Chemically modified heparins as inhibitors of heparan sulfate specific endo-beta-glucuronidase (heparanase) of metastatic melanoma cells

To determine the significance of the heparan sulfate (HS) degradative endo-beta-glucuronidase (heparanase) in tumor invasion and metastasis and to develop possible antimetastatic agents, we synthesized specific inhibitors of this enzyme. We previously found that heparanase activity correlates with the lung colonization abilities of murine B16 melanoma cells and is inhibited by heparin [Nakajima, M., Irimura, T., Di Ferrante, N., & Nicolson, G. L. (1984) J. Biol. Chem. 259, 2283-2290]. In this study, heparin was chemically modified in order to determine which portions of its structure are responsible for heparanase inhibitory activity and to obtain heparanase inhibitors that have minimal additional biological effects, such as anticoagulation. N-Sulfate groups and O-sulfate in heparin were removed separately, and the resultant free amino groups were acetylated or resulfated. Heparin was also reduced at the carboxyl groups of uronic acid. The heparanase inhibitory activities of these heparin derivatives were examined by high-speed gel-permeation chromatography and by the use of radioactive HS immobilized on agarose beads. The results indicated that although N-sulfate and O-sulfate groups on glucosamine residues, and carboxyl groups on uronic acid residues, are important for heparanase inhibition, they are not essential for full activity. When highly metastatic B16-BL6 melanoma cells were incubated with N-acetylated N-desulfated heparin, N-resulfated N- and O-desulfated heparin, or carboxyl-reduced heparin and injected intravenously to syngenic C57BL/6 mice, significant reductions in the numbers of experimental melanoma lung metastases occurred.     

Heparanases and tumor metastasis

The successful penetration of endothelial basement membranes is an important process in the formation of hematogenous tumor metastases. Heparan sulfate (HS) proteoglycan is a major constituent of endothelial basement membranes, and we have found that HS-degradative activities of metastatic B16 melanoma sublines correlate with their lung-colonizing potentials. The melanoma HS-degrading enzyme is a unique endo-beta-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites and is detectable in a variety of cultured human malignant melanomas. The treatment of B16 melanoma cells with heparanase inhibitors that have few other biological activities, such as N-acetylated N-desulfated heparin, results in significant reductions in the numbers of experimental lung metastases in syngeneic mice, indicating that heparanase plays an important role in melanoma metastasis. HS-degrading endoglycosidases are not tumor-specific and have been found in several normal tissues and cells. There are at least three types of endo-beta-D-glucuronidases based on their substrate specificities. Melanoma heparanase, an Mr approximately 96,000 enzyme with specificity for beta-D-glucuronosyl-N-acetylglucosaminyl linkages in HS, is different from platelet and mastocytoma endoglucuronidases. Elevated levels of heparanase have been detected in sera from metastatic tumor-bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and extent of metastases. The results suggest that heparanase is potentially a useful marker for tumor metastasis.     

Suramin interrupts androgen-inducible autocrine loop involving heparin binding growth factor in mouse mammary cancer (Shionogi carcinoma 115) cells

The androgen-dependent clonal cell line SC-3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)-autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC-3 cells, FGF receptor expression is upregulated by the SC-3-derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effect of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC-3 cells were studied. Suramin inhibited androgen-dependent growth of SC-3 cells in a concentration-dependent fashion: ～50% inhibition was observed at 25 μM. [³H]Thymidine incorporation into the cells stimulated with partially purified SC-3-derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF-induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC-3 cells with suramin decreased cell surface ¹²⁵I-bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 μM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor-1 mRNA to a similar extent, whereas it appeared not to affect the levels of β-actin mRNA. Moreover, suramin completely blocked androgen- or bFGF-induced accumulation of FGF receptor-1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC-3 cells through inhibition of an androgen-inducible autocrine loop involving SC-3-derived growth factor and FGF receptor. © 1993 Wiley-Liss, Inc.     

Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

Suramin sensitizing cells to ionizing radiation by inactivating DNA-dependent protein kinase

Here we report that suramin sensitizes LM217, MDA-MB-468, T98G and A431 cells to ionizing radiation. Suramin sensitized cells to X radiation in a dose-dependent fashion, and longer exposure to suramin before X irradiation resulted in more efficient sensitization. The dose-modifying factors calculated from the survival curves were 1.18 in LM217 cells and 1.37 in MDA-MB-468 cells. Suramin did not sensitize Scid cells that had no DNA-dependent protein kinase activity. Suramin inhibited DNA-dependent protein kinase activity in vitro and in vivo. The concentration of suramin resulting in 50% inhibition in vitro was 1.7 microM in LM217 cells and 2.4 microM in MDA-MB-468 cells. Exposure of LM217 and MDA-MB-468 cells to suramin did not affect the level of Ku70 (G22P1) or Ku80 (XRCC5), but it increased the level of DNA-PKcs(PRKDC). Suramin did not sensitize LM217 or MDA-MB-468 cells to UV radiation. Suramin's effects were not caused by accumulation of cells in a specific phase of the cell cycle. These results suggest that suramin sensitizes cells to ionizing radiation by inhibiting DNA-dependent protein kinase activity.     

Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A2 analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin-Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity.     

Suramin and suramin analogues inhibit merozoite surface protein-1 secondary processing and erythrocyte invasion by the malaria parasite Plasmodium falciparum

Malarial merozoites invade erythrocytes; and as an essential step in this invasion process, the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP142) is further cleaved to a 33-kDa N-terminal polypeptide (MSP133) and an 19-kDa C-terminal fragment (MSP119) in a secondary processing step. Suramin was shown to inhibit both merozoite invasion and MSP142 proteolytic cleavage. This polysulfonated naphthylurea bound directly to recombinant P. falciparum MSP142 (Kd = 0.2 microM) and to Plasmodium vivax MSP142 (Kd = 0.3 microM) as measured by fluorescence enhancement in the presence of the protein and by isothermal titration calorimetry. Suramin bound only slightly less tightly to the P. vivax MSP133 (Kd = 1.5 microM) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd approximately 15 mM) (NMR measurements). Several residues in MSP119 were implicated in the interaction with suramin using NMR measurements. A series of symmetrical suramin analogues that differ in the number of aromatic rings and substitution patterns of the terminal naphthylamine groups was examined in invasion and processing assays. Two classes of analogue with either two or four bridging rings were found to be active in both assays, whereas two other classes without bridging rings were inactive. We propose that suramin and related compounds inhibit erythrocyte invasion by binding to MSP1 and by preventing its cleavage by the secondary processing protease. The results indicate that enzymatic events during invasion are suitable targets for drug development and validate the novel concept of an inhibitor binding to a macromolecular substrate to prevent its proteolysis by a protease.     

Cell surface localization of heparanase on macrophages regulates degradation of extracellular matrix heparan sulfate

Extravasation of peripheral blood monocytes through vascular basement membranes requires degradation of extracellular matrix components including heparan sulfate proteoglycans (HSPGs). Heparanase, the heparan sulfate-specific endo-beta-glucuronidase, has previously been shown to be a key enzyme in melanoma invasion, yet its involvement in monocyte extravasation has not been elucidated. We examined a potential regulatory mechanism of heparanase in HSPG degradation and transmigration through basement membranes in leukocyte trafficking using human promonocytic leukemia U937 and THP-1 cells. PMA-treated cells were shown to degrade 35S-sulfated HSPG in endothelial extracellular matrix into fragments of an approximate molecular mass of 5 kDa. This was not found with untreated cells. The gene expression levels of heparanase or the enzyme activity of the amount of cell lysates were no different between untreated and treated cells. Immunocytochemical staining with anti-heparanase mAb revealed pericellular distribution of heparanase in PMA-treated cells but not in untreated cells. Cell surface heparanase capped into a restricted area on PMA-treated cells when they were allowed to adhere. Addition of a chemoattractant fMLP induced polarization of the PMA-treated cells and heparanase redistribution at the leading edge of migration. Therefore a major regulatory process of heparanase activity in the cells seems to be surface expression and capping of the enzyme. Addition of the anti-heparanase Ab significantly inhibited enzymatic activity and transmigration of the PMA-treated cells, suggesting that the cell surface redistribution of heparanase is involved in monocyte extravasation through basement membranes.     

Effect of suramin on the human pathogen Candida albicans: implications on the fungal development and virulence

Candida albicans is an opportunistic pathogen that is of growing medical importance because it causes superficial, mucosal and systemic infections in susceptible individuals. Here, the effect of suramin, a polysulfonated naphthylurea derivative, on C. albicans development and virulence was evaluated. Firstly, it was demonstrated that suramin (500 microM) arrested its growth, showing a fungicidal action dependent on cell number. Suramin treatment caused profound changes in the yeast ultrastructure as shown by transmission electron microscopy. The more important changes were the enlargement of the fungi cytoplasmic vacuoles, the appearance of yeasts with an empty cytoplasm resembling ghost cells and a reduction in cell wall thickness. Suramin also blocked the transformation of yeast cells to the germ-tube and the interaction between C. albicans and epithelial cells. In order to ascertain that the action of suramin on C. albicans growth is a general feature instead of being strain-specific, the effects of suramin on 14 oral clinical strains isolated from healthy children and HIV-positive infants were analyzed. Interestingly, the strains of C. albicans isolated from HIV-positive patients were more resistant to suramin than strains isolated from healthy patients. Altogether, the results produced here show that suramin interfered with essential fungal processes, such as growth, differentiation and interaction with host cells.     

Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice

Suramin is a polysulfonated derivative of urea and has been widely used both to treat infections and as a chemotherapeutic drug. Suramin has been shown to inhibit growth factor signaling pathways; however, its effect on apoptosis is unknown. Here we show that suramin inhibits apoptosis induced through death receptors in hepatoma and lymphoma cells. It also inhibits the proapoptotic effect of chemotherapeutic drugs. The antiapoptotic mechanism is specific to cell type and is caused by reduced activation, but not altered composition, of the death-inducing signaling complex (DISC), and by inhibition of the initiator caspases 8, 9 and 10. Suramin also shows similar effects in in vivo models: apoptotic liver damage induced by CD95 stimulation and endotoxic shock mediated by tumor-necrosis factor (TNF) are inhibited in mice, but necrotic liver damage is not inhibited in a rat model of liver transplantation. Thus, the antiapoptotic property of suramin in the liver may be therapeutically exploited.     

Suramin interaction with human alpha-thrombin: inhibitory effects and binding studies

Suramin is a hexasulfonated naphthylurea commonly used as antitrypanosomial drug and more recently for the treatment of malignant tumors. Here we show that suramin binds to human alpha-thrombin inhibiting both the hydrolysis of the synthetic substrate S-2238 (IC50 = 40 microM), and the thrombin-induced fibrinogen clotting (IC50 = 20 microM). The latter is completely reversed by albumin (30 mg mL(-1)) suggesting that, at therapeutic concentrations, suramin is unable to affect alpha-thrombin activity in the plasma. Kinetic analysis showed that suramin acts as a non-competitive inhibitor decreasing Vmax without changing the Km for S-2238 hydrolysis. Calorimetric studies revealed two distinct binding sites for suramin in alpha-thrombin. In addition, circular dichroism studies showed that suramin causes significant changes in alpha-thrombin tertiary structure, without affecting the secondary structure content. Interaction with alpha-thrombin resulted in an increased fluorescence emission of the drug. Complex formation was strongly affected by high ionic strength suggesting the involvement of electrostatic interactions. Altogether our data suggest that part of the biological activities of suramin might be related to alpha-thrombin inhibition at extra-vascular sites.     

Suramin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of NF-kappaB activity

AIM: Suramin is a symmetrical polysulfonated naphthylamine derivative of urea. There have been few studies on the effect of suramin on cytokines. We examined the effects of suramin on production of inflammatory cytokines. METHODS: We made an acute liver injury model treated with d-galactosamine (GalN) and lipopolysaccharide (LPS). Plasma AST, ALT, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels were measured. We compared with survival rate, histological found and NF-kappaB activity between with and without treatment of suramin. In macrophage like cell line, TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity was measured. RESULTS: The lethality of mice administered suramin with GalN/LPS was significantly decreased compared with that in mice without suramin. Changes of hepatic necrosis and apoptosis were slight in suramin-treated mice. Serum AST, ALT, TNF-alpha, IL-6 levels and NF-kappaB activity in the liver were significantly lower in mice administered suramin. In an in vitro model, suramin preincubation inhibited TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity. CONCLUSIONS: Suramin inhibits TNF-alpha and IL-6 production through the suppression of NF-kappaB activity from macrophages and shows therapeutic effects on acute liver damage.     

Suramin affects coupling of rhodopsin to transducin.

Suramin, a polysulfonated naphthylurea, is under investigation for the treatment of several cancers. It interferes with signal transduction through G(s), G(i), and G(o), but structural and kinetic aspects of the molecular mechanism are not well understood. Here, we have investigated the influence of suramin on coupling of bovine rhodopsin to G(t), where G-protein activation and receptor structure can be monitored by spectroscopic in vitro assays. G(t) fluorescence changes in response to rhodopsin-catalyzed nucleotide exchange reveal that suramin inhibits G(t) activation by slowing down the rate of complex formation between metarhodopsin-II and G(t). The metarhodopsin-I/-II photoproduct equilibrium, GTPase activity, and nucleotide uptake by G(t) are unaffected. Attenuated total reflection Fourier transform infrared spectroscopy shows that the structure of rhodopsin, metarhodopsin-II, and the metarhodopsin-II G(t) complex is also not altered. Instead, suramin dissociates G(t) from disk membranes in the dark, whereas metarhodopsin-II G(t) complexes are stable. F?rster resonance energy transfer suggests a suramin-binding site near Trp(207) on the G(t alpha) subunit (K(d) approximately 0.5 microM). The kinetic analyses and the structural data are consistent with a specific perturbation by suramin of the membrane attachment site on G(t alpha). Disruption of membrane anchoring may contribute to some of the effects of suramin exerted on other G-proteins.     

Decreased ganglioside neuraminidase activity in fibroblasts from mucopolysaccharidosis patients. Inhibition of the activity in vitro by sulfated glycosaminoglycans and other compounds

The neuraminidase activities towards the ganglioside substrates GD1a, GD3 and GM3 were found to be markedly diminished in homogenates of fibroblasts cultured from patients with various genetic mucopolysaccharidoses. Mixing normal and patients' fibroblast homogenates revealed this effect to be due to the presence of diffusible inhibitors. The neuraminidase acting on the trisaccharide sialyllactose, on the other hand, showed normal activity in all the cell lines tested. Experiments in vitro revealed the sulfated glycosaminoglycans chondroitin 4-sulfate and heparin, the polysaccharide dextran sulfate, and the trypanocidal drug suramin to be strongly inhibitory on the ganglioside GD1a neuraminidase activity of normal fibroblast homogenates. Regarding chondroitin 4-sulfate, this inhibition was of the non-competitive type. A disulfated tetrasaccharide prepared from chondroitin 4-sulfate, on the other hand, was not at all inhibitory. These and additional findings led us to propose a model for the interaction between enzyme and inhibitor, involving a 'clamping' mechanism by the polysulfated compounds. We conclude that the decreased ganglioside neuraminidase activities of mucopolysaccharidosis fibroblasts are due to an inhibition by the accumulated sulfated glycosaminoglycans and that such inhibition is responsible for the storage of certain gangliosides in the tissues of the patients.     

Heparin oligosaccharide sequence and size essential for inhibition of pulmonary artery smooth muscle cell proliferation

Heparin has a wide range of important biological activities including inhibition of pulmonary artery smooth muscle cell proliferation. To determine the minimum size of the heparin glycosaminoglycan chain essential for antiproliferative activity, porcine intestinal mucosal heparin was partially depolymerized with heparinase and fractionated to give oligosaccharides of different sizes. The structure of these oligosaccharides was fully characterized by 1D and 2D 1H NMR spectroscopy. These oligosaccharides were assayed for antiproliferative effects on cultured bovine pulmonary artery smooth muscle cells (PASMCs). The tetrasaccharide (4-mer) exhibited no heparin-like activity. Decasaccharides (10-mers) and dodecasaccharides (12-mers) displayed a reduced level of activity when compared to full-length heparin. Little effect on activity was observed in deca- and dodecasaccharides with one less 2-O-sulfo group. The 14-, 16-, and 18-mers showed comparable growth-inhibition effects on PAMSC as porcine intestinal mucosal heparin. These data suggest that a 14-mer is the minimum size of oligosaccharide that is essential for full heparin-like antiproliferative activity. Since the 14- to 18-mers have no 3-O-sulfo groups in their glucosamine residues, their full activity confirms that these 3-O-sulfonated glucosamine residues, which are required for heparin's anticoagulant activity, are not an essential requirement for antiproliferative activity.     

Inhibition of fibronectin binding to platelets by proteolytic fragments and synthetic peptides which support fibroblast adhesion

To define regions within fibronectin (Fn) recognized by platelet binding sites, inhibition of Fn binding by an Fn fragment and synthetic peptides has been analyzed. A highly purified 120-kDa chymotryptic fragment, which has cell attachment activity but did not bind to insolubilized heparin or gelatin, inhibited Fn binding to platelets with an ID50 approximately 3 microM. Previous work indicates that fibroblasts attach to an 11.5-kDa subfragment of this 120-kDa fragment, and that one of four 30-residue synthetic peptides containing sequences from this region supports cell attachment. Only the peptide containing the COOH terminus of the 11.5-kDa fragment inhibited Fn binding to platelets, with an ID50 approximately 10 microM and is the peptide which supports fibroblast attachment. Of the smaller peptides studied from this sequence, all peptides containing the Arg-Gly-Asp-Ser sequence, including the tetrapeptide itself, were active in inhibiting Fn binding to platelets (ID50 values approximately 10-20 microM). The same peptides support fibroblast attachment. Those which lacked this sequence including Gly-Asp-Ser-Pro and Thr-Gly-Arg-Gly (immediately adjacent tetrapeptides) lacked both activities. Further evidence for specificity of inhibition was provided by structurally modified peptides in which substitution of a Glu for Asp abolished inhibitory activity and substitution of Lys for Arg or Ala for Gly reduced activity 6- and 8-fold, respectively. In addition, Arg-Gly-Asp-Ser-containing peptides inhibited the rate and extent of thrombin-induced platelet aggregation. These data suggest that the Arg-Gly-Asp-Ser tetrapeptide contains a recognition specificity involved in the binding of Fn to platelets and that platelets share features of this recognition specificity with fibroblasts.     

Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.     

Understanding the mechanism of the antimitogenic activity of suramin

Fibroblast growth factors (FGFs) play crucial roles in the regulation of key cellular processes such as angiogenesis, differentiation, and tumor growth. Suramin, a polysulfonated naphthylurea, is known to be a potent inhibitor of FGF-induced angiogenesis. Using isothermal titration calorimetry, we demonstrate that human acidic fibroblast growth factor (hFGF-1) binds to suramin with high affinity in the nanomolar range. The suramin:hFGF-1 binding stoichiometry is estimated to be 2:1. Size-exclusion chromatography data reveal that suramin oligomerizes hFGF-1 to form a stable tetramer. Thermal unfolding experiments monitored by steady state fluorescence, and limited trypsin digestion analysis data suggest that suramin-induced oligomerization of hFGF-1 occurs in two steps. The first step involves the binding of suramin at specific sites on the protein. Two molecules of suramin appear to bind simultaneously to one molecule of hFGF-1. Binding of suramin possibly involves formation of solvent-exposed nonpolar surfaces in hFGF-1. In the second step, FGF appears to oligomerize through coalescence of the solvent-accessible nonpolar surfaces. Results of the NMR experiments reveal that suramin binds to residues in the heparin binding pocket as well as to residues involved in FGF receptor binding. On the basis of the results of this study, we propose a model to explain the molecular mechanism(s) underlying the antimitogenic activity of suramin. To our knowledge, this is the first study in which suramin interaction sites on FGF have been characterized.     

Structural basis for antagonism by suramin of heparin binding to vaccinia complement protein

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.     

Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxin-sensitive guanine-nucleotide-binding proteins.

The anti-helminthic drug suramin inhibited the basal high-affinity GTPase activity of both C6 BU1 glioma and NG 108-15 neuroblastoma x glioma hybrid-cell membranes with an IC50 (concentration causing half-maximal inhibition) value close to 30 micrograms/ml. This effect was shown to occur via a non-competitive mechanism in which the binding affinity of the G-proteins for GTP was not altered, but the maximal velocity of the subsequent hydrolysis was reduced. In NG 108-15 membranes, both opioid peptides and foetal-calf serum stimulated high-affinity GTPase activity in a pertussis-toxin-sensitive manner. These effects have previously been shown to be mediated by different G-proteins [McKenzie, Kelly, Unson, Spiegel & Milligan (1988) Biochem. J. 249, 653-659]. Suramin completely prevented the opioid-peptide-stimulated increase in GTP hydrolysis, but did not prevent the opioid peptide from binding to its receptor. Suramin, however, did not block the foetal-calf-serum-stimulated GTPase response. This selective action of suramin provides further evidence for distinct roles for two separate pertussis-toxin-sensitive G-proteins in signal transduction in NG 108-15 membranes and provides the first evidence for a selective effect of a drug on the functions of different G-proteins.