Giorgio Zoppetti

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.

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.

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.

ATR/Raman and Fractal Characterization of HPBCD/Progesterone Complex Solid Particles

PurposeCharacterization of hydroxypropyl-β-cyclodextrin/progesterone (HPBCD/P) complex solid particles obtained from an aqueous solution, by three different technological processes, with the aim of preparing ready-to-dissolve powders for injectable as well as solid oral formulations in progestinic therapy.MethodsHPBCD/P complex in the 2:1 molar ratio was prepared in aqueous solution and obtained as dry solid particles by freeze-drying, by spray-drying and by fluid-bed evaporation of the solvent. The particles were characterized by μ-FT-IR, μ-Raman and X-ray spectroscopy, by thermal analysis (differential scanning calorimetry-DSC and thermogravimetry-TGA), by Karl Fischer (KF) titration, by image and fractal analysis and by BET specific surface area analysis. The structure of the complex was also defined by comparison of FT-IR and Raman spectra of progesterone with those of pregnenolone and testosterone, structurally related. Dissolution tests were also performed.ResultsPowders of the complex obtained by the three different methods are different in size and shape. Particles obtained by freeze-drying are flat and angular, irregularly shaped without any relation to known geometrical solid figures. Particles obtained by spray-drying are spherically shaped and display a very small size (5-10 μm), with evident deformations and depression of the external surface, due to the rapid evaporation of the solvent. Particles obtained by fluid bed technique have intermediate sizes, display a tri-dimensional structure and irregular surface, with small and rounded protuberances. Fractal dimension of the particle contour was found close to one unit for the microspheres obtained by spray-drying. FT-IR and Raman spectra confirm the occurrence of the complexation by the shift of representative bands of the two carbonyl groups in positions 3 and 20 of the complexed progesterone. X-ray diffractograms indicate the amorphous nature of all the types of particles, also suggested by the absence of any melting peak of the drug in DSC thermograms. The samples contain different amounts of humidity: particles obtained by fluid-bed method demonstrated non-porous in BET analysis. Dissolution of different types of particles is complete after 3 min and only negligible differences could be appreciated among the three powders.Conclusions– μ-FT-IR, μ-Raman and X-ray spectroscopy, and the dissolution test did not reveal defined differences among the three different types of particles, confirming occurrence of the complex in the solid state. The spherical shape, the very small size and the low value of the contour fractal dimension allows better technological performance of the particles obtained by spray-drying: this drying process appears the most promising one to prepare dry particles of the HPBCD/P complex, in view of its formulation in the fast preparation of extemporaneous injectable solutions and solid oral formulations intended for sublingual delivery.

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.

Further Purification of Heparin Reduces Its Bleeding Effects in the Mesenteric Vessels of Rats

It has been suggested that unusually high hemorrhagic effects of some commercial unfractionated heparins or some low molecular weight heparins, evaluated on a template bleeding model in the rat, is partly due to heparin contaminants, especially EDTA.’ The aim of this study was to investigate whether removal of EDTA contaminants from a commercial unfractionated sodium heparin (H) led to a reduction of its hemorrhagic effects evaluated on the mesenteric microvascular system in rats. For this purpose, H (specific activity 176 IU/mg) containing 10,000 ppm of EDTA as determined by HPLC analysis using a reverse-phase column (Lichrosorb RP-8) was purified with an ethanol extraction procedure. H was submitted to repeated treatments with ethanol-water (75-25 vol.) under controlled conditions of temperature and stirring. The obtained solid was dried with absolute ethanol, yielding the purified heparin (PH) with a specific activity of 180 IUlmg. The ethanol-water solution was recovered by vacuum drying, yielding the extractable fraction (E). The ’H-NMR spectrum (300 mHz) of PH was the same as the original preparation, however without any EDTA signals and the HPLC profile indicated an EDTA content lower than 20 ppm. E consisted mainly of EDTA (sodium and calcium forms). EDTA was the major component (30%) of extracted contaminant and only a very small amount of the original H (0.5%) was present. The bleeding effects of H, PH, E, EDTA-Ca, recombined mixture of PH and E as well as “reconstructed” mixture of PH and EDTA were evaluated on mesenteric vessels of rats.* Male Sprague-Dawley rats weighing 350-400 g were anesthetized with urethan 1 g/kg i.p. on the exteriorized mesenteric vascular bed, superfused with Tyrode at 37°C. Six venules or six arterioles of selected diameter (250-300 pm) were sequentially resected every 15 min. The total hemorrhagic time (THT) and blood loss (BL) from each distal venular or proximal arteriolar segment was measured. Whole PHT (see text of FIG. 11, THT and BL evaluations, based on the overall data from the five post-treatment resections, were carried out after a single drug administration (0.75 mg/kg) via i.v. route. Control rats received an equivalent of saline.

The Interaction of Basic Fibroblast Growth Factor (bFGF) With Heparan Sulfate Proteoglycans

Basic fibroblast growth factor (bFGF) is an angiogenic molecule involved in several physiological and pathological processes as wound repair, embryonic development and tumor growth. In particular, bFGF promotes Chemotaxis, mitogenesis and production of proteases in endothelial cells in culture. On these cells bFGF acts by binding to specific tyrosine kinase receptors and to cell-associated heparan sulfate proteoglycans (HSPGs). The physiological significance of the interaction of bFGF with cell-associated and with soluble HSPGs is manyfold. HSPGS protect bFGF from inactivation in the extracellular environment and modulate the bioavailability of the growth factor. At the cell surface, soluble and cell-associated HSPGs seem to play contrasting roles in modulating the dimerization of bFGF and its interaction with tyrosine kinase receptors. Finally, HSPGs affect the internalization and the intracellular fate of bFGF, suggesting that bFGF-HSPGs complexes are involved in the intracellular delivery of bFGF. It derives that the bioavailability and the biological activity of bFGF strictly depend on the glycosaminoglycan milieu of the extracellular environment, indicating the possibility to modulate the biological activity of bFGF in vivo by using exogenous GAGs. The capacity of GAGs to bind to and to influence the biological activity of bFGF depends on size, degree of sulfation and disaccharide composition and heparin-related molecules with different structures are able to differently affect the biological activities of bFGF. In this chapter we will discuss the state of the knowledge of the physiological significance and of the biochemical bases of the interaction of bFGF with HSPGs and exogenous GAGs, in the view of a possible therapeutic use of heparin-related oligosaccharides as bFGF agonists or antagonists.