Mucin-Inspired Single-Chain Polymer (MIP) Fibers as Potent SARS-CoV-2 Inhibitors.

Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin-inspired nanostructure, produced via a synthetic methodology to prepare methacrylate-based dendronized polysulfates (MIP-1) on a multi gram-scale with high molecular weight (MW=450 kDa) and thiol end-functionalized mucin-inspired polymer (MIP) via RAFT polymerization. Cryo-electron tomography (Cryo-ET) analysis of MIP-1 confirmed a mucin-mimetic wormlike single-chain fiber structure (length=144±59 nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS-CoV-2 and its mutant strain, with a remarkable low half maximal (IC50 ) inhibitory concentration (IC50 =10.0 nM). Additionally, we investigate the impact of fiber length on SARS-CoV-2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.

Zona Pellucida like Domain Protein 1 (ZPLD1) Polymerization Is Regulated by Two Distinguished Hydrophobic Motifs.

Zona Pellucida Like Domain 1 Protein (ZPLD1) is a main component of the cupula, a gelatinous structure located in the labyrinth organ of the inner ear and involved in vestibular function. The N-glycosylated protein is likely able to organize high-molecular-weight polymers via its zona pellucida (ZP) module, which is common for many extracellular proteins that self-assemble into matrices. In this work, we confirmed that ZPLD1 can form multimers while setting up a cellular model leveraging Madin-Darby canine kidney (MDCK) cells to study protein polymerization. We identified two motifs within ZPLD1 which regulate its polymerization and follow previously published conserved regions, identified across ZP proteins. Mutational depletion of either one of these modules led to diminished or abnormal polymer formation outside of the cells, likely due to altered processing at the plasma membrane. Further, intracellular polymer formation was observed. Proteolytic cleavage during secretion, separating the regulatory motif located distinct of the ZP module from the mature monomer, seems to be necessary to enable polymerization. While the molecular interactions of the identified motifs remain to be proven, our findings suggest that ZPLD1 is a polymer forming ZP protein following an orchestrated mechanism of protein polymerization to finally build up a gelatinous hydrogel.

Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems.

The counterions neutralizing the charges on polyelectrolytes such as DNA or heparin may dissociate in water and greatly influence the interaction of such polyelectrolytes with biomolecules, particularly proteins. In this Review we give an overview of studies on the interaction of proteins with polyelectrolytes and how this knowledge can be used for medical applications. Counterion release was identified as the main driving force for the binding of proteins to polyelectrolytes: Patches of positive charge become multivalent counterions of the polyelectrolyte and lead to the release of counterions from the polyelectrolyte and a concomitant increase in entropy. This is shown from investigations on the interaction of proteins with natural and synthetic polyelectrolytes. Special emphasis is paid to sulfated dendritic polyglycerols (dPGS). The Review demonstrates that we are moving to a better understanding of charge-charge interactions in systems of biological relevance. Research along these lines will aid and promote the design of synthetic polyelectrolytes for medical applications.

Polysulfates Block SARS-CoV-2 Uptake through Electrostatic Interactions*.

Here we report that negatively charged polysulfates can bind to the spike protein of SARS-CoV-2 via electrostatic interactions. Using a plaque reduction assay, we compare inhibition of SARS-CoV-2 by heparin, pentosan sulfate, linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). Highly sulfated LPGS is the optimal inhibitor, with an IC50 of 67 µg mL-1 (approx. 1.6 µm). This synthetic polysulfate exhibits more than 60-fold higher virus inhibitory activity than heparin (IC50 : 4084 µg mL-1 ), along with much lower anticoagulant activity. Furthermore, in molecular dynamics simulations, we verified that LPGS can bind more strongly to the spike protein than heparin, and that LPGS can interact even more with the spike protein of the new N501Y and E484K variants. Our study demonstrates that the entry of SARS-CoV-2 into host cells can be blocked via electrostatic interactions, therefore LPGS can serve as a blueprint for the design of novel viral inhibitors of SARS-CoV-2.

Platelet-derived growth factor receptor ß activation and regulation in murine myelofibrosis.

There is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor ß (PDGFRß) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRß signaling in myelofibrosis is sparse. Using the Gata-1low mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an in situ proximity ligation assay to provide a detailed characterization of PDGFRß signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRß and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRß-PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRß tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRß by protein tyrosine phosphatases as endogenous PDGFRß antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRß-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRß-T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1low mice. In vitro, T-cell protein tyrosine phosphatase (Ptpn2) knockdown increased PDGFRß phosphorylation at Y751 and Y1021, leading to enhanced downstream signaling in fibroblasts. Furthermore, Ptpn2 knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis.

Rational Design of a DNA-Scaffolded High-Affinity Binder for Langerin.

Binders of langerin could target vaccines to Langerhans cells for improved therapeutic effect. Since langerin has low affinity for monovalent glycan ligands, highly multivalent presentation has previously been key for targeting. Aiming to reduce the amount of ligand required, we rationally designed molecularly defined high-affinity binders based on the precise display of glycomimetic ligands (Glc2NTs) on DNA-PNA scaffolds. Rather than mimicking langerin's homotrimeric structure with a C3-symmetric scaffold, we developed readily accessible, easy-to-design bivalent binders. The method considers the requirements for bridging sugar binding sites and statistical rebinding as a means to both strengthen the interactions at single binding sites and amplify the avidity enhancement provided by chelation. This gave a 1150-fold net improvement over the affinity of the free ligand and provided a nanomolar binder (IC50 =300 nM) for specific internalization by langerin-expressing cells.

Sulfated Dendritic Polyglycerol Is a Potent Complement Inhibitor.

The complement system is a powerful mechanism of the innate immune defense system. Dysregulation may contribute to several diseases. Heparin is a known regulator of the complement system, but its application is limited due to its anticoagulative activity. A promising alternative is the synthetic analogue dendritic polyglycerol sulfate (dPGS). Although dPGS-mediated inhibition of the classical and alternative pathway has been roughly described previously, here we analyzed the effects of dPGS regarding the three pathways at different levels of the proteolytic cascades for the first time. Regarding the final outcome (membrane attack complex formation), IC50 values for dPGS varied between the alternative (900 nM), the classical (300 nM), and the lectin pathway (60 nM). In a backward approach, processing of proteins C5 and C3 via the respective convertase was analyzed by ELISA to narrow down dPGS targets. A dose-dependent reduction of C5a and C3a levels was detected. Further, the analysis via surface plasmon resonance revealed novel dPGS binding proteins; the pro-inflammatory anaphylatoxins C3a and C5a and the classical pathway activator C1q showed down to nanomolar binding affinities. The fully synthetic multivalent polymer dPGS seems to be a promising candidate for the further development to counteract excessive complement activation in disease.

Design and Synthesis of PEG-Oligoglycerol Sulfates as Multivalent Inhibitors for the Scavenger Receptor LOX-1.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a cell surface scavenger receptor. The protein is involved in binding and internalization of oxidized low-density lipoprotein (oxLDL), which leads under pathophysiological circumstances to plaque formation in arteries and initiation of atherosclerosis. A structural feature of LOX-1 relevant to oxLDL binding is the "basic spine" motif consisting of linearly aligned arginine residues stretched over the dimer surface. Inhibition of LOX-1 can be done by blocking these positively charged motifs. Here we report on the design, synthesis, and evaluation of a series of novel LOX-1 inhibitors having different numbers of sulfates and polyethylene glycerol (PEG) spacer. Two molecules, compounds 6b and 6d, showed binding affinity in the low nM range, i.e. 45.8 and 47.4 nM, respectively. The in vitro biological studies reveal that these molecules were also able to block the interaction of LOX-1 with its cognate ligands oxLDL, aged RBC, and bacteria.

Interactions of Fullerene-Polyglycerol Sulfates at Viral and Cellular Interfaces.

Understanding the mechanism of interactions of nanomaterials at biointerfaces is a crucial issue to develop new antimicrobial vectors. In this work, a series of water-soluble fullerene-polyglycerol sulfates (FPS) with different fullerene/polymer weight ratios and varying numbers of polyglycerol sulfate branches are synthesized, characterized, and their interactions with two distinct surfaces displaying proteins involved in target cell recognition are investigated. The combination of polyanionic branches with a solvent exposed variable hydrophobic core in FPS proves to be superior to analogs possessing only one of these features in preventing interaction of vesicular stomatitis virus coat glycoprotein (VSV-G) with baby hamster kidney cells serving as a model of host cell. Interference with L-selectin-ligand binding is dominated by the negative charge, which is studied by two assays: a competitive surface plasmon resonance (SPR)-based inhibition assay and the leukocyte cell (NALM-6) rolling on ligands under flow conditions. Due to possible intrinsic hydrophobic and electrostatic effects of synthesized compounds, pico- to nanomolar half maximal inhibitory concentrations (IC50 ) are achieved. With their highly antiviral and anti-inflammatory properties, together with good biocompatibility, FPS are promising candidates for the future development towards biomedical applications.

Biodegradable Polyglycerol Sulfates Exhibit Promising Features for Anti-inflammatory Applications.

Inflammatory processes are beneficial responses to overcome injury or illness. Knowledge of the underlying mechanisms allows for a specific treatment. Thus, synthetic systems can be generated for a targeted interaction. In this context, dendritic polyglycerol sulfates (dPGS) have been investigated as anti-inflammatory compounds. Biodegradable systems are required to prevent compound accumulation in the body. Here we present biodegradable analogs of dPGS based on hyperbranched poly(glycidol- co-caprolactone) bearing a hydrophilic sulfate outer shell (hPG- co-PCLS). The copolymers were investigated regarding their physical and chemical properties. The cytocompatibility was confirmed using A549, Caco-2, and HaCaT cells. Internalization of hPG- co-PCLS by A549 and Caco-2 cells was observed as well. Moreover, we demonstrated that hPG- co-PCLS acted as a competitive inhibitor of the leukocytic cell adhesion receptor L-selectin. Further, a reduction of complement activity was observed. These new biodegradable dPGS analogs are therefore attractive for therapeutic applications regarding inflammatory diseases.

Reducing Macro- and Microheterogeneity of N-Glycans Enables the Crystal Structure of the Lectin and EGF-Like Domains of Human L-Selectin To Be Solved at 1.9†ŠResolution.

L-Selectin, a cell-adhesion receptor on the surface of most leukocytes, contains seven N-glycosylation sites. In order to obtain the crystal structure of human L-selectin, we expressed a shortened version of L-selectin comprising the C-type lectin and EGF-like domains (termed LE) and systematically analysed mutations of the three glycosylation sites (Asn22, Asn66 and Asn139) in order to reduce macroheterogeneity. After we further removed microheterogeneity, we obtained crystals that diffracted X-rays up to 1.9 Šfrom a variant (LE010) with exchanges N22Q and N139Q and one GlcNAc2 Man5 N-glycan chain attached to Asn66. Crystal-structure analysis showed that the terminal mannose of GlcNAc2 Man5 of one LE010 molecule was coordinated to Ca2+ in the binding site of a symmetry-related LE010. The orientation of the lectin and EGF-like domain was similar to the described "bent" conformation of E- and P-selectins. The Ca2+ -binding site reflects the binding mode seen in E- and P-selectin structures co-crystallised with ligands.

Mannose-Functionalized Hyperbranched Polyglycerol Loaded with Zinc Porphyrin: Investigation of the Multivalency Effect in Antibacterial Photodynamic Therapy.

The antibacterial photodynamic activity of hyperbranched polyglycerol (hPG) loaded with zinc porphyrin photosensitizers and mannose units was investigated. hPG, with a MW of 19.5 kDa, was functionalized with about 15 molecules of the photosensitizer {5,10,15-tris(3-hydroxyphenyl)-20-[4-(prop-2-yn-1-ylamino)tetrafluorophenyl]porphyrinato}-zinc(II) by using copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC). These nanoparticle conjugates were functionalized systematically with increasing loadings of mannose in the range of approximately 20 to 110 groups. With higher mannose loadings (ca. 58-110 groups) the water-insoluble zinc porphyrin photosensitizer could thus be transferred into a water-soluble form. Targeting of the conjugates was proven in binding studies to the mannose-specific lectin concanavalin A (Con A) by using surface plasmon resonance (SPR). The antibacterial phototoxicity of the conjugates on Staphylococcus aureus (as a typical Gram-positive germ) was investigated in phosphate-buffered saline (PBS). It was shown that conjugates with approximately 70-110 mannose units exhibit significant antibacterial activity, whereas conjugates with approximately 20-60 units did not induce bacterial killing at all. These results give an insight into the multivalency effect in combination with photodynamic therapy (PDT). On addition of serum to the bacterial cultures, a quenching of this antibacterial phototoxicity was observed. In fluorescence studies with the conjugates in the presence of increasing bovine serum albumin (BSA) concentrations, protein-conjugate associations could be identified as a plausible cause for this quenching.

Noncharged and Charged Monodendronised Perylene Bisimides as Highly Fluorescent Labels and their Bioconjugates.

A series of water-soluble, hydroxylated and sulphated, polyglycerol (PG) dendronised, monofunctional perylene bisimides (PBIs) were synthesised in three generations. Their photophysical properties were determined by absorption and emission spectroscopy and their suitability as potential biolabels examined by biological in vitro studies after bioconjugation. It could be shown that the photophysical properties of the PBI labels can be improved by increasing the sterical demand and ionic charge of the attached dendron. Thereby, charged labels show superior suppression of aggregation over charge neutral labels owing to electrostatic repulsion forces on the PG-dendron. The ionic charges also enabled a reduction in dendron generation while retaining the labels' outstanding fluorescence quantum yields (FQYs) up to 100 %. These core-unsubstituted perylene derivatives were successfully applied as fluorescent labels upon bioconjugation to the therapeutic antibody cetuximab. The dye-antibody conjugates showed a strongly enhanced aggregation tendency compared to the corresponding free dyes. Biological evaluation by receptor-binding, cellular uptake, and cytotoxicity studies revealed that labelling did not affect the antibody's function, which renders the noncharged and charged dendronised PBIs suitable candidates as fluorescent labels in biological imaging.

Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography.

l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin.

Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration.

Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-LewisX (SLeX ) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepared in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical degree of polymerization, as they are derived from the same precursor polymer. Binding assays to selectins, to activated endothelial cells, and to macrophages show that polyHPMA with SLeX is an excellent binder to E-, L-, and P-selectins. However, mimetic P4 can also achieve close to comparable binding affinities in in vitro measurements and surprisingly, it also significantly inhibits the migration of macrophages; this provides new perspectives for the therapy of severe inflammatory diseases.

Structural requirements of mono- and multivalent L-selectin blocking aptamers for enhanced receptor inhibition in vitro and in vivo.

L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting. FROM THE CLINICAL EDITOR: During inflammation, leukocytes extravasate from blood vessels under chemotaxic signals. The presence of L-selectin on endothelium acts as a mediator for the extravasation process. In this study, the authors investigated an L-selectin specific blocking DNA aptamer in various forms, as inhibitors to leukocyte binding and extravasation. This new approach confirmed the potential use of aptamers in clinical setting.

Size dependence of steric shielding and multivalency effects for globular binding inhibitors.

Competitive binding inhibitors based on multivalent nanoparticles have shown great potential for preventing virus infections. However, general design principles of highly efficient inhibitors are lacking as the quantitative impact of factors such as virus concentration, inhibitor size, steric shielding, or multivalency effects in the inhibition process is not known. Based on two complementary experimental inhibition assays we determined size-dependent steric shielding and multivalency effects. This allowed us to adapt the Cheng-Prusoff equation for its application to multivalent systems. Our results show that the particle and volume normalized IC50 value of an inhibitor at very low virus concentration predominantly depends on its multivalent association constant, which itself exponentially increases with the inhibitor/virus contact area and ligand density. Compared to multivalency effects, the contribution of steric shielding to the IC50 values is only minor, and its impact is only noticeable if the multivalent dissociation constant is far below the virus concentration, which means if all inhibitors are bound to the virus. The dependence of the predominant effect, either steric shielding or multivalency, on the virus concentration has significant implications on the in vitro testing of competitive binding inhibitors and determines optimal inhibitor diameters for the efficient inhibition of viruses.

Effects of dendritic polyglycerol sulfate on articular chondrocytes.

INTRODUCTION: Inflammatory processes driven by cytokines play a crucial role during osteoarthritis (OA) progression. Dendritic polyglycerol sulfate (dPGS) was analyzed in vitro for its effects on articular chondrocytes, cartilage and cytokines involved in the OA process. METHODS: The metabolic activity of cultured human articular chondrocytes stimulated for 24 h with dPGS (10(-3)-10(-6) mol/L) was monitored using AlamarBlue(®) assay. The dPGS uptake was studied using fluorescence labeled nanoparticles. Further, chondrocytes were either treated with 10(-6) M dPGS, TNFα (10 ng/mL) alone or with a combination of both. The influence on extracellular matrix components, pro- and anti-inflammatory cytokines, matrix metalloproteinase (MMP)1 and the anaphylatoxin receptor C3aR was analyzed by RTD-PCR, flow cytometry and ELISA. RESULTS: Even at higher dosages (10(-3) mol/L), dPGS did not influence chondrocytes viability. Uptake of dPGS was successfully monitored in human articular chondrocytes and synovial fibroblasts, penetration into cartilage chips was up to ~50 µm. Cellular treatment with dPGS had no effect on synthesis of pro-inflammatory cytokines TNFα and IL-6, but expression of the anti-inflammatory IL-10 was upregulated. Cotreatment with TNFα and dPGS reduced the TNFα level, while IL-1ß, IL-6 and IL-10 expression did not change. Collagen type II gene expression was significantly reduced after preincubating cells with dPGS, but remained unaffected at the protein level. CONCLUSION: Results indicate that dPGS could play a role in regulation of cytokines associated with the inflammatory aspect of OA progression.

Detecting and Quantifying Biomolecular Interactions of a Dendritic Polyglycerol Sulfate Nanoparticle Using Fluorescence Lifetime Measurements.

Interactions of nanoparticles with biomaterials determine the biological activity that is key for the physiological response. Dendritic polyglycerol sulfates (dPGS) were found recently to act as an inhibitor of inflammation by blocking selectins. Systemic application of dPGS would present this nanoparticle to various biological molecules that rapidly adsorb to the nanoparticle surface or lead to adsorption of the nanoparticle to cellular structures such as lipid membranes. In the past, fluorescence lifetime measurements of fluorescently tagged nanoparticles at a molecular and cellular/tissue level have been proven to reveal valuable information on the local nanoparticle environment via characteristic fluorescent lifetime signatures of the nanoparticle bound dye. Here, we established fluorescence lifetime measurements as a tool to determine the binding affinity to fluorescently tagged dPGS (dPGS-ICC; ICC: indocarbocyanine). The binding to a cell adhesion molecule (L-selectin) and a human complement protein (C1q) to dPGS-ICC was evaluated by the concentration dependent change in the unique fluorescence lifetime signature of dPGS-ICC. The apparent binding affinity was found to be in the nanomolar range for both proteins (L-selectin: 87 ± 4 nM and C1q: 42 ± 12 nM). Furthermore, the effect of human serum on the unique fluorescence lifetime signature of dPGS-ICC was measured and found to be different from the interactions with the two proteins and lipid membranes. A comparison between the unique lifetime signatures of dPGS-ICC in different biological environments shows that fluorescence lifetime measurements of unique dPGS-ICC fluorescence lifetime signatures are a versatile tool to probe the microenvironment of dPGS in cells and tissue.

Synthesis of multivalent carbohydrate mimetics with aminopolyol end groups and their evaluation as L-selectin inhibitors.

In this article a series of divalent and trivalent carbohydrate mimetics on the basis of an enantiopure aminopyran and of serinol is described. These aminopolyols are connected by amide bonds to carboxylic acid derived spacer units either by Schotten-Baumann acylation or by coupling employing HATU as reagent. The O-sulfation employing the SO3·DMF complex was optimized. It was crucial to follow this process by 700 MHz (1)H NMR spectroscopy to ensure full conversion and to use a refined neutralization and purification protocol. Many of the compounds could not be tested as L-selectin inhibitor by SPR due to their insolubility in water, nevertheless, a divalent and a trivalent amide showed surprisingly good activities with IC50 values in the low micromolar range.