Functional Linkers Support Targeting of Multivalent Tweezers to Taspase1.

Taspase 1 is a unique protease not only pivotal for embryonic development but also implicated in leukemias and solid tumors. As such, this enzyme is a promising while still challenging therapeutic target, and with its protein structure featuring a flexible loop preceding the active site a versatile model system for drug development. Supramolecular ligands provide a promising complementary approach to traditional small-molecule inhibitors. Recently, the multivalent arrangement of molecular tweezers allowed the successful targeting of Taspase 1's surface loop. With this study we now want to take the next logic step und utilize functional linker systems that not only allow the implementation of novel properties but also engage in protein surface binding. Consequently, we chose two different linker types differing from the original divalent assembly: a backbone with aggregation-induced emission (AIE) properties to enable monitoring of binding and a calix[4]arene scaffold initially pre-positioning the supramolecular binding units. With a series of four AIE-equipped ligands with stepwise increased valency we demonstrated that the functionalized AIE linkers approach ligand binding affinities in the nanomolar range and allow efficient proteolytic inhibition of Taspase 1. Moreover, implementation of the calix[4]arene backbone further enhanced the ligands' inhibitory potential, pointing to a specific linker contribution.

Tuning Nanobodies' Bioactivity: Coupling to Ultrasmall Gold Nanoparticles Allows the Intracellular Interference with Survivin.

Nanobodies are highly affine binders, often used to track disease-relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease-relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin's multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein-protein interactions. However, as Survivin harbors no classical "druggable" binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X-ray scattering of the Survivin-SuN complex locates the proposed interaction interface between the C-terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2 nm) gold nanoparticles (SuN-N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability.

A Fluorophore-Labeled Lysine Dendrimer with an Oxo-Anion-Binding Motif for Tracking Gene Transfection.

A transfection vector based on a peptide dendrimer (1) has been developed and its abilities for DNA binding and transport have been investigated. By attaching a fluorophore to the vector system (1*), several steps in the transfection process could be monitored directly. As DLS and AFM studies showed, the labeled vector 1* condensed DNA into tightly packed aggregates able to enter eukaryotic cells. Co-localization experiments revealed that the ligand/plasmid complex is taken up by the endosomal pathway followed by an endosomal escape or lysosomal degradation. Afterwards, the plasmid DNA seems to enter the nucleus due to a breakdown of the nuclear envelope during mitosis, as only cells that have recently undergone mitosis showed H2B-GFP expression.

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.

Cationic Solution and Solid-State Emitters - Robust Imaging Agents for Cells, Bacteria, and Protists.

A library of eight different cationic emitters with emission properties in solution and in solid-state (solution and solid-state emitters - SSSE) is presented. These compounds, bearing either ammonium or pyridinium groups, have been investigated regarding their photophysical properties as well as their potential application in biological imaging. Besides high quantum yields as well as a high degree of stability during the imaging process, it was additionally revealed that a broad range of biological targets can be addressed, such as different bacterial strains, human cells as well as protists. The reported SSSE approach employing the mentioned robust emitters for biological imaging, will contribute to a rapid and facile way to design and apply affordable emitters with outstanding properties. Additionally, these emitters will overcome the drawbacks of classical luminophores and agents featuring well-known aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ) properties.

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.

Deoxyestrone-based lipofection agents with solution- and solid-state emission properties.

In this contribution, three deoxyestrone-based emissive lipofection agents are reported. Because of a centrally incorporated terephthalonitrile motif, these ligands can be classified as solution and solid-state emitters (SSSEs). With the attachment of tobramycin, these amphiphilic structures are able to form lipoplexes, mediating gene transfection of HeLa and HEK 293T cells.

Exploiting Vitamin D Receptor and Its Ligands to Target Squamous Cell Carcinomas of the Head and Neck.

Vitamin D (VitD) and its receptor (VDR) have been intensively investigated in many cancers. As knowledge for head and neck cancer (HNC) is limited, we investigated the (pre)clinical and therapeutic relevance of the VDR/VitD-axis. We found that VDR was differentially expressed in HNC tumors, correlating to the patients' clinical parameters. Poorly differentiated tumors showed high VDR and Ki67 expression, whereas the VDR and Ki67 levels decreased from moderate to well-differentiated tumors. The VitD serum levels were lowest in patients with poorly differentiated cancers (4.1 ± 0.5 ng/mL), increasing from moderate (7.3 ± 4.3 ng/mL) to well-differentiated (13.2 ± 3.4 ng/mL) tumors. Notably, females showed higher VitD insufficiency compared to males, correlating with poor differentiation of the tumor. To mechanistically uncover VDR/VitD's pathophysiological relevance, we demonstrated that VitD induced VDR nuclear-translocation (VitD < 100 nM) in HNC cells. RNA sequencing and heat map analysis showed that various nuclear receptors were differentially expressed in cisplatin-resistant versus sensitive HNC cells including VDR and the VDR interaction partner retinoic acid receptor (RXR). However, RXR expression was not significantly correlated with the clinical parameters, and cotreatment with its ligand, retinoic acid, did not enhance the killing by cisplatin. Moreover, the Chou-Talalay algorithm uncovered that VitD/cisplatin combinations synergistically killed tumor cells (VitD < 100 nM) and also inhibited the PI3K/Akt/mTOR pathway. Importantly, these findings were confirmed in 3D-tumor-spheroid models mimicking the patients' tumor microarchitecture. Here, VitD already affected the 3D-tumor-spheroid formation, which was not seen in the 2D-cultures. We conclude that novel VDR/VitD-targeted drug combinations and nuclear receptors should also be intensely explored for HNC. Gender-specific VDR/VitD-effects may be correlated to socioeconomic differences and need to be considered during VitD (supplementation)-therapies.

Selective Disruption of Survivin's Protein-Protein Interactions: A Supramolecular Approach Based on Guanidiniocarbonylpyrrole.

Targeting specific protein binding sites to interfere with protein-protein interactions (PPIs) is crucial for the rational modulation of biologically relevant processes. Survivin, which is highly overexpressed in most cancer cells and considered to be a key player of carcinogenesis, features two functionally relevant binding sites. Here, we demonstrate selective disruption of the Survivin/Histone H3 or the Survivin/Crm1 interaction using a supramolecular approach. By rational design we identified two structurally related ligands (LNES and LHIS ), capable of selectively inhibiting these PPIs, leading to a reduction in cancer cell proliferation.

Recognition of a Flexible Protein Loop in Taspase 1 by Multivalent Supramolecular Tweezers.

Many natural proteins contain flexible loops utilizing well-defined complementary surface regions of their interacting partners and usually undergo major structural rearrangements to allow perfect binding. The molecular recognition of such flexible structures is still highly challenging due to the inherent conformational dynamics. Notably, protein-protein interactions are on the other hand characterized by a multivalent display of complementary binding partners to enhance molecular affinity and specificity. Imitating this natural concept, we here report the rational design of advanced multivalent supramolecular tweezers that allow addressing two lysine and arginine clusters on a flexible protein surface loop. The protease Taspase 1, which is involved in cancer development, carries a basic bipartite nuclear localization signal (NLS) and thus interacts with Importin α, a prerequisite for proteolytic activation. Newly established synthesis routes enabled us to covalently fuse several tweezer molecules into multivalent NLS ligands. The resulting bi- up to pentavalent constructs were then systematically compared in comprehensive biochemical assays. In this series, the stepwise increase in valency was robustly reflected by the ligands' gradually enhanced potency to disrupt the interaction of Taspase 1 with Importin α, correlated with both higher binding affinity and inhibition of proteolytic activity.

Computational model predicts protein binding sites of a luminescent ligand equipped with guanidiniocarbonyl-pyrrole groups.

The 14-3-3 protein family, one of the first discovered phosphoserine/phosphothreonine binding proteins, has attracted interest not only because of its important role in the cell regulatory processes but also due to its enormous number of interactions with other proteins. Here, we use a computational approach to predict the binding sites of the designed hybrid compound featuring aggregation-induced emission luminophores as a potential supramolecular ligand for 14-3-3ζ in the presence and absence of C-Raf peptides. Our results suggest that the area above and below the central pore of the dimeric 14-3-3ζ protein is the most probable binding site for the ligand. Moreover, we predict that the position of the ligand is sensitive to the presence of phosphorylated C-Raf peptides. With a series of experiments, we confirmed the computational prediction of two C 2 related, dominating binding sites on 14-3-3ζ that may bind to two of the supramolecular ligand molecules.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.

Umbelliferone Decorated Water-soluble Zinc(II) Phthalocyanines - In Vitro Phototoxic Antimicrobial Anti-cancer Agents.

In this contribution we report on the synthesis, characterization and application of water-soluble zinc(II) phthalocyanines, which are decorated with four or eight umbelliferone moieties for photodynamic therapy (PDT). These compounds are linked peripherally to zinc(II) phthalocyanine by a triethylene glycol linker attached to pyridines, leading to cationic pyridinium units, able to increase the water solubility of the system. Beside their photophysical properties they were analyzed concerning their cellular distribution in human hepatocyte carcinoma (HepG2) cells as well as their phototoxicity towards HepG2 cells, Gram-positive (S. aureus strain 3150/12 and B. subtilis strain DB104) and Gram-negative bacteria (E. coli strain UTI89 and E. coli strain Nissle 1917). At low light doses and concentrations, they exhibit superb antimicrobial activity against Gram-positive bacteria as well as anti-tumor activity against HepG2. They are even capable to inactivate Gram-negative bacteria, whereas the dark toxicity remains low. These unique water-soluble compounds can be regarded as all-in-one type photosensitizers with broad applications ranges in the future.

Nanoparticle decoration impacts airborne fungal pathobiology.

Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.

Smart Glycopolymeric Nanoparticles for Multivalent Lectin Binding and Stimuli-Controlled Guest Release.

The synthesis and self-assembly of a polymer featuring a self-complementary supramolecular binding motif guanidiniocarbonyl pyrrole carboxylate zwitterion (GCP-zwitterion) bearing lactose moieties are reported. The GCP-zwitterion acts as a cross-linker to facilitate self-assembly of the polymeric chain into nanoparticles (NPs) at neutral pH in an aqueous medium. The formation of polymeric NPs can be controlled by addition of external stimuli (acid or base), which disfavors self-assembly of the GCP-zwitterion because of protonation or deprotonation of the GCP units in the polymer chain. The small-sized (<40 nm) NPs have a hydrophobic cavity and accessible lactose units on the outer shell for multivalent lectin binding. The multivalent interaction between NPs and the lectin peanut agglutinin was confirmed by agglutination experiments. In addition, the stimuli-responsive property of NPs was exploited for the uptake and release of a hydrophobic guest Nile red. Furthermore, the selectivity toward different cell lines (HEK 296T, HeLa, and Hep2G) was tested, and a cellular uptake of cargo-loaded NPs was found for Hep2G cells bearing the lactose-specific asialogylcoprotein receptor, whereas all other cells showed no NP interaction.

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.

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.

The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.

A Branched Tripeptide with an Anion-Binding Motif as a New Delivery Carrier for Efficient Gene Transfection.

Branched and dendrimeric cationic peptides have shown better transfection efficiency than linear peptides, owing to their superior capacity for inducing DNA condensation. We have designed and synthesized two analogously guanidinocarbonylpyrrole-substituted (GCP-substituted) branched cationic tripeptides that provide extremely strong electrostatic attraction towards DNA. Both ligands 1 and 2 can bind to DNA and form condensed complexes, owing to the branched structure and high positive charges, as demonstrated by isothermal titration calorimetry (ITC), ζ potential and atomic force microscopy (AFM). After the replacement of the carboxylate group by an amide group, binding of ligand 2 to DNA shows exothermic enthalpy and positive entropy changes relative to ligand 1. Rational interpretation would suggest that ligand 2 might aid the translocation of plasmid pF143 to HEK 293T cells, showing high gene transfection efficiency. This work therefore provides a facile way, by modifying a branched cationic tripeptide with GCP, to turn a peptide even a tripeptide into an efficient gene transfection vector.

MTOR inhibitor-based combination therapies for pancreatic cancer.

BACKGROUND: Although the mechanistic target of rapamycin (MTOR) kinase, included in the mTORC1 and mTORC2 signalling hubs, has been demonstrated to be active in a significant fraction of patients with pancreatic ductal adenocarcinoma (PDAC), the value of the kinase as a therapeutic target needs further clarification. METHODS: We used Mtor floxed mice to analyse the function of the kinase in context of the pancreas at the genetic level. Using a dual-recombinase system, which is based on the flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies, we generated a novel cellular model, allowing the genetic analysis of MTOR functions in tumour maintenance. Cross-species validation and pharmacological intervention studies were used to recapitulate genetic data in human models, including primary human 3D PDAC cultures. RESULTS: Genetic deletion of the Mtor gene in the pancreas results in exocrine and endocrine insufficiency. In established murine PDAC cells, MTOR is linked to metabolic pathways and maintains the glucose uptake and growth. Importantly, blocking MTOR genetically as well as pharmacologically results in adaptive rewiring of oncogenic signalling with activation of canonical extracellular signal-regulated kinase and phosphoinositide 3-kinase-AKT pathways. We provide evidence that interfering with such adaptive signalling in murine and human PDAC models is important in a subgroup. CONCLUSIONS: Our data suggest developing dual MTORC1/TORC2 inhibitor-based therapies for subtype-specific intervention.

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.