Impact of Peptide Sequences on Their Structure and Function: Mimicking of Virus-Like Nanoparticles for Nucleic Acid Delivery.

We rationally designed a series of amphiphilic hepta-peptides enriched with a chemically conjugated guanidiniocarbonylpyrrole (GCP) unit at the lysine side chain. All peptides are composed of polar (GCP) and non-polar (cyclohexyl alanine) residues but differ in their sequence periodicity, resulting in different secondary as well as supramolecular structures. CD spectra revealed the assembly of ß-sheet-, α-helical and random structures for peptides 1, 2 and 3, respectively. Consequently, this enabled the formation of distinct supramolecular assemblies such as fibres, nanorod-like or spherical aggregates. Notably, all three cationic peptides are equipped with the anion-binding GCP unit and thus possess a nucleic acid-binding centre. However, only the helical (2) and the unstructured (3) peptide were able to assemble into small virus-like DNA-polyplexes and effectively deliver DNA into cells. Notably, as both peptides (2 and 3) were also capable of siRNA-delivery, they could be utilized to downregulate expression of the caner-relevant protein Survivin.

Functional Disruption of the Cancer-Relevant Interaction between Survivin and Histone H3 with a Guanidiniocarbonyl Pyrrole Ligand.

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.

From Supramolecular Vesicles to Micelles: Controllable Construction of Tumor-Targeting Nanocarriers Based on Host-Guest Interaction between a Pillar[5]arene-Based Prodrug and a RGD-Sulfonate Guest.

The targeting ability, drug-loading capacity, and size of the drug nanocarriers are crucial for enhancing the therapeutic index for cancer therapy. Herein, the morphology and size-controllable fabrication of supramolecular tumor-targeting nanocarriers based on host-guest recognition between a novel pillar[5]arene-based prodrug WP5-DOX and a Arg-Gly-Asp (RGD)-modified sulfonate guest RGD-SG is reported. The amphiphilic WP5-DOX⊃RGD-SG complex with a molar ratio of 5:1 self-assembles into vesicles, whereas smaller-sized micelles can be obtained by changing the molar ratio to 1:3. This represents a novel strategy of controllable construction of supramolecular nanovehicles with different sizes and morphologies based on the same host-guest interactions by using different host-guest ratios. Furthermore, in vitro and in vivo studies reveal that both these prodrug nanocarriers could selectively deliver doxorubicin to RGD receptor-overexpressing cancer cells, leading to longer blood retention time, enhanced antitumor efficacy, and reduced systematic toxicity in murine tumor model, suggesting their potential application for targeted drug delivery.

Dimensional control of supramolecular assemblies of diacetylene-derived peptide gemini amphiphile: from spherical micelles to foamlike networks.

Peptide amphiphiles capable of assembling into multidimensional nanostructures have attracted much attention over the past decade due to their potential applications in materials science. Herein, a novel diacetylene-derived peptide gemini amphiphile with a fluorenylmethyloxycarbonyl (Fmoc) group at the N-terminus is reported to hierarchically assemble into spherical micelles, one-dimensional nanorods, two-dimensional foamlike networks and lamellae. Solvent polarity shows a remarkable effect on the self-assembled structures by changing the balance of four weak noncovalent interactions (hydrogen-bonding, π-π stacking, hydrophobic interaction, and electrostatic repulsion). We also show the time-evolution not only from spherical micelles to helical nanofibers in aqueous solution, but also from branched wormlike micelles to foamlike networks in methanol solution. In this work, the presence of the Fmoc group plays a key role in the self-assembly process. This work provides an efficient strategy for precise morphological control, aiding the future development in materials science.

Formation of Twisted ß-Sheet Tapes from a Self-Complementary Peptide Based on Novel Pillararene-GCP Host-Guest Interaction with Gene Transfection Properties.

Small peptides capable of assembling into well-defined nanostructures have attracted extensive attention due to their interesting applications as biomaterials. This work reports the first example of a pillararene functionalized with a guanidiniocarbonyl pyrrole (GCP)-conjugated short peptide segment. The obtained amphiphilic peptide 1 spontaneously self-assembles into a supramolecular ß-sheet in aqueous solution based on host-guest interaction between pillararene and GCP unit as well as hydrogen-bonding between the peptide strands. Interestingly, peptide 1 at low concentration shows transitions from small particles to "pearl necklace" assemblies, and finally to branched fibers in a time-dependent process. At higher concentration, it directly assembles into twisted ß-sheet tapes. Notably, without pillararene moiety, the control peptide A forms α-helix structure with morphology changing from particles to bamboo-like assemblies depending on concentration, indicating a significant role of the pillararene-GCP host-guest interaction for the secondary structure formation. Moreover, peptide 1 can serve as an efficient gene transfection vector.

Rational Design, Binding Studies, and Crystal-Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14-3-3ζ Adapter Protein.

14-3-3 Proteins play a central role in signalling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14-3-3 proteins plays a key role in their activity. Here, we present, to the best of our knowledge, the first example of a small molecule binding to the 14-3-3ζ dimerisation interface. This compound was designed by rational in silico optimisation of a peptidic ligand identified from biochemical screening of a peptidic library, and the binding was characterised by UV/Vis spectroscopy, microscale thermophoresis, multiscale simulations, and X-ray crystallography.

Correction: A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates.

Correction for 'A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates' by Debabrata Maity et al., Chem. Commun., 2017, 53, 208-211.

Peptide-Based Probes with an Artificial Anion-Binding Motif for Direct Fluorescence "Switch-On" Detection of Nucleic Acid in Cells.

This work reports two new peptide-based fluorescence probes (1 and 2) for the detection of ds-DNA at physiological pH. Probes 1 and 2 contain a fluorophore, either amino-naphthalimide or diethyl-aminocoumarin, respectively, and two identical peptide arms each equipped with a guanidiniocarbonylpyrrole (GCP) anion-binding motif. These probes show "switch-on" fluorescence response upon binding to ds-DNA, whereby they can differentiate between various types of polynucleotides. For instance, they exhibit more pronounced fluorescence response for AT-rich polynucleotides than GC-rich polynucleotides, and both give only negligible response to ds-RNA. The fluorimetric response of 1 is proportional to the AT-basepair content in DNA, whereas the fluorescence of 2 is sensitive to the secondary structure of the polynucleotide. Fluorescence experiments, thermal melting experiments and circular dichroism studies suggest that 1 interacts with ds-DNA in a combined intercalation and minor groove binding, whereas 2 interacts mainly with the outer surface of DNA/RNA. As 1 and 2 have a very low cytotoxicity, 1 can be applied for the imaging of nuclear DNA in cells.

A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates.

We report a fluorescence probe 1, which contains a naphthalimide fluorophore with two symmetric peptidic arms equipped with a tailor-made anion-binding motif, the guanidiniocarbonyl pyrrole moiety, for the detection of nucleoside triphosphates. Upon binding to nucleoside triphosphates, especially ATP, 1 shows significant turn-on fluorescence response. Probe 1 can also be applied for the imaging of ATP in cells.

pH-Controlled Formation of a Stable ß-Sheet and Amyloid-like Fibers from an Amphiphilic Peptide: The Importance of a Tailor-Made Binding Motif for Secondary Structure Formation.

The new amphiphilic peptide 1 is composed of alternating cyclohexyl side chains and guanidiniocarbonyl pyrrole (GCP) groups. In contrast to analogue 2, which contains lysine instead of the GCP groups and only exists as a random coil owing to charge repulsion, peptide 1 forms a stable ß-sheet at neutral pH in aqueous medium. The weakly basic GCP groups (pKa ≈7) are key for secondary structure formation as they stabilize the ß-sheet through mutual interactions (formation of a "GCP zipper"). The ß-sheets further aggregate into left-handed helically twisted fibers. However, ß-sheet formation is completely reversible as a function of pH. At low pH (ca. 4), peptide 1 is unstructured (random coil) as all GCP units are protonated. Only round colloidal particles are observed. The amyloid nature of the fibers formed at neutral pH was confirmed by staining experiments with Congo Red and thioflavin T. Furthermore, at millimolar concentrations, peptide 1 forms a stable hydrogel.

Use of an Octapeptide-Guanidiniocarbonylpyrrole Conjugate for the Formation of a Supramolecular ß-Helix that Self-Assembles into pH-Responsive Fibers.

Peptides that adopt ß-helix structures are predominantly found in transmembrane protein domains or in the lipid bilayer of vesicles. Constructing a ß-helix structure in pure water has been considered difficult without the addition of membrane mimics. Herein, we report such an example; peptide 1 self-assembles into a supramolecular ß-helix in pure water based on charge interactions between the individual peptides. Peptide 1 further showed intriguing transitions from small particles to helical fibers in a time-dependent process. The fibers can be switched to vesicles by changing the pH value.

Two-Component Self-Assembly: Hierarchical Formation of pH-Switchable Supramolecular Networks by π-π Induced Aggregation of Ion Pairs.

Two-component self-assembly is a promising approach to construct functional nanomaterials. Interaction of a flexible guanidiniocarbonyl pyrrole tetra-cation (1) with naphthalene diimide dicarboxylic acid (NDIDC) in aqueous DMSO leads to the formation of supramolecular networks. First, the carboxylate groups of NDIDC bind to the guanidiniocarbonyl pyrrole cations of 1 in a 1:2 stoichiometry. Further π-π induced aggregation then leads to 3D networks, as established by dynamic light scattering studies (DLS), NMR, fluorescence titration, viscosity measurements, AFM, and TEM microscopy. Due to ion pairing, the resulting aggregates can be switched between the monomers and the aggregates reversibly using external stimuli like protonation or deprotonation. At high concentration, a stable colloidal solution is formed, which shows an extensive Tyndall effect. Increasing the concentrations even further leads to formation of a supramolecular gel.

A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells.

Incorporation of a Non-Natural Arginine Analogue into a Cyclic Peptide Leads to Formation of Positively Charged Nanofibers Capable of Gene Transfection.

Functionalization of the tetracationic cyclic peptide (Ka)4 with a single guanidiniocarbonyl pyrrole (GCP) moiety, a weakly basic but highly efficient arginine analogue, completely alters the self-assembly properties of the peptide. In contrast to the nonfunctionalized peptide 2, which does not self-assemble, GCP-containing peptide 1 forms cationic nanofibers of micrometer length. These aggregates are efficient gene transfection vectors. DNA binds to their cationic surface and is efficiently delivered into cells.

Discovery of potent inhibitors of human ß-tryptase from pre-equilibrated dynamic combinatorial libraries.

Pre-equilibrated dynamic combinatorial libraries based on acyl hydrazone interchange of peptide-derived hydrazides and di- and tri-aldehydes have been used to discover potent inhibitors with nanomolar affinities for ß-tryptase. To identify potent inhibitors the activity of the full library containing 95 members was compared with those of sub-libraries in which individual building blocks were missing. The most active library members contain a rigid central aromatic scaffold with three cationic peptide arms. The arms of the best inhibitors also contained a tailor-made GCP oxoanion binding motif attached to a lysine side chain. The most potent tri-armed hydrazones with peptide arms GKWR or GKWK(GCP) were shown to inhibit ß-tryptase (Kica. 10-20 nM) reversibly, non-competitively and selectively (compared to related serine proteases, e.g. trypsin and chymotrypsin), most likely by binding to the protein surface, also in agreement with molecular modelling calculations. These new inhibitors are one order of magnitude more efficient than related tetravalent inhibitors obtained from previous work on a split-mix-combinatorial library and were identified with significantly less effort, demonstrating the usefulness of this approach for the identification of enzyme inhibitors in general.

Preparation and antimalarial activity of a novel class of carbohydrate-derived, fused thiochromans.

A novel class of fused thiochroman derivatives has been prepared by an efficient and versatile synthetic procedure involving nucleophilic displacement of the side-chain iodo substituent in 2-deoxy-2-C-iodomethyl glucosides by thiophenolate ions, and subsequent intramolecular C-glycoside formation. A range of aromatic substituents is tolerated, and the subsequent facile selective oxidation of the sulfur to the sulfoxide or sulfone level expands the range and molecular diversity of the series of compounds. A selection of the sulfoxide and sulfone derivatives bearing lipophilic substituents on the aromatic portion were found to have antimalarial activities in the low micromolar range.

A fluorescent light-up probe as an inhibitor of intracellular ß-tryptase.

A pyrene-functionalized peptidic inhibitor binds to and inhibits ß-tryptase in a non-competitive and reversible manner even in cells. Upon protein binding a fluorescence increase of the two pyrene fluorophores is observed which allows using as a fluorescent light-up probe for this enzyme.