Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles.

Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.

Lysine-selective molecular tweezers are cell penetrant and concentrate in lysosomes.

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.

Supramolecular Enhancement of a Natural 14-3-3 Protein Ligand.

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.

Specific inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers.

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.

New Tools to Probe the Protein Surface: Ultrasmall Gold Nanoparticles Carry Amino Acid Binders.

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.

Molecular Lysine Tweezers Counteract Aberrant Protein Aggregation.

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.

The molecular tweezer CLR01 reduces aggregated, pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy.

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.