Inhibition of Human Metapneumovirus Binding to Heparan Sulfate Blocks Infection in Human Lung Cells and Airway Tissues

Human metapneumovirus (HMPV), a recently discovered paramyxovirus, infects nearly 100% of the world population and causes severe respiratory disease in infants, the elderly, and immunocompromised patients. We previously showed that HMPV binds heparan sulfate proteoglycans (HSPGs) and that HMPV binding requires only the viral fusion (F) protein. To characterize the features of this interaction critical for HMPV binding and the role of this interaction in infection in relevant models, we utilized sulfated polysaccharides, heparan sulfate mimetics, and occluding compounds. Iota-carrageenan demonstrated potent anti-HMPV activity by inhibiting binding to lung cells mediated by the F protein. Furthermore, analysis of a minilibrary of variably sulfated derivatives of Escherichia coli K5 polysaccharide mimicking the HS structure revealed that the highly O-sulfated K5 polysaccharides inhibited HMPV infection, identifying a potential feature of HS critical for HMPV binding. The peptide dendrimer SB105-A10, which binds HS, reduced binding and infection in an F-dependent manner, suggesting that occlusion of HS at the target cell surface is sufficient to prevent infection. HMPV infection was also inhibited by these compounds during apical infection of polarized airway tissues, suggesting that these interactions take place during HMPV infection in a physiologically relevant model. These results reveal key features of the interaction between HMPV and HS, supporting the hypothesis that apical HS in the airway serves as a binding factor during infection, and HS modulating compounds may serve as a platform for potential antiviral development

Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide Derivatives *

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds

Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist

Angiogenesis plays a key role in various physiological and pathological conditions, including inflammation and tumor growth. The bone morphogenetic protein (BMP) antagonist gremlin has been identified as a novel pro-angiogenic factor. Gremlin promotes neovascular responses via a BMP-independent activation of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2). BMP antagonists may act as covalent or non-covalent homodimers or in a monomeric form, while VEGFRs ligands are usually dimeric. However, the oligomeric state of gremlin and its role in modulating the biological activity of the protein remain to be elucidated.Here we show that gremlin is expressed in vitro and in vivo both as a monomer and as a covalently linked homodimer. Mutagenesis of amino acid residue Cys141 prevents gremlin dimerization leading to the formation of gremlinC141A monomers. GremlinC141A monomer retains a BMP antagonist activity similar to the wild-type dimer, but is devoid of a significant angiogenic capacity. Notably, we found that gremlinC141A mutant engages VEGFR2 in a non-productive manner, thus acting as receptor antagonist. Accordingly, both gremlinC141A and wild-type monomers inhibit angiogenesis driven by dimeric gremlin or VEGF-A165. Moreover, by acting as a VEGFR2 antagonist, gremlinC141A inhibits the angiogenic and tumorigenic potential of murine breast and prostate cancer cells in vivo.In conclusion, our data show that gremlin exists in multiple forms endowed with specific bioactivities and provide new insights into the molecular bases of gremlin dimerization. Furthermore, we propose gremlin monomer as a new inhibitor of VEGFR2 signalling during tumor growth

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p < 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p < 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p < 0.0001) or urgent (20.4% vs. 38.5%; p < 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p < 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

: Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against SARS-CoV-2, the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively-charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations also showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Taken together, our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan co-receptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy

Sulfated Escherichia coli K5 polysaccharide derivatives inhibit dengue virus infection of human microvascular endothelial cells by interacting with the viral envelope protein E domain III

Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface. However, heparan sulfate proteoglycans (HSPGs) were highly expressed on these cells and pre-treatment of HMEC-1 cells with heparinase II or with glycosaminoglycans reduced DENV infectivity up to 90%, suggesting that DENV uses HSPGs as attachment receptor on microvascular endothelial cells. Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range. The highly sulfated K5-OS(H) and K5-N,OS(H) inhibited virus attachment and subsequent entry into microvascular endothelial cells by interacting with the viral envelope (E) protein, as shown by surface plasmon resonance (SPR) analysis using the receptor-binding domain III of the E protein.status: publishe

Sulfated Derivatives of Escherichia coli K5 Capsular Polysaccharide Are Potent Inhibitors of Human Cytomegalovirus▿

To date, there are few drugs licensed for the treatment of human cytomegalovirus (HCMV) infections, most of which target the viral DNA polymerase and suffer from many drawbacks. Thus, there is still a strong need for new anti-HCMV compounds with novel mechanisms of action. In this study, we investigated the anti-HCMV activity of chemically sulfated derivatives of Escherichia coli K5 capsular polysaccharide. These compounds are structurally related to cellular heparan sulfate and have been previously shown to be effective against some enveloped and nonenveloped viruses. We demonstrated that two derivatives, i.e., K5-N,OS(H) and K5-N,OS(L), are able to prevent cell infection by different strains of HCMV at concentrations in the nanomolar range while having no significant cytotoxicity. Studies performed to elucidate the mechanism of action of their anti-HCMV activity revealed that these compounds do not interact with either the host cell or the viral particle but need a virus-cell interaction to exert antiviral effects. Furthermore, these K5 derivatives were able to inhibit the attachment step of HCMV infection, as well as the viral cell-to-cell spread. Since the mode of inhibition of these compounds appears to differ from that of the available anti-HCMV drugs, sulfated K5 derivatives could represent the basis for the development of a novel class of potent anti-HCMV compounds. Interestingly, our studies highlight that small variations of the K5 derivatives structure can modulate the selectivity and potency of their activities against different viruses, including viruses belonging to the same family

Sulfated K5 Escherichia coli Polysaccharide Derivatives as Wide-Range Inhibitors of Genital Types of Human Papillomavirus▿

Genital human papillomaviruses (HPV) represent the most common sexually transmitted agents and are classified into low or high risk by their propensity to cause genital warts or cervical cancer, respectively. Topical microbicides against HPV may be a useful adjunct to the newly licensed HPV vaccine. A main objective in the development of novel microbicides is to block HPV entry into epithelial cells through cell surface heparan sulfate proteoglycans. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was integrated with innovative biochemical and biological assays to prepare a collection of sulfated K5 derivatives with a backbone structure resembling the heparin/heparan biosynthetic precursor and to test them for their anti-HPV activity. Surface plasmon resonance assays revealed that O-sulfated K5 with a high degree of sulfation [K5-OS(H)] and N,O-sulfated K5 with a high [K5-N,OS(H)] or low [K5-N,OS(L)] sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low levels of sulfation, prevented the interaction between HPV-16 pseudovirions and immobilized heparin. In cell-based assays, K5-OS(H), K5-N,OS(H), and K5-N,OS(L) inhibited HPV-16, HPV-18, and HPV-6 pseudovirion infection. Their 50% inhibitory concentration was between 0.1 and 0.9 μg/ml, without evidence of cytotoxicity. These findings provide insights into the design of novel, safe, and broad-spectrum microbicides against genital HPV infections