Comprehensive Characterization of the Viscoelastic Properties of Bovine Submaxillary Mucin (BSM) Hydrogels and the Effect of Additives.

This study presents a comprehensive characterization of the viscoelastic and structural properties of bovine submaxillary mucin (BSM), which is widely used as a commercial source to conduct mucus-related research. We conducted concentration studies of BSM and examined the effects of various additives, NaCl, CaCl2, MgCl2, lysozyme, and DNA, on its rheological behavior. A notable connection between BSM concentration and viscoelastic properties was observed, particularly under varying ionic conditions. The rheological spectra could be well described by a fractional Kelvin-Voigt model with a minimum of model parameters. A detailed proteomics analysis provided insight into the protein, especially mucin composition within BSM, showing MUC19 as the main component. Cryo-scanning electron microscopy enabled the visualization of the porous BSM network structure. These investigations give us a more profound comprehension of the BSM properties, especially those pertaining to viscoelasticity, and how they are influenced by concentration and environmental conditions, aspects relevant to the field of mucus research.

Exploring Mechanisms of Lipid Nanoparticle-Mucus Interactions in Healthy and Cystic Fibrosis Conditions.

Mucus forms the first defense line of human lungs, and as such hampers the efficient delivery of therapeutics to the underlying epithelium. This holds particularly true for genetic cargo such as CRISPR-based gene editing tools which cannot readily surmount the mucosal barrier. While lipid nanoparticles (LNPs) emerge as versatile non-viral gene delivery systems that can help overcome the delivery challenge, many knowledge gaps remain, especially for diseased states such as cystic fibrosis (CF). This study provides fundamental insights into Cas9 mRNA or ribonucleoprotein-loaded LNP-mucus interactions in healthy and diseased states by assessing the impact of the genetic cargo, mucin sialylation, mucin concentration, ionic strength, pH, and polyethylene glycol (PEG) concentration and nature on LNP diffusivity leveraging experimental approaches and Brownian dynamics (BD) simulations. Taken together, this study identifies key mucus and LNP characteristics that are critical to enabling a rational LNP design for transmucosal delivery.

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on sputum viscoelastic properties, airway infection and inflammation in patients with cystic fibrosis.

BACKGROUND: Recent studies demonstrated that the triple combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and reduces pulmonary exacerbations in cystic fibrosis (CF) patients with at least one F508del allele. However, effects of ETI on downstream consequences of CFTR dysfunction, i.e. abnormal viscoelastic properties of airway mucus, chronic airway infection and inflammation have not been studied. The aim of this study was to determine the longitudinal effects of ETI on airway mucus rheology, microbiome and inflammation in CF patients with one or two F508del alleles aged ≥12 years throughout the first 12 months of therapy. METHODS: In this prospective observational study, we assessed sputum rheology, the microbiome, inflammation markers and proteome before and 1, 3 and 12 months after initiation of ETI. RESULTS: In total, 79 patients with CF and at least one F508del allele and 10 healthy controls were enrolled in this study. ETI improved the elastic modulus and viscous modulus of CF sputum at 3 and 12 months after initiation (all p<0.01). Furthermore, ETI decreased the relative abundance of Pseudomonas aeruginosa in CF sputum at 3 months and increased the microbiome α-diversity at all time points. In addition, ETI reduced interleukin-8 at 3 months (p<0.05) and free neutrophil elastase activity at all time points (all p<0.001), and shifted the CF sputum proteome towards healthy. CONCLUSIONS: Our data demonstrate that restoration of CFTR function by ETI improves sputum viscoelastic properties, chronic airway infection and inflammation in CF patients with at least one F508del allele over the first 12 months of therapy; however, levels close to healthy were not reached.

Respiratory viruses interacting with cells: the importance of electrostatics.

The COVID-19 pandemic has rekindled interest in the molecular mechanisms involved in the early steps of infection of cells by viruses. Compared to SARS-CoV-1 which only caused a relatively small albeit deadly outbreak, SARS-CoV-2 has led to fulminant spread and a full-scale pandemic characterized by efficient virus transmission worldwide within a very short time. Moreover, the mutations the virus acquired over the many months of virus transmission, particularly those seen in the Omicron variant, have turned out to result in an even more transmissible virus. Here, we focus on the early events of virus infection of cells. We review evidence that the first decisive step in this process is the electrostatic interaction of the spike protein with heparan sulfate chains present on the surface of target cells: Patches of cationic amino acids located on the surface of the spike protein can interact intimately with the negatively charged heparan sulfate chains, which results in the binding of the virion to the cell surface. In a second step, the specific interaction of the receptor binding domain (RBD) within the spike with the angiotensin-converting enzyme 2 (ACE2) receptor leads to the uptake of bound virions into the cell. We show that these events can be expressed as a semi-quantitative model by calculating the surface potential of different spike proteins using the Adaptive Poison-Boltzmann-Solver (APBS). This software allows visualization of the positive surface potential caused by the cationic patches, which increased markedly from the original Wuhan strain of SARS-CoV-2 to the Omicron variant. The surface potential thus enhanced leads to a much stronger binding of the Omicron variant as compared to the original wild-type virus. At the same time, data taken from the literature demonstrate that the interaction of the RBD of the spike protein with the ACE2 receptor remains constant within the limits of error. Finally, we briefly digress to other viruses and show the usefulness of these electrostatic processes and calculations for cell-virus interactions more generally.

Functionalized Fullerene for Inhibition of SARS-CoV-2 Variants.

As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C60 fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4 nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.

An optimized protocol for assessment of sputum macrorheology in health and muco-obstructive lung disease.

Background: Airway mucus provides important protective functions in health and abnormal viscoelasticity is a hallmark of muco-obstructive lung diseases such as cystic fibrosis (CF). However, previous studies of sputum macrorheology from healthy individuals and patients with CF using different experimental protocols yielded in part discrepant results and data on a systematic assessment across measurement settings and conditions remain limited. Objectives: The aim of this study was to develop an optimized and reliable protocol for standardized macrorheological measurements of airway mucus model systems and native human sputum from healthy individuals and patients with muco-obstructive lung disease. Methods: Oscillatory rheological shear measurements were performed using bovine submaxillary mucin (BSM) at different concentrations (2% and 10% solids) and sputum samples from healthy controls (n = 10) and patients with CF (n = 10). Viscoelastic properties were determined by amplitude and frequency sweeps at 25°C and 37°C with or without solvent trap using a cone-plate geometry. Results: Under saturated atmosphere, we did not observe any temperature-dependent differences in 2% and 10% BSM macrorheology, whereas in the absence of evaporation control 10% BSM demonstrated a significantly higher viscoelasticity at 37°C. Similarly, during the measurements without evaporation control at 37°C we observed a substantial increase in the storage modulus G' and the loss modulus G″ of the highly viscoelastic CF sputum but not in the healthy sputum. Conclusion: Our data show systematically higher viscoelasticity of CF compared to healthy sputum at 25°C and 37°C. For measurements at the higher temperature using a solvent trap to prevent evaporation is essential for macrorheological analysis of mucus model systems and native human sputum. Another interesting finding is that the viscoelastic properties are not much sensitive to the applied experimental deformation and yield robust results despite their delicate consistency. The optimized protocol resulting from this work will facilitate standardized quantitative assessment of abnormalities in viscoelastic properties of airway mucus and response to muco-active therapies in patients with CF and other muco-obstructive lung diseases.

Synthetic α-Helical Peptides as Potential Inhibitors of the ACE2 SARS-CoV-2 Interaction.

During viral cell entry, the spike protein of SARS-CoV-2 binds to the α1-helix motif of human angiotensin-converting enzyme 2 (ACE2). Thus, alpha-helical peptides mimicking this motif may serve as inhibitors of viral cell entry. For this purpose, we employed the rigidified diproline-derived module ProM-5 to induce α-helicity in short peptide sequences inspired by the ACE2 α1-helix. Starting with Ac-QAKTFLDKFNHEAEDLFYQ-NH2 as a relevant section of α1, a series of peptides, N-capped with either Ac-ßHAsp-[ProM-5] or Ac-ßHAsp-PP, were prepared and their α-helicities were investigated. While ProM-5 clearly showed a pronounced effect, an even increased degree of helicity (up to 63 %) was observed in sequences in which non-binding amino acids were replaced by alanine. The binding affinities of the peptides towards the spike protein, as determined by means of microscale thermophoresis (MST), revealed only a subtle influence of the α-helical content and, noteworthy, led to the identification of an Ac-ßHAsp-PP-capped peptide displaying a very strong binding affinity (KD =62 nM).

Design and Functional Analysis of Heterobifunctional Multivalent Phage Capsid Inhibitors Blocking the Entry of Influenza Virus.

Multiple conjugation of virus-binding ligands to multivalent carriers is a prominent strategy to construct highly affine virus binders for the inhibition of viral entry into host cells. In a previous study, we introduced rationally designed sialic acid conjugates of bacteriophages (Qß) that match the triangular binding site geometry on hemagglutinin spike proteins of influenza A virions, resulting in effective infection inhibition in vitro and in vivo. In this work, we demonstrate that even partially sialylated Qß conjugates retain the inhibitory effect despite reduced activity. These observations not only support the importance of trivalent binding events in preserving high affinity, as supported by computational modeling, but also allow us to construct heterobifunctional modalities. Capsids carrying two different sialic acid ligand-linker structures showed higher viral inhibition than their monofunctional counterparts. Furthermore, capsids carrying a fluorescent dye in addition to sialic acid ligands were used to track their interaction with cells. These findings support exploring broader applications as multivalent inhibitors in the future.

Atomistic insight into the essential binding event of ACE2-derived peptides to the SARS-CoV-2 spike protein.

The pathogenic agent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters into human cells through the interaction between the receptor binding domain (RBD) of its spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor. Efforts have been made towards finding antivirals that block this interaction, therefore preventing infection. Here, we determined the binding affinity of ACE2-derived peptides to the RBD of SARS-CoV-2 experimentally and performed MD simulations in order to understand key characteristics of their interaction. One of the peptides, p6, binds to the RBD of SARS-CoV-2 with nM affinity. Although the ACE2-derived peptides retain conformational flexibility when bound to SARS-CoV-2 RBD, we identified residues T27 and K353 as critical anchors mediating the interaction. New ACE2-derived peptides were developed based on the p6-RBD interface analysis and expecting the native conformation of the ACE2 to be maintained. Furthermore, we found a correlation between the helicity in trifluoroethanol and the binding affinity to RBD of the new peptides. Under the hypothesis that the conservation of peptide secondary structure is decisive to the binding affinity, we developed a cyclized version of p6 which had more helicity than p6 and approximately half of its KD value.

Inhibition of SARS-CoV-2 Replication by a Small Interfering RNA Targeting the Leader Sequence.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide and led to approximately 4 million deaths as of August 2021. Despite successful vaccine development, treatment options are limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed eight small interfering RNAs (siRNAs) targeting the highly conserved 5'-untranslated region (5'-UTR) of SARS-CoV-2. The most promising candidate identified in initial reporter assays, termed siCoV6, targets the leader sequence of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS-CoV-2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS-CoV-2 alpha variant and has perfect homology against all sequences of the delta variant that were analyzed by bioinformatic means. Interestingly, the siRNA was even highly active in virus replication assays with the SARS-CoV-1 family member. This work thus identified a very potent siRNA with a broad activity against various SARS-CoV viruses that represents a promising candidate for the development of new treatment options.

Polyglycerol-Based Mucus-Inspired Hydrogels.

The mucus layer is a hydrogel network that covers mucosal surfaces of the human body. Mucus has important protective properties that are related to its unique rheological properties, which are based on mucins being the main glycoprotein constituents. Mucin macromolecules entangle with one another and form a physical network that is instrumental for many important defense functions. Mucus derived from various human or animal sources is poorly defined and thus not suitable for many application purposes. Herein, a synthetic route is fabricated to afford a library of compositionally defined mucus-inspired hydrogels (MIHs). MIHs are synthesized by thiol oxidation to render disulfide bonds between the crosslinker ethoxylated trimethylolpropane tri(3-mercaptopropionate) (THIOCURE ETTMP 1300) and the linear precursors, dithiolated linear polyglycerol (LPG(SH)2 ) or polyethylene glycol (PEG(SH)2 ) of different molecular weights. The mixing ratio of linear polymers versus crosslinker and the length of the linear polymer are varied, thus delivering a library of compositionally defined mucin-inspired constructs. Their viscoelastic properties are determined by frequency sweeps at 25 and 37 °C and compared to the corresponding behavior of native human mucus. Here, MIHs composed of a 10:1 ratio of LPG(SH)2 and ETTMP 1300 are proved to be the best comparable to human airway mucus rheology.

Evaluation of Multivalent Sialylated Polyglycerols for Resistance Induction in and Broad Antiviral Activity against Influenza A Viruses.

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.

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.

Quantification of Multivalent Interactions between Sialic Acid and Influenza A Virus Spike Proteins by Single-Molecule Force Spectroscopy.

Multivalency is a key principle in reinforcing reversible molecular interactions through the formation of multiple bonds. The influenza A virus deploys this strategy to bind strongly to cell surface receptors. We performed single-molecule force spectroscopy (SMFS) to investigate the rupture force required to break individual and multiple bonds formed between synthetic sialic acid (SA) receptors and the two principal spike proteins of the influenza A virus (H3N2): hemagglutinin (H3) and neuraminidase (N2). Kinetic parameters such as the rupture length (χß) and dissociation rate (koff) are extracted using the model by Friddle, De Yoreo, and Noy. We found that a monovalent SA receptor binds to N2 with a significantly higher bond lifetime (270 ms) compared to that for H3 (36 ms). By extending the single-bond rupture analysis to a multibond system of n protein-receptor pairs, we provide an unprecedented quantification of the mechanistic features of multivalency between H3 and N2 with SA receptors and show that the stability of the multivalent connection increases with the number of bonds from tens to hundreds of milliseconds. Association rates (kon) are also provided, and an estimation of the dissociation constants (KD) between the SA receptors to both proteins indicate a 17-fold higher binding affinity for the SA-N2 bond with respect to that of SA-H3. An optimal designed multivalent SA receptor showed a higher binding stability to the H3 protein of the influenza A virus than to the monovalent SA receptor. Our study emphasizes the influence of the scaffold on the presentation of receptors during multivalent binding.

Adaptive Flexible Sialylated Nanogels as Highly Potent Influenza A Virus Inhibitors.

Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand-receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC50 values in low picomolar concentrations and also blocks the virus entry into MDCK-II cells.

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1-4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle7.

Force Spectroscopy Shows Dynamic Binding of Influenza Hemagglutinin and Neuraminidase to Sialic Acid.

The influenza A virus infects target cells through multivalent interactions of its major spike proteins, hemagglutinin (HA) and neuraminidase (NA), with the cellular receptor sialic acid (SA). HA is known to mediate the attachment of the virion to the cell, whereas NA enables the release of newly formed virions by cleaving SA from the cell. Because both proteins target the same receptor but have antagonistic functions, virus infection depends on a properly tuned balance of the kinetics of HA and NA activities for viral entry to and release from the host cell. Here, dynamic single-molecule force spectroscopy, based on scanning force microscopy, was employed to determine these bond-specific kinetics, characterized by the off rate koff, rupture length xß and on rate kon, as well as the related free-energy barrier ΔG and the dissociation constant KD. Measurements were conducted using surface-immobilized HA and NA of the influenza A virus strain A/California/04/2009 and a novel, to our knowledge, synthetic SA-displaying receptor for functionalization of the force probe. Single-molecule force spectroscopy at force loading rates between 100 and 50,000 pN/s revealed most probable rupture forces of the protein-SA bond in the range of 10-100 pN. Using an extension of the widely applied Bell-Evans formalism by Friddle, De Yoreo, and co-workers, it is shown that HA features a smaller xß, a larger koff and a smaller ΔG than NA. Measurements of the binding probability at increasing contact time between the scanning force microscopy force probe and the surface allow an estimation of KD, which is found to be three times as large for HA than for NA. This suggests a stronger interaction for NA-SA than for HA-SA. The biological implications in regard to virus binding to the host cell and the release of new virions from the host cell are discussed.