Pasqua Oreste

Interaction of HIV-1 Tat Protein with Heparin ROLE OF THE BACKBONE STRUCTURE, SULFATION, AND SIZE

Human immunodeficiency virus type 1 (HIV-1) Tat protein is released from infected cells. Extracellular Tat enters the cell where it stimulates the transcriptional activity of HIV-long terminal repeat (LTR) and of endogenous genes. Heparin modulates the angiogenic (Albini, A., Benelli, R., Presta, M., Rusnati, M., Ziche, M., Rubartelli, A., Paglialunga, G., Bussolino, F., and Noonan, D. (1996) Oncogene 12, 289-297) and transcriptional (Mann, D. A., and Frankel, A. D. (1991) EMBO J. 10, 1733-1739) activity of extracellular Tat. Here we demonstrate that heparin binds specifically to recombinant HIV-1 Tat produced as glutathione S-transferase (GST) fusion protein and immobilized on glutathione-agarose beads. Heparin and heparan sulfate (HS), but not dermatan sulfate, chondroitin sulfates A and C, hyaluronic acid, and K5 polysaccharide, competed with 3H-labeled heparin for binding to immobilized GST-Tat and inhibited HIV-LTR transactivation induced by extracellular GST-Tat. Selective 2-O-, 6-O-, total-O-desulfation, or N-desulfation/N-acetylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-O-desulfated heparins also showed a reduced capacity to inhibit the transactivating activity of GST-Tat. Very low molecular weight heparins showed a significant decrease in their capacity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kDa heparin was affinity chromatographed on immobilized GST-Tat to isolate binding and non-binding subfractions, the Tat-bound fraction was ≥1,000 times more potent than the unbound fraction in inhibiting the transactivating activity of GST-Tat. The results demonstrate that Tat interacts in a size-dependent manner with heparin/HS and that high affinity Tat-heparin interaction requires at least some 2-O-, 6-O-, and N-positions to be sulfated. The Tat binding activity of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating the possibility to design specific heparin/HS-like structures with Tat-antagonist activity.

Multiple Interactions of HIV-I Tat Protein with Size-defined Heparin Oligosaccharides

Tat protein, a transactivating factor of the human immunodeficiency virus type I, acts also as an extracellular molecule. Heparin affects the bioavailability and biological activity of extracellular Tat (Rusnati, M., Coltrini, D., Oreste, P., Zoppetti, G., Albini, A., Noonan, D., D’Adda di Fagagna, F., Giacca, M., and Presta, M. (1997) J. Biol. Chem. 272, 11313–11320). Here, a series of homogeneously sized, 3H-labeled heparin fragments were evaluated for their capacity to bind to free glutathioneS-transferase (GST)-Tat protein and to immobilized GST-Tat. Hexasaccharides represent the minimum sized heparin fragments able to interact with GST-Tat at physiological ionic strength. Also, the affinity of binding increases with increasing the molecular size of the oligosaccharides, with large fragments (≥18 saccharides) approaching the affinity of full-size heparin. 6-Mer heparin binds GST-Tat with a dissociation constant (K d ) equal to 0.7 ± 0.4 μm and a molar oligosaccharide:GST-Tat ratio of about 1:1. Interaction of GST-Tat with 22-mer or full-size heparin is consistent instead with two-component binding. At subsaturating concentrations, a single molecule of heparin interacts with 4–6 molecules of GST-Tat with high affinity (K d values in the nanomolar range of concentration); at saturating concentrations, heparin binds GST-Tat with lower affinity (K d values in the micromolar range of concentration) and a molar oligosaccharide:GST-Tat ratio of about 1:1. In agreement with the binding data, a positive correlation exists between the size of heparin oligosaccharides and their capacity to inhibit cell internalization, long terminal repeat-transactivating activity of extracellular Tat in HL3T1 cells, and its mitogenic activity in murine adenocarcinoma T53 Tat-less cells. The data demonstrate that the modality of heparin-Tat interaction is strongly affected by the size of the saccharide chain. The possibility of establishing multiple interactions increases the affinity of large heparin fragments for Tat protein and the capacity of the glycosaminoglycan to modulate the biological activity of extracellular Tat.

Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5☆

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

Sulfated K5 Escherichia coli polysaccharide derivatives: A novel class of candidate antiviral microbicides.

Antiviral microbicides, topical agents that prevent sexually transmitted infections, mainly work by blocking the interaction between viral proteins and cell surface components. In many instances, virus-cell interaction is mediated by cell surface heparan sulfate proteoglycans (HSPGs). HSPGs are exploited as attachment receptors by three sexually transmitted viruses: Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) and Human Papilloma Virus (HPV). Since these viruses can either infect or co-infect humans, virus/HSPGs interaction is a preferential target for the development of wide-spectrum antiviral microbicides. Several polyanionic compounds prevent HIV, HSV and HPV infections in cell culture models by acting as heparan sulfate (HS)-antagonists. However, three promising polyanionic compounds recently failed to pass phase III clinical trials designed to establish their efficacy in preventing HIV acquisition. In this scenario, new polyanionic compounds must be added to the pipeline of candidate microbicides and their development as effective drugs reconsidered. The capsular K5 polysaccharide from Escherichia coli has the same structure as the heparin/HS biosynthetic precursor. Chemical and enzymatic modifications have led to the synthesis of K5 derivatives with different degrees of sulfation and charge distribution and devoid of anticoagulant activity and cell toxicity. Recently attracting attention as candidate microbicides, they potently inhibit a broad spectrum of HIV-1 strains and genital types of HPV and HSV-1 and 2 in vitro. With a focus on the K5 derivatives, this article reviews the literature on polyanions as antiviral microbicides and discusses the possible therapeutic implications of this novel class of compounds.

Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists

The HIV‐1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat‐antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O‐sulfated K5 with high sulfation degree (K5‐OS(H)) and N,O‐sulfated K5 with 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 sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV‐LTR‐transactivation. Also, K5‐OS(H) and K5‐N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor‐2 on endothelial cell (EC) surface. Finally, K5‐OS(H) inhibits αvβ3 integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5‐OS(H) and K5‐N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti‐AIDS therapies.

Broad spectrum inhibition of Hiv-1 infection by sulfated K5 escherichia coli polysaccharide derivatives

Objective: HIV-1 entry into CD4 cells represents a main target for developing novel antiretroviral agents and microbicides. Design: Sulfated derivatives of the Escherichia coli K5 polysaccharide have a backbone structure resembling the heparin precursor, but are devoid of the anticoagulant activity. The derivatives were chemically sulfated in the N position after N-deacetylation, in the O position, or in both sites. Methods: HIV replication in human T cell blasts, monocyte-derived macrophages and cell lines was studied in the presence of sulfated K5 derivatives. Results: O-sulfated [K5-OS(H)] and N,O-sulfated [K5-N,OS(H)] K5 derivatives with high degree of sulfation inhibited the replication of an HIV strain using CXCR4 as entry co-receptor (X4 virus) in both cell lines and T-cell blasts. K5 derivatives also strongly inhibited the multiplication of CCR5-dependent HIV (R5 virus) in cell lines, T-cell blasts and primary monocyte-derived macrophages. Their 50% inhibitory concentration was between 0.07 and 0.46 μM, without evidence of cytotoxicity even at the maximal concentration tested (9 μM). In addition, both K5-N,OS(H) and K5-OS(H) potently inhibited the replication of several primary HIV-1 isolates in T-cell blasts, with K5-N,OS(H) being more active than K5-OS(H) on dual tropic R5X4 strains. K5 derivatives inhibited the early steps of virion attachment and/or entry. Conclusions: Because K5 derivatives are unlikely to penetrate into cells they may represent potential topical microbicides for the prevention of sexual HIV-1 transmission.

Heparan Sulfate Proteoglycans Mediate the Angiogenic Activity of the Vascular Endothelial Growth Factor Receptor-2 Agonist Gremlin

Objective—Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs. Methods and Results—Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N-, 2-O, and 6-O-sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase1/2 and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O-sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo. Conclusion—HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist.

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

ABSTRACT 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.

Antiangiogenic Activity of Semisynthetic Biotechnological Heparins. Low-Molecular-Weight-Sulfated Escherichia coli K5 Polysaccharide Derivatives as Fibroblast Growth Factor Antagonists

Objective— Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. Methods and Results— Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and αvβ3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/αvβ3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. Conclusions— LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/αvβ3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.

Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin IDENTIFICATION OF THE HEPARIN-BINDING MOTIF OF p17 AS A TARGET FOR THE DEVELOPMENT OF MULTITARGET ANTAGONISTS

Background: HIV-1 p17 binds heparin and heparan sulfate proteoglycans of the cell surface. Results: Heparin/p17 interaction occurs through heparin sulfate groups and a linear basic motif of p17 N terminus, also involved in p17/CXCR1 interaction. Conclusion: Targeting the basic motif inhibits p17-receptors interaction and consequent biological activities. Significance: Heparin-like molecules represent template for the development of new treatments of p17-dependent/AIDS-associated pathologies. Once released by HIV+ cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (Kd = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.