Mg2+-ATPase as a membrane ecto-enzyme of human granulocytes. Inhibitors, activators and response to phagocytosis.

(1) The Mg2+-ATPase of purified human granulocytes is located at the plasma membrane. Thus, no additional enzyme activity was detected when the cells were disrupted. Moreover, the Mg2+-ATPase activity of intact cells was inhibited by such poorly permeant reagents as diazotized sulfanilic acid and suramin. Finally, the enzyme activity of cell homogenates was recovered in particulate fractions. (2)The surface Mg2+-ATPase of human granulocytes had an apparent Km of 50 microns for ATP and displayed substrate inhibition. (3) The enzyme was not affected by ouabain, but was inhibited by N-ethyl malemide, sodium meta-periodate, suramin and diazotized sulfanilic acid. The enzyme was activated by cytochalasins B and D and by UDP. Activation by UDP was characterized by changes in the enzyme's apparent Km and V and by belief of substrate inhibition. (4)Internalization of surface membranes subsequent to phagocytosis of suitable particles did not result in depletion of Mg2+-ATPase from the cell surface. The enzyme activity did not decrease after exposure to several varieties of paraffin oil emulsion particles, even if the challenged cells had been pretreated with colchicine of cytochalasin B. (5) Since suramin, which inhibited Mg2+-ATPase, had no effect upon other granulocyte functions such as chemotaxis, superoxide anion generation, or phagocytosis, it is unlikely that the enzyme plays a major role in these functions.     

Structural and thermodynamic analysis of thrombin:suramin interaction in solution and crystal phases

Suramin is a hexasulfonated naphthylurea which has been recently characterized as a non-competitive inhibitor of human alpha-thrombin activity over fibrinogen, although its binding site and mode of interaction with the enzyme remain elusive. Here, we determined two X-ray structure of the thrombin:suramin complex, refined at 2.4 Å resolution. While a single thrombin:suramin complex was found in the asymmetric unit cell of the crystal, some of the crystallographic contacts with symmetrically related molecules are mediated by both the enzyme and the ligand. Molecular dynamics simulations with the 1:1 complex demonstrate a large rearrangement of suramin in the complex, but with the protein scaffold and the more extensive protein–ligand regions keep unchanged. Small-angle X-ray scattering measurements at high micromolar concentration demonstrate a suramin-induced dimerization of the enzyme. These data indicating a dissimilar binding mode in the monomeric and oligomeric states, with a monomeric, 1:1 complex to be more likely to exist at the thrombin physiological, nanomolar concentration range. Collectively, close understanding on the structural basis for interaction is given which might establish a basis for design of suramin analogues targeting thrombin.     

Kinetics of inhibition of sperm beta-acrosin activity by suramin

Sperm beta-acrosin activity is inhibited by suramin, a polysulfonated naphthylurea compound with therapeutic potential as a combined antifertility agent and microbicide. A kinetic analysis of enzyme inhibition suggests that three and four molecules of suramin bind to one molecule of ram and boar beta-acrosins respectively. Surface charge distribution models of boar beta-acrosin based on its crystal structure indicate several positively charged exosites that represent potential 'docking' regions for suramin. It is hypothesised that the spatial arrangement and distance between these exosites determines the capacity of beta-acrosin to bind suramin.     

Mapping of suramin binding sites on the group IIA human secreted phospholipase A2

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A₂ (hsPLA₂GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein, namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA₂GIIA, and indicates that suramin represents a novel class of phospholipase A₂ inhibitor.     

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.     

The propeptide domain of lysyl oxidase induces phenotypic reversion of ras-transformed cells

Lysyl oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, lysyl oxidase reverts ras-mediated transformation, and lysyl oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces lysyl oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of lysyl oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. beta-aminopropionitrile, the selective inhibitor of lysyl oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense lysyl oxidase demonstrated that lysyl oxidase gene expression mediated phenotypic reversion. Since lysyl oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the lysyl oxidase propeptide, and not the lysyl oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Akt-dependent induction of NF-kappaB activity. Thus, the lysyl oxidase propeptide, which is released during extracellular proteolytic processing of pro-lysyl oxidase, functions to inhibit ras-dependent cell transformation.     

Malic enzyme type VII isoenzyme as an indicator of suramin resistance in Trypanosoma evansi

This study analysed the suramin sensitivity of 29 stocks of Trypanosoma evansi isolated from Egypt, Sudan, and Indonesia and compared the results with the isoenzyme banding patterns of 20 soluble enzymes in these stocks of T. evansi. The results showed that the type VII banding pattern of malic enzyme was found only in T. evansi stocks which were highly resistant to suramin.     

The cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei. Kinetic properties and comparison with homologous enzymes

The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5-10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on Km and Vmax. The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.     

Modulation of adenylyl cyclase activity in young and adult rat brain cortex. Identification of suramin as a direct inhibitor of adenylyl cyclase

Adenylyl cyclase (AC) in brain cortex from young (12-day-old) rats exhibits markedly higher activity than in adult (90-day-old) animals. In order to find some possibly different regulatory features of AC in these two age groups, here we modulated AC activity by dithiothreitol (DTT), Fe(2+), ascorbic acid and suramin. We did not detect any substantial difference between the effects of all these tested agents on AC activity in cerebrocortical membranes from young and adult rats, and the enzyme activity was always about two-fold higher in the former preparations. Nevertheless, several interesting findings have come out of these investigations. Whereas forskolin- and Mn(2+)-stimulated AC activity was significantly enhanced by the addition of DTT, increased concentrations of Fe(2+) ions or ascorbic acid substantially suppressed the enzyme activity. Lipid peroxidation induced by suitable combinations of DTT/Fe(2+) or by ascorbic acid did not influence AC activity. We have also observed that PKC- or protein tyrosine kinase-mediated phosphorylation apparently does not play any significant role in different activity of AC determined in cerebrocortical preparations from young and adult rats. Our experiments analysing the presumed modulatory role of suramin revealed that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme activity was diminished to the same extent by suramin in membranes from both tested age groups. Our present data show that AC is regulated similarly in brain cortex from both young and adult rats, but its overall activity is much lower in adulthood.     

ATP: a mediator for HCl-induced TRPV1 activation in esophageal mucosa

In esophageal mucosa, HCl causes TRPV1-mediated release of calcitonin gene-related peptide (CGRP) and substance P (SP) from submucosal neurons and of platelet-activating factor (PAF) from epithelial cells. CGRP and SP release was unaffected by PAF antagonists but reduced by the purinergic antagonist suramin. ATP caused CGRP and SP release from esophageal mucosa, confirming a role of ATP in the release. The human esophageal epithelial cell line HET-1A was used to identify epithelial cells as the site of ATP release. HCl caused ATP release from HET-1A, which was reduced by the TRPV1 antagonist 5-iodoresiniferatoxin. Real-time PCR demonstrated the presence of mRNA for several P2X and P2Y purinergic receptors in epithelial cells. HCl also increased activity of lyso-PAF acetyl-CoA transferase (lyso-PAF AT), the enzyme responsible for production of PAF. The increase was blocked by suramin. ATP caused a similar increase, confirming ATP as a mediator for the TRPV1-induced increase in enzyme activity. Repeated exposure of HET-1A cells to HCl over 2 days caused upregulation of mRNA and protein expression for lyso-PAF AT. Suramin blocked this response. Repeated exposure to ATP caused a similar mRNA increase, confirming ATP as a mediator for upregulation of the enzyme. Thus, HCl-induced activation of TRPV1 causes ATP release from esophageal epithelial cells that causes release of CGRP and SP from esophageal submucosal neurons and activation of lyso-PAF AT, the enzyme responsible for the production of PAF in epithelial cells. Repeated application of HCl or of ATP causes upregulation of lyso-PAF AT in epithelial cells.     

Inhibition of the activities of DNA primase-polymerase alpha complex from KB cells by hexasodium sym-bis(m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4 ,6,8-trisulfonate)carbamide

A trypanocidal drug suramin [hexasodium sym-bis(m-amino-benzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4, 6, 8-trisulfonate)carbamide] was found to be a potent inhibitor of the activities of DNA primase and polymerase alpha from human KB cells. The mechanism of the inhibition by suramin was, however, quite different by these two polymerases. In the case of DNA primase, suramin inhibited competitively the incorporation of a nucleotide substrate, GTP, on the template polydeoxycytidylate, while the polymerase alpha was inhibited competitively by the drug with respect to the template primer (activated DNA). The observed inhibitory effect of suramin on nucleic acid synthesis seems to explain yet unknown mechanism of trypanocidal action of the drug.     

Suramin suppresses growth,alkaline-phosphatase and telomerase activity of human osteosarcoma cells in vitro

Neoadjuvant chemotherapy in osteosarcoma improves the survival dramatically, but there is currents drug resistance in about 25% of patients, leading researchers to investigate alternative therapy forms. Suramin has in the last two decades been used as salvage therapy in some cancers. This study was undertaken to investigate suramin as a possible salvage therapy in osteosarcoma. The effect of suramin on three human osteosarcoma cell lines (MG-63, HOS and SaOS-2) and three primary osteosarcoma cell lines isolated from biopsies was investigated. Suramin significantly inhibited cell proliferation, determined by 3H-thymidine incorporation, of osteosarcoma cells at a dose ranging from 250 to 500 microg/ml. Suramin decreased the secretion of alkaline-phosphatase after stimulation by 1,25-dihydroxy-Vitamin D(3) up to 50% and decreased telomerase activity by up to 40%. The data demonstrate that suramin has marked in vitro effects on human osteosarcoma cells supporting further clinical investigation.     

Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status

The major ectonucleoside triphosphate phosphohydrolase in the chicken gizzard smooth muscle membranes is an ecto-ATPase, an integral membrane glycoprotein belonging to the E-ATPase (or E-NTPDase) family. The gizzard ecto-ATPase is distinguished by its unusual kinetic properties, temperature dependence, and response to a variety of modulators. Compounds that promote oligomerization of the enzyme protein, i.e., concanavalin A, chemical cross-linking agent, and eosin iodoacetamide, increase its activity. Compounds that inhibit some ion-motive ATPases, e.g., sulfhydryl reagents, xanthene derivatives, NBD-halides, and suramin, also inhibit the gizzard ecto-ATPase, but not another E-ATPase, the chicken liver ecto-ATP-diphosphohydrolase, which contains the same conserved regions as the ecto-ATPase. Furthermore, inhibition of the gizzard ecto-ATPase by these compounds as well as detergents is not prevented by preincubation of the membranes with the substrate, ATP, indicating that their interaction with the enzyme occurs at a locus other than the catalytic site. On the other hand, the inhibitory effect of these compounds, except suramin, is abolished or reduced if the membranes are preincubated with concanavalin A. It is concluded that these structurally unrelated modulators exert their effect by interfering with the oligomerization of the ecto-ATPase protein. Our findings suggest that, under physiological conditions, the gizzard smooth muscle ecto-ATPase may exhibit a range of activities determined by membrane events that affect the status of oligomerization of the enzyme.     

Biochemical and structural investigations of Bothropstoxin-II, a myotoxic Asp49 phospholipase A2 from Bothrops jararacussu venom

Bothropstoxin-II a calcium-dependent enzyme from Bothrops jararacussu venom causes tissue damage and several haemostatic disorders including platelet aggregation. In order to elucidate the structural determinants of its multiple pharmacological activities, we have studied the effects of suramin on Bothropstoxin-II and present details concerning the mode of binding.     

Effect of the lysosomotropic preparation suramin on the ultrastructural and functional characteristics of rat liver cells in acute toxic hepatitis

A study was made of the influence of suramin and of a combination of suramin and CCl4 on the ultrastructure of hepatocytes, structural and functional state of lysosomes and functional activity of the rat's liver. The latter was characterized by alanine aminotransferase level in blood. Increasing amounts of autophagosomes as well as lysosomal fusion disturbances were registered 24 and 48 hours after a single suramin administration. The combined suramin and CCl4 administration resulted even in a higher damage of hepatocytes. A decrease in the intralysosomal rate of proteolysis in hepatocytes, was also shown in addition to the impairment of liver functional activity. Some ultrastructural features of compensation of the insufficiency of the hepatocyte vacuolar apparatus were noted during suramin and CCl4 administration. Changes in the nucleolar structure were noticed in hepatocytes administered both suramin and CCl4. It is suggested that these changes may involve a compensatory increase in nucleologenesis due to the decrease in the activity of the protein-synthesizing apparatus in hepatocytes.     

Suramin prevents import of acyl-CoA oxidase into rat liver peroxisomes.

The inhibitory effect of suramin on the import of [35S]acyl-CoA oxidase into purified rat liver peroxisomes was investigated in vitro. The import of acyl-CoA oxidase was inhibited completely by 10 microM suramin, whilst the latency of catalase remained unchanged. The important value decreased 60% by pretreatment of peroxisomes with 10 microM suramin, but it did not decrease by pretreatment of translation products. Polysulfonate compounds which have two clusters of negative charges, such as Cibacron blue F3GA and Trypan blue, as well as suramin, inhibited the import, whilst mono- and disulfonate compounds did not.     

Spectroscopic studies on the complex formation of suramin with bovine and human serum albumin.

The binding of suramin to bovine and human serum albumin was investigated by gel filtration and spectroscopic measurements. Besides some low-affinity binding sites suramin has, on the bovine serum albumin molecule one and on the human serum albumin molecule two, high-affinity binding sites. Spectroscopic measurements reveal that there are large differences between the albumins in the mechanism of binding to the high-affinity binding sites. Further, it is suggested that high concentrations of suramin provoke an unfolding of the albumin moleculse. In order to explain the unusual behaviour of suramin in connection with the displacement of other ligands from the albumin binding the fluorescence probe 1-anilino-8-naphthalenesulfonic acid (ANS) was employed as a reporter group molecule for fluorescence as well as circular dichroism measurements. By these measurements it could be shown that suramin greatly influences the microorganization of both albumin molecules. In the case of these measurements large differences between bovine and human serum albumin were also found.     

The suramin-treated rat as a model of mucopolysaccharidosis: reversibility of biochemical and morphological changes in the liver

Rats treated with the trypanocidal drug suramin, a potent inhibitor of several lysosomal enzymes, develop a storage disorder which mimics the features of mucopolysaccharidosis (Constantopoulos et al. 1983). In this paper we have examined the reversibility of the biochemical and pathological changes induced in the liver of the suramin-treated rat. Rats were injected with a single intravenous dose of suramin (250 mg/kg) and allowed to survive for periods of up to 6 months. The liver was examined for suramin content, pathological changes, biochemical storage of glycosaminoglycans (GAGs) and for the blockade of the relevant hydrolytic enzymes. GAG storage in the liver peaked at approximately 14 days after administration of suramin when there was a five-fold increase in the GAG content. Thereafter GAGs decreased in parallel with the fall of suramin concentrations so that within 6 months the content had returned to control levels. The activity of most of the enzymes tested had also returned to control levels within 6 months. The pathological changes which are evident in the liver 1-2 weeks after administration of the drug had diminished considerably within 6 months. These results indicate that significant reversibility of both the biochemical and pathological changes induced by suramin occurs and they support the suitability of the suramin treated rat as a model to assess the value of therapeutic treatments of mucopolysaccharidosis.     

Suramin, a novel antitumor compound

Suramin, a polyanionic compound originally synthesized for use as an antiparasitic agent, has recently entered clinical trials for the treatment of a variety of human cancers refractory to conventional modalities of therapy. This is based on suramin's ability to bind and to inactivate growth factor and enzyme systems critical to cellular homeostasis and proliferation. In addition, this compound possesses adrenocorticolytic properties in vivo and exerts significant cytostatic and cytocidal effects against a variety of human tumor cell lines in vitro. Pilot studies using suramin have thus far been conducted in adrenocortical carcinoma, prostate cancer refractory to conventional hormonal manipulation and nodular lymphomas.