The protein Survivin is highly upregulated in most cancers and considered to be a key player in carcinogenesis. We explored a supramolecular approach to address Survivin as a drug target by inhibiting the protein–protein interaction of Survivin and its functionally relevant binding partner Histone H3. Ligand L1 is based on the guanidiniocarbonyl pyrrole cation and serves as a highly specific anion binder in order to target the interaction between Survivin and Histone H3. NMR titration confirmed binding of L1 to Survivin's Histone H3 binding site. The inhibition of the Survivin–Histone H3 interaction and consequently a reduction of cancer cell proliferation were demonstrated by microscopic and cellular assays.

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.

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 K D 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 K D 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.

Das Protein Survivin ist bei den meisten Krebsarten überexprimiert und gilt als einer der Hauptakteure der Krebsentstehung. Wir haben einen supramolekularen Ansatz genutzt, um Survivin als Wirkstoff‐Ziel zu adressieren, indem wir die Protein‐Protein‐Interaktion von Survivin und seinem funktionell relevanten Bindungspartner Histon H3 inhibieren. Ligand L1 basiert auf dem Guanidiniumcarbonylpyrrol‐Kation und dient als hochspezifischer Anionenbinder. Durch NMR‐Titrationen konnte die Bindung von L1 an die Histon H3‐Bindungsstelle von Survivin bestätigt werden. Die Hemmung der Survivin‐Histon‐H3‐Interaktion und folglich eine Verringerung der Proliferation von Krebszellen wurde durch mikroskopische und zelluläre Assays nachgewiesen.

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