 remodeled the early oligomerization of Aβ42 by compacting the structures of dimers and tetramers and as a consequence eliminated higher-order oligomers. Unexpectedly, the negative-control derivative, CLR03, which lacks the hydrophobic arms of the tweezer structure, was found to facilitate early Aβ oligomerization. Our study provides an example of IMS as a powerful tool to study and better understand the interaction between small molecule modulators and Aβ oligomerization, which is not attainable by other methods, and provides important insights into therapeutic development of molecular tweezers for AD treatment. (Graph Presented). © 2015 American Chemical Society.
view abstract 10.1021/acs.jpcb.5b00692
- Hybridization of an Aβ-specific antibody fragment with aminopyrazole-based β-sheet ligands displays striking enhancement of target affinity
Hellmert, M. and Müller-Schiffmann, A. and Peters, M.S. and Korth, C. and Schrader, T.
Organic and Biomolecular Chemistry 13 (2015)Determining Aβ levels in body fluids remains a powerful tool in the diagnostics of Alzheimer's disease. This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aβ to make diagnostic procedures more sensitive. A monoclonal antibody IC16 was generated to an N-terminal epitope of Aβ and the variable regions of the heavy and light chains were cloned as a recombinant protein (scFv). A 6 × histidine tag was fused to the C-terminus of IC16-scFv allowing hybridization with a small organic β-sheet binder via Ni-NTA complexation. On the other hand, a multivalent nitrilotriacetic acid (NTA)-equipped trimeric aminopyrazole (AP) derivative was synthesized based on a cyclam platform; and experimental evidence was obtained for efficient Ni2+-mediated complex formation with the histidine-tagged antibody species. In a proof of principle experiment the hybrid molecule showed a strong increase in affinity towards Aβ. Thus, the specific binding power of recombinant antibody fragments to their β-sheet rich targets can be conveniently enhanced by non-covalent hybridization with small organic β-sheet binders. © 2015 The Royal Society of Chemistry.
view abstract 10.1039/c4ob02411g
- Molecular Tweezers Inhibit Islet Amyloid Polypeptide Assembly and Toxicity by a New Mechanism
Lopes, D.H.J. and Attar, A. and Nair, G. and Hayden, E.Y. and Du, Z. and McDaniel, K. and Dutt, S. and Bandmann, H. and Bravo-Rodriguez, K. and Mittal, S. and Klärner, F.-G. and Wang, C. and Sanchez-Garcia, E. and Schrader, T. and Bitan, G.
ACS Chemical Biology 10 (2015)In type-2 diabetes (T2D), islet amyloid polypeptide (IAPP) self-associates into toxic assemblies causing islet β-cell death. Therefore, preventing IAPP toxicity is a promising therapeutic strategy for T2D. The molecular tweezer CLR01 is a supramolecular tool for selective complexation of K residues in (poly)peptides. Surprisingly, it inhibits IAPP aggregation at substoichiometric concentrations even though IAPP has only one K residue at position 1, whereas efficient inhibition of IAPP toxicity requires excess CLR01. The basis for this peculiar behavior is not clear. Here, a combination of biochemical, biophysical, spectroscopic, and computational methods reveals a detailed mechanistic picture of the unique dual inhibition mechanism for CLR01. At low concentrations, CLR01 binds to K1, presumably nucleating nonamyloidogenic, yet toxic, structures, whereas excess CLR01 binds also to R11, leading to nontoxic structures. Encouragingly, the CLR01 concentrations needed for inhibition of IAPP toxicity are safe in vivo, supporting its development toward disease-modifying therapy for T2D. © 2015 American Chemical Society.
view abstract 10.1021/acschembio.5b00146
- Suitable clothes for tailoring proteins
Wettig, B. and Heid, C. and Schrader, T.
Nachrichten aus der Chemie 63 (2015)
view abstract 10.1002/nadc.201590361
- The Lys-Specific Molecular Tweezer, CLR01, Modulates Aggregation of the Mutant p53 DNA Binding Domain and Inhibits Its Toxicity
Herzog, G. and Shmueli, M.D. and Levy, L. and Engel, L. and Gazit, E. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Segal, D.
Biochemistry 54 (2015)The tumor suppressor p53 plays a unique role as a central hub of numerous cell proliferation and apoptotic pathways, and its malfunction due to mutations is a major cause of various malignancies. Therefore, it serves as an attractive target for developing novel anticancer therapeutics. Because of its intrinsically unstable DNA binding domain, p53 unfolds rapidly at physiological temperature. Certain mutants shift the equilibrium toward the unfolded state and yield high-molecular weight, nonfunctional, and cytotoxic β-sheet-rich aggregates that share tinctorial and conformational similarities with amyloid deposits found in various protein misfolding diseases. Here, we examined the effect of a novel protein assembly modulator, the lysine (Lys)-specific molecular tweezer, CLR01, on different aggregation stages of misfolded mutant p53 in vitro and on the cytotoxicity of the resulting p53 aggregates in cell culture. We found that CLR01 induced rapid formation of β-sheet-rich, intermediate-size p53 aggregates yet inhibited further p53 aggregation and reduced the cytotoxicity of the resulting aggregates. Our data suggest that aggregation modulators, such as CLR01, could prevent the formation of toxic p53 aggregates. © 2015 American Chemical Society.
view abstract 10.1021/bi501092p
- Toxicity Inhibitors Protect Lipid Membranes from Disruption by Aβ42
Malishev, R. and Nandi, S. and Kolusheva, S. and Levi-Kalisman, Y. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Jelinek, R.
ACS Chemical Neuroscience 6 (2015)Although the precise molecular factors linking amyloid β-protein (Aβ) to Alzheimer's disease (AD) have not been deciphered, interaction of Aβ with cellular membranes has an important role in the disease. However, most therapeutic strategies targeting Aβ have focused on interfering with Aβ self-assembly rather than with its membrane interactions. Here, we studied the impact of three toxicity inhibitors on membrane interactions of Aβ42, the longer form of Aβ, which is associated most strongly with AD. The inhibitors included the four-residue C-terminal fragment Aβ(39-42), the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and the lysine-specific molecular tweezer, CLR01, all of which previously were shown to disrupt different steps in Aβ42 self-assembly. Biophysical experiments revealed that incubation of Aβ42 with each of the three modulators affected membrane interactions in a distinct manner. Interestingly, EGCG and CLR01 were found to have significant interaction with membranes themselves. However, membrane bilayer disruption was reduced when the compounds were preincubated with Aβ42, suggesting that binding of the assembly modulators to the peptide attenuated their membrane interactions. Importantly, our study reveals that even though the three tested compounds affect Aβ42 assembly differently, membrane interactions were significantly inhibited upon incubation of each compound with Aβ42, suggesting that preventing the interaction of Aβ42 with the membrane contributes substantially to inhibition of its toxicity by each compound. The data suggest that interference with membrane interactions is an important factor for Aβ42 toxicity inhibitors and should be taken into account in potential therapeutic strategies, in addition to disruption or remodeling of amyloid assembly. © 2015 American Chemical Society.
view abstract 10.1021/acschemneuro.5b00200
- Affinity polymers tailored for the protein A binding site of immunoglobulin G proteins
Latza, P. and Gilles, P. and Schaller, T. and Schrader, T.
Chemistry - A European Journal 20 (2014)Rational design in combination with a screening process was used to develop affinity polymers for a specific binding site on the surface of immunoglobulin G (IgG) proteins. The concept starts with the identification of critical amino acid residues on the protein interface and their topological arrangement. Appropriate binding monomers were subsequently synthesized. Together with a sugar monomer (2-5 equiv) for water solubility and a dansyl monomer (0.5 equiv) as a fluorescent label, they were subjected in aqueous solution to linear radical copolymerization in various compositions (e.g., azobisisobutyronitrile (AIBN), homogeneous water/DMF mixtures). After ultrafiltration and lyophilization, colorless dry water-soluble powders were obtained. NMR spectroscopic and gel permeation chromatography (GPC) characterization indicated molecular weights between 30 and 500 kD and confirmed retention of monomer composition as well as the absence of monomers. In a competitive enzyme-linked immunosorbent assay (ELISA) screen of the polymer libraries (20-50 members), few copolymers qualified as strong and selective binders for the protein A binding site on the Fc fragment of the antibody. Their monomer composition precisely reflected the critical amino acids found at the interface. The simple combination of a charged and a nonpolar binding monomer sufficed for selective submicromolar IgG recognition by the synthetic polymer. Affinities were confirmed by fluorescence titrations; they increased with decreasing salt load but remained largely unaltered at lowered pH. Other proteins, including those of similar size and isoelectric point (pI), were bound 10-1000 times less tightly. This example indicates that interaction domains in other proteins may also be targeted by synthetic polymers if their comonomer composition reflects the nature and arrangement of amino acid residues on the protein surface. Some like it hot: Designed affinity polymers recognize the hot spot on a protein surface by virtue of their carefully selected binding monomers. Here, it is the protein A binding site on the Fc fragment of immunoglobulin G proteins (see figure), which is essential for medicinal antibody purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/chem.201402399
- Molecular basis for preventing α-synuclein aggregation by a molecular tweezer
Acharya, S. and Safaie, B.M. and Wongkongkathep, P. and Ivanova, M.I. and Attar, A. and Klärner, F.-G. and Schrader, T. and Loo, J.A. and Bitan, G. and Lapidus, L.J.
Journal of Biological Chemistry 289 (2014)Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys- 12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein ispredominantlymonomeric.Theresultscan be successfully modeled using a kineticschemein which two aggregation- pronemonomerscanformanencountercomplexthat leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high.Takentogether, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
view abstract 10.1074/jbc.M113.524520
- Molecular Tweezers Targeting Transthyretin Amyloidosis
Ferreira, N. and Pereira-Henriques, A. and Attar, A. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Gales, L. and Saraiva, M.J. and Almeida, M.R.
Neurotherapeutics 11 (2014)Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the "molecular tweezers", CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer's disease and Parkinson's disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis. © 2014 The Author(s).
view abstract 10.1007/s13311-013-0256-8
- Safety and pharmacological characterization of the molecular tweezer CLR01-A broad-spectrum inhibitor of amyloid proteins' toxicity
Attar, A. and Chan, W.C. and Klärner, F.-G. and Schrader, T. and Bitan, G.
BMC Pharmacology and Toxicology 15 (2014)Background: The "molecular tweezer" CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer's disease and other amyloidoses, here we studied its safety and pharmacokinetics. Methods: Toxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer's disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5-72 h post administration. Results: Examination of CLR01's effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound's putative "processspecific" mechanism of action. A single-injection of 100 mg/kg CLR01 in mice - 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin. The brain penetration of CLR01 was found to be 1-3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h. Conclusion: Estimation of brain levels compared to amyloid ß-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01. The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses. © 2014 Attar et al.
view abstract 10.1186/2050-6511-15-23
- Aromatic interactions by molecular tweezers and clips in chemical and biological systems
Klärner, F.-G. and Schrader, T.
Accounts of Chemical Research 46 (2013)Noncovalent interactions involving aromatic rings, such as π-stacking and CH-π, occur throughout a range of fundamental processes including self-assembly and (bio)catalysis. Molecular clips and tweezers possess a central parallel or torus-shaped cavity with a surrounding belt of convergent aromatic rings; hence these structures exploit multiple aromatic interactions in a positively cooperative manner. Both clips and tweezers demonstrate selective binding of cationic or neutral guests that bear acceptor groups. The electrostatic surface potentials (ESP) explain this unexpected behavior: calculated ESPs were highly negative inside the tweezer or clip cavity, providing complementary profiles to the positive ESP plots of their preferred guest molecules. This Account presents more complex systems that use aromatic clips and tweezers to alter the reactivities of included guest species, to distinguish between guest enantiomers, and to interfere with biological processes such as enzymatic activity and protein aggregation.Napthalene tweezers show potential applications in organocatalysis. When pyridinium moieties are bound within the spacious cavity of naphthyl-spaced tweezers, the resulting complex significantly influences the first step of single-electron reductions of (bi)pyridinium salts. In addition, the environment within the tweezer cavity strongly accelerates the Menshutkin reaction (the alkylation of pyridine derivatives).Introduction of phosphonate, phosphate, or sulfate anions into the central aromatic bridge renders clips and tweezers water-soluble. Larger systems form extremely tight intertwined dimers that rely on the nonclassical hydrophobic effect for their stability. Smaller clips and tweezers with a simple benzene bridge remain monomeric in buffered aqueous solution and display a complementary binding profile. While the clips with parallel sidewalls prefer flat aromatic cations such as pyridinium salts, the torus-shaped tweezers bind to basic amino acids lysine and arginine via a threading process. These mutually exclusive binding modes make water-soluble clips and tweezers valuable tools for probing critical biological interactions with positively charged amino acid side chains and cofactors.Molecular clips and tweezers can be employed for the complete inhibition of dehydrogenases. The clip extracts NAD+ from its Rossman fold, while the tweezer complexes access strategic lysine residues around the active site. Our new enzyme inhibitors recognize the protein surface and thus offer additional targets for medicinal chemistry. Finally, the ability of molecular tweezers to cap critical lysine residues can be used to interfere with the pathology of protein misfolding diseases such as Alzheimer's disease, because many of them involve noncovalent interactions with these critical residues during their early stages. When the key protein produces a β-sheet-rich nucleus, this structure undergoes spontaneous polymerization into highly toxic oligomers, ultimately leading to mature fibrils. The benzene-spaced phosphate tweezer forms a specific complex with lysine residues 16 and 28 in Aβ42 and thus prevents the formation of misfolded oligomers rich in β-sheets. This entirely new process-specific mechanism that prevents pathologic protein aggregation also operates in many other related amyloidogenic proteins. © 2012 American Chemical Society.
view abstract 10.1021/ar300061c
- Ditopic arginine-aspartate binders recognize RGD loops
Gersthagen, T. and Hofmann, J. and Klärner, F.-G. and Schmuck, C. and Schrader, T.
European Journal of Organic Chemistry (2013)A synthetic strategy was developed for the construction of unsymmetrical arginine tweezers with an additional carboxylate binding site. The ditopic receptor molecules 2 and 3 use two arms to bind arginine-glycine-aspartate (RGD) peptides. However, contrary to expectations from molecular mechanics simulations, the tweezer cavity remains empty, and arginine is bound through a simple guanidinium/phosphate ion-pair formation. On the other hand, carboxylate recognition by the guanidiniopyrrole moiety operates very efficiently, leading to complexation of ionic amino acids in both linear and cyclic RGD peptides with concentrations in the low micromolar range in a buffered aqueous solution (Kd = 20-30 μM). Truncation of the whole guanidiniopyrrole arm leads to the simple unsymmetrical monophosphate tweezer 1, which lacks the aspartate binder, but restores the arginine inclusion. Tweezer 1 displays a remarkable affinity and selectivity towards cyclic RGD peptides, presumably by a combination of arginine inclusion and additional cooperative hydrogen bonds between its phenolic OH group and the peptide backbone (NMR evidence, K d = 15-30 μM). Comparison with symmetrical diphosphate tweezers 1a and 2-4 shows that in unsymmetrical tweezers the spacer group must guarantee an open cavity. Both binding sites of the ditopic molecular tweezers bind arginine-glycine-aspartate (RGD) loops (Kd ≈ 20 μM in buffer), but not by inclusion inside the cavity of the tweezers. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/ejoc.201201052
- Linker effects on amino acid and peptide recognition by molecular tweezers
Dutt, S. and Wilch, C. and Gersthagen, T. and Wölper, C. and Sowislok, A.A. and Klärner, F.-G. and Schrader, T.
European Journal of Organic Chemistry 2013 (2013)Transition from monotopic symmetrical to ditopic unsymmetrical molecular recognition frequently occurs when a general, powerful, but unspecific receptor molecule is transformed into a specific ditopic host. Especially in water, this endeavor is accompanied by great challenges, comprising, among other things, host-guest orientation, orthogonal recognition modes, and the nature of the linker unit. This work presents a case study on a powerful general host for basic amino acids and peptides. The symmetrical molecular tweezer skeleton was systematically desymmetrized and modified with various common linker units, and the profound influence of these changes on the molecular recognition profile was studied in detail by NMR spectroscopy, fluorescence titrations, X-ray crystallography, and molecular simulations. A number of diverse effects were revealed that could be attributed to the chemical nature of the different linkers. In general, long alkyl tethers block the cavity of the tweezers by van der Waals contacts to CH groups around its entrance; alkoxyalkyl tethers likewise lower tweezer affinities for basic amino acids by competing self-inclusion. As a general trend, affinities for linkers with ester and carboxylate moieties were substantially higher than those for tethers with ethers and alcohols, likely because the electron-rich carbonyl group keeps the cavity open. Additional hydrogen bonds between the linker unit and suitable amino acid or peptide guests greatly support the complexation process; finally, high solvent polarity and salt load shift the binding equilibrium from external ion pairing to guest inclusion. Unsymmetrical molecular tweezers with an acetyl linker bind tightly to AcArgOMe by ditopic recognition and simultaneous inclusion within the aromatic cavity (100 ns MD simulation). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/ejoc.201301211
- Molecular tweezers modulate 14-3-3 protein-protein interactions
Bier, D. and Rose, R. and Bravo-Rodriguez, K. and Bartel, M. and Ramirez-Anguita, J.M. and Dutt, S. and Wilch, C. and Klärner, F.-G. and Sanchez-Garcia, E. and Schrader, T. and Ottmann, C.
Nature Chemistry 5 (2013)Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins-a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)-in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. © 2013 Macmillan Publishers Limited. All rights reserved.
view abstract 10.1038/nchem.1570
- Molecular tweezers with varying anions: A comparative study
Dutt, S. and Wilch, C. and Gersthagen, T. and Talbiersky, P. and Bravo-Rodriguez, K. and Hanni, M. and Sánchez-García, E. and Ochsenfeld, C. and Klärner, F.-G. and Schrader, T.
Journal of Organic Chemistry 78 (2013)Selective binding of the phosphate-substituted molecular tweezer 1a to protein lysine residues was suggested to explain the inhibition of certain enzymes and the aberrant aggregation of amyloid petide Aβ42 or α-synuclein, which are assumed to be responsible for Alzheimer's and Parkinson's disease, respectively. In this work we systematically investigated the binding of four water-soluble tweezers 1a-d (substituted by phosphate, methanephosphonate, sulfate, or O-methylenecarboxylate groups) to amino acids and peptides containing lysine or arginine residues by using fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry (ITC). The comparison of the experimental results with theoretical data obtained by a combination of QM/MM and ab initio1H NMR shift calculations provides clear evidence that the tweezers 1a-c bind the amino acid or peptide guest molecules by threading the lysine or arginine side chain through the tweezers' cavity, whereas in the case of 1d the guest molecule is preferentially positioned outside the tweezer's cavity. Attractive ionic, CH-π, and hydrophobic interactions are here the major binding forces. The combination of experiment and theory provides deep insight into the host-guest binding modes, a prerequisite to understanding the exciting influence of these tweezers on the aggregation of proteins and the activity of enzymes. © 2013 American Chemical Society.
view abstract 10.1021/jo4009673
- Transport of supramolecular drugs across the cell membrane by calcium phosphate nanoparticles
Rotan, O. and Sokolova, V. and Gilles, P. and Hu, W. and Dutt, S. and Schrader, T. and Epple, M.
Materialwissenschaft und Werkstofftechnik 44 (2013)Many target sites of synthetic supramolecular drug molecules are located inside cells. Since larger and highly charged molecules are typically not able to cross the cell membrane on their own, an efficient carrier is needed. Calcium phosphate nanoparticles were loaded with different artificial protein and DNA binders, i. e. a polyfunctional anionic polymer, a cationic calixarene dimer and amphiphilic molecular tweezers. The loading of calcium phosphate nanoparticles with these molecules was quantitatively determined by UV spectroscopy. As visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), the functionalized calcium phosphate nanoparticles were easily taken up by HeLa cells together with their cargo. In contrast, the dissolved molecules alone were not able to penetrate the cell membrane. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/mawe.201300085
- Unraveling the function of paralogs of the aldehyde dehydrogenase super family from Sulfolobus solfataricus
Esser, D. and Kouril, T. and Talfournier, F. and Polkowska, J. and Schrader, T. and Brasen, C. and Siebers, B.
Extremophiles 17 (2013)Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles, e.g., in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus, five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and alpha-ketoglutaric semialdehyde dehydrogenase (alpha-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)(+)-dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD(+), SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)(+)). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and alpha-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in gamma-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO-SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218, methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD(+)- and CoA-dependent oxidation of methylmalonate semialdehyde, malonate semialdehyde as well as propionaldehyde (PA). For MSDH, a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.
view abstract 10.1007/s00792-012-0507-3
- A Novel "Molecular Tweezer" Inhibitor of α-Synuclein Neurotoxicity in Vitro and in Vivo
Prabhudesai, S. and Sinha, S. and Attar, A. and Kotagiri, A. and Fitzmaurice, A.G. and Lakshmanan, R. and Ivanova, M.I. and Loo, J.A. and Klärner, F.-G. and Schrader, T. and Stahl, M. and Bitan, G. and Bronstein, J.M.
Neurotherapeutics 9 (2012)Summary: Aggregation of α-synuclein (α-syn) is implicated as being causative in the pathogenesis of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Despite several therapies that improve symptoms in these disorders, none slow disease progression. Recently, a novel "molecular tweezer" (MT) termed CLR01 has been described as a potent inhibitor of assembly and toxicity of multiple amyloidogenic proteins. Here we investigated the ability of CLR01 to inhibit assembly and toxicity of α-syn. In vitro, CLR01 inhibited the assembly of α-syn into β-sheet-rich fibrils and caused disaggregation of pre-formed fibrils, as determined by thioflavin T fluorescence and electron microscopy. α-Syn toxicity was studied in cell cultures and was completely mitigated by CLR01 when α-syn was expressed endogenously or added exogenously. To determine if CLR01 was also protective in vivo, we used a novel zebrafish model of α-syn toxicity (α-syn-ZF), which expresses human, wild-type α-syn in neurons. α-Syn-ZF embryos developed severe deformities due to neuronal apoptosis and most of them died within 48 to 72 h. CLR01 added to the water significantly improved zebrafish phenotype and survival, suppressed α-syn aggregation in neurons, and reduced α-syn-induced apoptosis. α-Syn expression was found to inhibit the ubiquitin proteasome system in α-syn-ZF neurons, resulting in further accumulation of α-syn. Treatment with CLR01 almost completely mitigated the proteasome inhibition. The data suggest that CLR01 is a promising therapeutic agent for the treatment of Parkinson's disease and other synucleinopathies. © 2012 The American Society for Experimental NeuroTherapeutics, Inc. (outside the U.S.).
view abstract 10.1007/s13311-012-0105-1
- Artificial signal transduction with primary and secondary messengers
Bernitzki, K. and Maue, M. and Schrader, T.
Chemistry - A European Journal 18 (2012)The complete, entirely artificial, signal-transduction process was realized with a pair of tailored transmembrane units that were equipped with receptor- and reactive sites at both amphiphilic ends. Thus, docking of the primary messenger, transmission of the signal, and release of the secondary messenger could all be imitated in a single experimental setup. The system imitates the signaling principle of receptor tyrosine kinases and employs bisphosphonate head-groups for oligoamine-recognition and a pair of thiol nucleophiles and pyridine disulfide tail-groups for intravesicle SN2 displacement. This system operates in a unidirectional fashion, does not suffer from intervesicle competition, and is highly sensitive towards the lipid composition of the membrane and the nature of the primary messenger. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/chem.201200623
- Comparison of three amyloid assembly inhibitors: The sugar scyllo- inositol, the polyphenol epigallocatechin gallate, and the molecular tweezer CLR01
Sinha, S. and Du, Z. and Maiti, P. and Klärner, F.-G. and Schrader, T. and Wang, C. and Bitan, G.
ACS Chemical Neuroscience 3 (2012)(Chemical Presented). Many compounds have been tested as inhibitors or modulators of amyloid β-protein (Aβ) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimer's disease (AD). These compounds typically were either designed to break apart β-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aβ, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958-16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aβ aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aβ using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aβ monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved. © 2012 American Chemical Society.
view abstract 10.1021/cn200133x
- Dimeric Calixarenes: A New Family of Major-Groove Binders
Hu, W. B. and Blecking, C. and Kralj, M. and Suman, L. and Piantanida, I. and Schrader, T.
Chemistry-a European Journal 18 (2012)A new class of potent DNA binding agents is presented. Dimeric calixarenes with cationic groups at their upper rims and flexible alkyl bridges can be synthesized from triply acyl-protected calixarene tetramines in relatively short synthetic sequences (35 steps). The compounds attach themselves to double-stranded nucleic acids in a noncovalent fashion, with micro- to nanomolar affinities. Guanidinium headgroups with their extended hydrogen-bonding fingers are more powerful than ammonium groups, and the benzylamine series is superior to the anilinium series (see below). The new ligands easily distinguish between RNA and various DNA types, and produce characteristic changes in UV/Vis, fluorescence, CD, as well as NMR spectra. Especially extended oligonucleotides of more than 100 base pairs are bound with affinities increasing from RNA (10 mu M Kd)<AT-rich (1 mu M)<GC-rich DNA double strands (10010 nM). Ethidium bromide displacement studies confirm this order. CE50 values are remarkably low (14 mu M), and are more than 300 times lower than that of spermine, which is a typical backbone binder. Stoichiometries are rather high (one calixarene dimer per two BP), suggesting a potential aggregation of bound ligands inside the major groove. Most UV/Vis melting curves display an inverted shape, and start from drastically enhanced absorption intensities for the DNA complexes. DAPI displacement studies prove that up to one equivalent of calixarene dimer can be accommodated in the dye-loaded DNA. RNA complexation by calixarene dimers is accompanied by a drastic CD spectral transition from the typical A-form to a perfect B-signature, providing further experimental evidence for major-groove binding. The orientation of the ligands can be deduced from NMR titrations and is reproduced in Monte-Carlo simulations on 1:1 complexes in water.
view abstract 10.1002/chem.201100634
- Discovery and structure activity relationship of small molecule inhibitors of toxic β-amyloid-42 fibril formation
Kroth, H. and Ansaloni, A. and Varisco, Y. and Jan, A. and Sreenivasachary, N. and Rezaei-Ghaleh, N. and Giriens, V. and Lohmann, S. and López-Deber, M.P. and Adolfsson, O. and Pihlgren, M. and Paganetti, P. and Froestl, W. and Nagel-Steger, L. and Willbold, D. and Schrader, T. and Zweckstetter, M. and Pfeifer, A. and Lashuel, H.A. and Muhs, A.
Journal of Biological Chemistry 287 (2012)Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
view abstract 10.1074/jbc.M112.357665
- Isolated β-turn model systems investigated by combined IR/UV spectroscopy
Schwing, K. and Fricke, H. and Bartl, K. and Polkowska, J. and Schrader, T. and Gerhards, M.
ChemPhysChem 13 (2012)The functionality of bioactive molecules sensitively depends on their structure. For the investigation of intrinsic structural properties, molecular beam experiments combined with laser spectroscopy have proven to be a suitable tool. Herein we present an analysis of the two isolated tripeptide model systems Ac-Phe-Tyr(Me)-NHMe and Boc-Phe-Tyr(Me)-NHMe. For this purpose, mass-selective combined IR/UV spectroscopy is applied to both substances in a molecular beam experiment. The comparison of the experimental data with DFT calculations, including different functionals as well as dispersion corrections, allows an assignment of both tripeptide models to β-turns formed independently from the protection groups and supported by the interaction of the two aromatic chromophores. Taking shape: Structural assignments are made for two isolated tripeptide model systems by the application of molecular beam experiments and combined IR/UV spectroscopy in combination with DFT calculations (see picture). For each of the peptides a β-turn conformer is formed without the influence of an additional environment and independently from the chosen protecting group. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/cphc.201100805
- Protection of primary neurons and mouse brain from Alzheimer's pathology by molecular tweezers
Attar, A. and Ripoli, C. and Riccardi, E. and Maiti, P. and Li Puma, D.D. and Liu, T. and Hayes, J. and Jones, M.R. and Lichti-Kaiser, K. and Yang, F. and Gale, G.D. and Tseng, C.-H. and Tan, M. and Xie, C.-W. and Straudinger, J.L. and Klärner, F.-G. and Schrader, T. and Frautschy, S.A. and Grassi, C. and Bitan, G.
Brain 135 (2012)Alzheimer's disease is a devastating cureless neurodegenerative disorder affecting >35 million people worldwide. The disease is caused by toxic oligomers and aggregates of amyloid β protein and the microtubule- associated protein tau. Recently, the Lys-specific molecular tweezer CLR01 has been shown to inhibit aggregation and toxicity of multiple amyloidogenic proteins, including amyloid β protein and tau, by disrupting key interactions involved in the assembly process. Following up on these encouraging findings, here, we asked whether CLR01 could protect primary neurons from Alzheimer's disease-associated synaptotoxicity and reduce Alzheimer's disease-like pathology in vivo. Using cell culture and brain slices, we found that CLR01 effectively inhibited synaptotoxicity induced by the 42-residue isoform of amyloid β protein, including ∼80% inhibition of changes in dendritic spines density and long-term potentiation and complete inhibition of changes in basal synaptic activity. Using a radiolabelled version of the compound, we found that CLR01 crossed the mouse blood-brain barrier at ∼2% of blood levels. Treatment of 15-month-old triple-transgenic mice for 1 month with CLR01 resulted in a decrease in brain amyloid β protein aggregates, hyperphosphorylated tau and microglia load as observed by immunohistochemistry. Importantly, no signs of toxicity were observed in the treated mice, and CLR01 treatment did not affect the amyloidogenic processing of amyloid β protein precursor. Examining induction or inhibition of the cytochrome P450 metabolism system by CLR01 revealed minimal interaction. Together, these data suggest that CLR01 is safe for use at concentrations well above those showing efficacy in mice. The efficacy and toxicity results support a process-specific mechanism of action of molecular tweezers and suggest that these are promising compounds for developing disease-modifying therapy for Alzheimer's disease and related disorders. © 2012 The Author.
view abstract 10.1093/brain/aws289
- Protein recognition: Calixarene connection
Nature Chemistry 4 (2012)
view abstract 10.1038/nchem.1386
- A modular synthetic route to dimeric calixarenes: A new family of DNA major groove binders
Blecking, C.J. and Hu, W. and Zadmard, R. and Dasgupta, A. and Schrader, T.
Synthesis (2011)We describe here a general strategy for the synthesis of dimeric anilinium/guanidinium calixarenes with aliphatic and heteroaromatic bridges. These compounds display a remarkably high affinity towards double-stranded (ds) DNA and act by a rare mechanism, i.e., insertion into the wide major groove of the nucleic acids. © Georg Thieme Verlag Stuttgart New York.
view abstract 10.1055/s-0030-1259980
- Artificial synthetic receptors as regulators of protein activity
Dutt, S. and Wilch, C. and Schrader, T.
Chemical Communications 47 (2011)This article discusses most recent work and progress in the direction of a rational design of small molecule receptors that efficiently interfere with the biological function of a particular receptor or enzyme - some of which are therapeutically relevant. More specifically, the following topics are highlighted here: the inhibition of voltage-dependent potassium channels of the Kv1.x family by designed porphyrin and calixarene ligands, the structural and functional recovery of the tetramerization domain of mutated P53 protein by tailored calixarene ligands and the control over LDH activity by supramolecular signaling. Finally a new way to modulate NAD+- dependent enzymatic activities by molecular clips and tweezers is presented. © 2011 The Royal Society of Chemistry.
view abstract 10.1039/c0cc05812b
- Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins
Sinha, S. and Lopes, D.H.J. and Du, Z. and Pang, E.S. and Shanmugam, A. and Lomakin, A. and Talbiersky, P. and Tennstaedt, A. and McDaniel, K. and Bakshi, R. and Kuo, P.-Y. and Ehrmann, M. and Benedek, G.B. and Loo, J.A. and Klärner, F.-G. and Schrader, T. and Wang, C. and Bitan, G.
Journal of the American Chemical Society 133 (2011)Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses. © 2011 American Chemical Society.
view abstract 10.1021/ja206279b
- Non covalent inclusion of nucleosides and nucleotides in water-soluble molecular clips
Bastkowski, F. and Polkowska, J. and Schrader, T. and Klärner, F.-G.
Heterocycles 82 (2011)The dimethylene-bridged molecular clips having naphthalene sidewalls and bearing either lithium phosphate or lithium methanephosphonate groups in the central benzene-spacer-unit bind various nucleosides and nucleotides in buffered aqueous solution at pH = 7.2. The binding constants (Ka) and the complexation-induced 1H NMR shifts of the guest signals (Δδmax) were determined by NMR titration experiments. The host-guest complexes of the phosphate-substituted clip with caffeine and theophylline (Ka = 31400 or 16800 M-1) are more stable than those with cytidine and uridine (Ka = 5240 or 5390 M -1) and with adenosine and guanosine (Ka = 1470 or 1120 M-1). In the case of the phosphonate-substituted clip 2 the selectivity in the complex formation toward one type of nucleoside is, however, less pronounced. The complexes of the nucleotides such as AMP, GMP, CMP, or UMP with both clips are less stable than the corresponding complexes of the nucleosides. To understand the observed selectivities in the complex formation we discuss attractive and repulsive electrostatic interactions on the basis of electrostatic potential surfaces (EPS) calculated for host and guest molecules by quantum chemical methods and hydrophobic effects which contribute to the complex stability to a large extent. The observed large complexation-induced 1H NMR shifts (Δδmax) of the guest signals provide good evidence that in each complex the nucleobase is encapsulated inside the clip cavity. © The Japan Institute of Heterocyclic Chemistry.
view abstract 10.3987/COM-10-S(E)112
- Rational design of β-sheet ligands against Aβ42- induced toxicity
Hochdörffer, K. and März-Berberich, J. and Nagel-Steger, L. and Epple, M. and Meyer-Zaika, W. and Horn, A.H.C. and Sticht, H. and Sinha, S. and Bitan, G. and Schrader, T.
Journal of the American Chemical Society 133 (2011)A β-sheet-binding scaffold was equipped with long-range chemical groups for tertiary contacts toward specific regions of the Alzheimer's Aβ fibril. The new constructs contain a trimeric aminopyrazole carboxylic acid, elongated with a C-terminal binding site, whose influence on the aggregation behavior of the Aβ42 peptide was studied. MD simulations after trimer docking to the anchor point (F19/F20) suggest distinct groups of complex structures each of which featured additional specific interactions with characteristic Aβ regions. Members of each group also displayed a characteristic pattern in their antiaggregational behavior toward Aβ. Specifically, remote lipophilic moieties such as a dodecyl, cyclohexyl, or LPFFD fragment can form dispersive interactions with the nonpolar cluster of amino acids between I31 and V36. They were shown to strongly reduce Thioflavine T (ThT) fluorescence and protect cells from Aβ lesions (MTT viability assays). Surprisingly, very thick fibrils and a high β-sheet content were detected in transmission electron microscopy (TEM) and CD spectroscopic experiments. On the other hand, distant single or multiple lysines which interact with the ladder of stacked E22 residues found in Aβ fibrils completely dissolve existing β-sheets (ThT, CD) and lead to unstructured, nontoxic material (TEM, MTT). Finally, the triethyleneglycol spacer between heterocyclic β-sheet ligand and appendix was found to play an active role in destabilizing the turn of the U-shaped protofilament. Fluorescence correlation spectroscopy (FCS) and sedimentation velocity analysis (SVA) provided experimental evidence for some smaller benign aggregates of very thin, delicate structure (TEM, MTT). A detailed investigation by dynamic light scattering (DLS) and other methods proved that none of the new ligands acts as a colloid. The evolving picture for the disaggregation mechanism by these new hybrid ligands implies transformation of well-ordered fibrils into less structured aggregates with a high molecular weight. In the few cases where fibrillar components remain, these display a significantly altered morphology and have lost their acute cellular toxicity. © 2011 American Chemical Society.
view abstract 10.1021/ja107675n
- Artificial RGD receptor molecules
Gersthagen, T. and Schmuck, C. and Schrader, T.
Supramolecular Chemistry 22 (2010)Integrins play a pivotal role in cell-cell adhesion, signalling and apoptosis. Many extracellular proteins use the RGD sequence (arginine-glycine- aspartate) as a key to dock onto and unlock their respective binding partners at the cell membrane (αVβ3-, a IIbβ3- and a5β1-integrin). Here, the RGD signal is transduced into the cytoplasm and triggers a variety of biological events such as blood coagulation, cell-matrix binding, cell differentiation and angiogenesis. A misfunction of this recognition system causes severe diseases, rendering the RGD recognition system an attractive drug target. Inhibition of RGD-integrin interactions can be reached in two different ways, by blocking integrins with RGD mimetics or by capping RGD-containing proteins by artificial RGD receptors. This review provides an overview over the very young history of artificial RGD receptor development, beginning with early research in arginine recognition, over the discovery of the first primitive RGD receptor until the present state of research and future prospects. © 2010 Taylor & Francis.
view abstract 10.1080/10610278.2010.514613
- Combining independent drug classes into superior, synergistically acting hybrid molecules
Müller-Schiffmann, A. and Andreyeva, A. and Rönicke, R. and Bartnik, D. and Brener, O. and Kutzsche, J. and Horn, A.H.C. and Hellmert, M. and Polkowska, J. and Gottmann, K. and Reymann, K.G. and Funke, S.A. and Nagel-Steger, L. and Moriscot, C. and Schoehn, G. and Sticht, H. and Willbold, D. and Schrader, T. and Korth, C.
Angewandte Chemie - International Edition 49 (2010)More than the sum of its parts: Novel hybrid compounds consisting of an organic β-sheet-breaking moiety and a signaling, D-enantiomeric Aβ-recognizing peptide moiety have been designed (see picture). The compounds, which were chemically synthesized and characterized by several techniques, combine rational design and drug selection from libraries and inhibit Aβ oligomerization and Aβ-induced synaptic pathology. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract 10.1002/anie.201004437
- Development and evaluation of a fluorescence microplate assay for quantification of heparins and other sulfated carbohydrates
Lühn, S. and Schrader, T. and Sun, W. and Alban, S.
Journal of Pharmaceutical and Biomedical Analysis 52 (2010)Due to their complex composition, quantification of heparins is difficult. On the one hand there are many biological tests, which only indirectly detect effects of the antithrombin-binding material. On the other hand direct quantitative methods are available but they are often insensitive, challenging, time-consuming or expensive. The aim of this study was to develop a sensitive, rapid, simple as well as inexpensive direct quantification assay suitable for routine analysis. Based on Polymer-H, a novel heparin complexing, fluorescent labeled synthetic polymer (λ(ex) 320 nm, λ(em) 510 nm), a microplate assay was developed and optimized. The specificity of the assay was evaluated by structure-assay response relationships studies using structurally defined glucan sulfates, heparins, and other natural and synthetic sulfated carbohydrates. The fluorescence intensity of Polymer-H (7.5 μg/ml) showed to be concentration-dependently amplified by heparins as well as by other sulfated carbohydrates. The best sensitivity, accuracy and linearity were observed in a range from 0.63 to 5.0 μg/ml heparins. No differences in the fluorescence between various heparins were observed, so that only one calibration curve is needed. In addition, all types of carbohydrates with a degree of sulfation (DS) > ∼1.2 and a Mr > 3000 can be quantified as well. By own calibration curves also other sulfated carbohydrates like fondaparinux or other glycosaminoglycans (DS > 0.4) can be determined. © 2009 Elsevier B.V. All rights reserved.
view abstract 10.1016/j.jpba.2009.12.014
- Effect of molecular clips and tweezers on enzymatic reactions by binding coenzymes and basic amino acids
Klärner, F.-G. and Schrader, T. and Polkowska, J. and Bastkowski, F. and Talbiersky, P. and Kuchenbrandt, M.C. and Schaller, T. and De Groot, H. and Kirsch, M.
Pure and Applied Chemistry 82 (2010)The tetramethylene-bridged molecular tweezers bearing lithium methanephos - phonate or dilithium phosphate substituents in the central benzene or naphthalene spacer-unit and the dimethylene-bridged clips containing naphthalene or anthracene sidewalls substituted by lithium methanephosphonate, dilithium phosphate, or sodium sulfate groups in the central benzene spacer-unit are water-soluble. The molecular clips having planar naphthalene sidewalls bind flat aromatic guest molecules preferentially, for example, the nicotinamide ring and/or the adenine-unit in the nucleotides NAD(P)+, NMN, or AMP, whereas the benzene- spaced molecular tweezers with their bent sidewalls form stable host-guest complexes with the aliphatic side chains of basic amino acids such as lysine and argenine. The phosphonate- substituted tweezer and the clips having an extended central naphthalene spacer-unit or extended anthracene and benzo[k]fluoranthene sidewalls, respectively, form highly stable self-assembled dimers in aqueous solution, evidently due to non-classical hydrophobic interactions. The phosphate-substituted molecular clip containing naphthalene sidewalls inhibits the enzymatic, ADH-catalyzed ethanol oxidation by binding the cofactor NAD+ in a competitive reaction. Surprisingly, tweezer-bearing phosphate substituents in the central benzene spacer-unit are more efficient inhibitors for the ethanol oxidation than the correspondingly substituted naphthalene clip, even though the tweezer does not bind the cofactor NAD+ within the limits of detection. The phosphate-substituted naphthalene clip is, however, a highly efficient inhibitor of the enzymatic oxidation of glucose-6-phosphate (G6P) with NADP+ catalyzed by glucose-6-phosphate dehydrogenase (G6PD), whereas the phosphonate- substituted clip only functions as an inhibitor by forming a complex with the cofactor. Detailed kinetic, thermodynamic, and computational modeling studies provide insight into the mechanism of these novel enzyme inhibition reactions. © 2010 IUPAC.
view abstract 10.1351/PAC-CON-09-10-02
- Modulation of aggregate size- and shape-distributions of the amyloid-β peptide by a designed β-sheet breaker
Nagel-Steger, L. and Demeler, B. and Meyer-Zaika, W. and Hochdörffer, K. and Schrader, T. and Willbold, D.
European Biophysics Journal 39 (2010)A peptide with 42 amino acid residues (Aβ42) plays a key role in the pathogenesis of the Alzheimer's disease. It is highly prone to self aggregation leading to the formation of fibrils which are deposited in amyloid plaques in the brain of diseased individuals. In our study we established a method to analyze the aggregation behavior of the Aβ peptide with a combination of sedimentation velocity centrifugation and enhanced data evaluation software as implemented in the software package UltraScan. Important information which becomes accessible by this methodology is the s-value distribution and concomitantly also the shape-distribution of the Aβ peptide aggregates generated by self-association. With this method we characterized the aggregation modifying effect of a designed bsheet breaker molecule. This compound is built from three head-to-tail connected aminopyrazole moieties and represents a derivative of the already described Tripyrazole. By addition of this compound to a solution of the Aβ42 peptide the maximum of the s-value distribution was clearly shifted to smaller s-values as compared to solutions where only the vehicle DMSO was added. This shift to smaller s-values was stable for at least 7 days. The information about size- and shape-distributions present in aggregated Aβ42 solutions was confirmed by transmission electron microscopy and by measurement of amyloid formation by thioflavin T fluorescence. © European Biophysical Societies' Association 2009.
view abstract 10.1007/s00249-009-0416-2
- RAFT polymers for protein recognition
Tominey, A.F. and Liese, J. and Wei, S. and Kowski, K. and Schrader, T. and Kraft, A.
Beilstein Journal of Organic Chemistry 6 (2010)A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end, N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of the added hydrophobic comonomer. © 2010 Tominey et al; licensee Beilstein-Institut.
view abstract 10.3762/bjoc.6.66
molecular clips and tweezers