Inhibition of Neutrophil Serine Proteinases by Suramin*

(Received for publication, May 22, 1996, and in revised form, December 26, 1996)

Martine Cadène Dagger , Jérôme Duranton Dagger , Anne North Dagger , Mustapha Si-Tahar §, Michel Chignard § and Joseph G. Bieth Dagger

From the Dagger  Laboratoire d'Enzymologie, INSERM Unité 392, Université Louis Pasteur de Strasbourg, F-67400 Illkirch, France and § Unité de Pharmacologie Cellulaire, Unité Associée I P/INSERM U 285, Institut Pasteur, F-75015 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and proteinase 3. The complexes it forms with these enzymes are partially active on synthetic substrates, but full inhibition takes place when elastase activity is measured with fibrous elastin or when cathepsin G activity is measured using platelet aggregation. One molecule of elastase binds four molecules of suramin with a Ki of 2 × 10-7 M as determined by enzyme inhibition or intrinsic fluorescence enhancement of suramin. The binding curves show no sign of cooperativity or anticooperativity. The Ki for the complexes with cathepsin G and proteinase 3 are 8 × 10-8 and 5 × 10-7 M, respectively. Ionic strength increases the Ki of the elastase-suramin complex in a way that suggests that four of the six sulfonate groups of suramin form ionic interactions with basic residues of the enzyme and that at saturation almost all arginines of elastase form salt bridges with suramin. The neutrophil proteinase-inhibitory activity of suramin might be used to prevent tissue destruction and thrombus formation in diseases where massive infiltration and activation of neutrophils take place.


INTRODUCTION

Suramin, a hexasulfonated naphtylurea with a symmetrical structure (e.g. Ref. 1), was synthesized 75 years ago and has been used in the treatment of trypanosomiasis and onchocerciasis (2). In the last decade, many new and therapeutically significant properties of this compound were described (for a review, see Ref. 3). It has thus been tried, although unsuccessfully, in patients with acquired immunodeficiency syndrome (4). Suramin has, however, proven to be useful as an anti-tumor drug in patients with corticosurrenal carcinoma (5), metastatic adrenal cancer (6), carcinomas of the kidneys (7), and carcinomas of the prostate gland (8).

Suramin has multiple modes of action. Although the mechanism of its trypanocide action is not well understood, it is probably based on the polyanion's ability to inhibit glycolytic enzymes of the parasite, thus interfering with its metabolism. Its use in acquired immunodeficiency syndrome was based on its potential inhibition of retroviral reverse transcriptase (9) and on its ability to decrease the binding of human immunodeficiency virus to T cells (10). The polysulfate is thought to exert its anti-tumoral action by impairing the binding of growth factors to the surface receptors of malignant cells (11, 12) and by inhibiting angiogenesis (13). It may also have anti-invasive properties, due to the inhibition of heparanases (1).

Here we report on a new property of suramin: its inhibitory action on three neutrophil serine proteinases, namely NE,1 cat G, and PR3, which are thought to play a pathogenic function in pulmonary emphysema, cystic fibrosis, and rheumatoid arthritis (14) and whose inhibition is therefore of therapeutic importance.


EXPERIMENTAL PROCEDURES

NE and cat G were isolated from purulent sputum and active site-titrated as described by Boudier and Bieth (15). PR3 was obtained from Merck through the courtesy of Dr. P. Davies and Dr. J. L. Humes and active site-titrated with titrated alpha 1PI (16). Bovine pancreatic trypsin (Choay, France) and chymotrypsin (Boehringer, Germany), titrated as described previously (15), were found to be 60 and 85% active, respectively, and were used without further purification. Suramin (hexasodium sym-bis[m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphtylamino-4,6,8-trisulfonate] carbamide) was a gift from Dr. Möller and Dr. Mardin (Bayer, Germany). The chemical structure of this compound may be found in Ref 1. [14C]Serotonin was from Amersham (United Kingdom). Remazol brilliant blue elastin was from Elastin Products Company (Owensville, MO). The p-nitroanilide and thiobenzylester substrates came from Bachem (Switzerland) and Enzyme Systems Products (Dublin, CA), respectively. Fibrinogen, cytochalasin B, prostacyclin, and 4,4'-dithiodipyridine were from Sigma.

Kinetics of Hydrolysis of Synthetic Substrates

Kinetics were monitored with a thermostated UVICON 941 spectrophotometer equipped with a PC microcomputer. The progress curves were recorded for 0.3-3 min, depending upon the reaction velocity.

The computer checked the linearity of the progress curves using linear regression analysis and calculated the initial reaction rates. Due to the great sensitivity of the spectrophotometer, less than 2% of the substrate was hydrolyzed during the rate measurements. The hydrolysis of p-nitroanilides was recorded at 410 nm, whereas that of the thiobenzylester was monitored at 324 nm in the presence of dithiodipyridine (17). All substrates except Suc-Ala3-pNA were dissolved in dimethylformamide whose final concentration in the reaction medium was 2% (v/v). Suc-Ala3-pNA was dissolved in N-methylpyrrolidone whose final concentration was 2.5% (v/v) during the measurement of Ki as a function of ionic strength and 1% (v/v) in all other experiments. The buffer was 50 mM Hepes, 100 mM NaCl, pH 7.4, unless otherwise stated. To determine the kinetic constants kcat and Km, initial rates were measured as a function of substrate concentration and the data were fitted to the Michaelis-Menten rate equation.

Nonlinear regression analysis (Enzfitter software) was used to calculate the best estimates of the enzymatic and fluorescence titration parameters.

Elastolysis

The effect of suramin on the elastolytic activity of NE was assessed as follows. Constant concentrations of NE were reacted with increasing concentrations of suramin in a total volume of 250 µl of 50 mM Hepes, 100 mM NaCl, pH 7.4. After 4 min, 200-µl aliquots were withdrawn from these mixtures and added to 2.3 ml of continuously stirred remazol brilliant blue elastin suspensions. After 15 and 30 min at 25 °C, 450-µl aliquots were withdrawn from these suspensions, diluted with 550 µl of ice-cold 0.75 M acetate buffer, pH 4.5, and centrifuged at 14,000 × g during 5 min. The absorbances of the supernatants were read at 595 nm to assay the concentration of soluble elastin peptides. The final concentrations of NE and remazol brilliant blue elastin were 2.5 µM and 3 mg/ml, respectively.

Preparation of Washed Human Platelets

Human blood from healthy volunteers was collected over citrate-phosphate-dextrose. The platelet-rich plasma obtained after blood centrifugation (180 × g, 20 min, 37 °C) was incubated with 1 µM [14C]serotonin (5 × 10-2 mCi/ml) for 30 min. Platelets were then isolated by successive centrifugations in the presence of prostacyclin (0.5 µM) and finally resuspended at 4 × 108 cells/ml in Tyrode's buffer composed of 137 mM NaCl, 2.68 mM KCl, 11.9 mM NaHCO3, 0.42 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, 5.5 mM glucose, 5 mM Hepes, 0.35% BSA, pH = 7.4.

Purification of Human Polymorphonuclear Neutrophils

Two volumes of blood, collected as for platelet preparation, were mixed with one volume of 3% (w/v) dextran in saline and allowed to sediment for 30 min. This step was followed by Ficoll-Hypaque gradient and hypotonic lysis of erythrocytes. After they had been washed, the neutrophils were resuspended in a final volume of Hanks' balanced salt solution such that the cell concentration was 107 cells/ml. The viability of recovered neutrophils was 98.7 ± 2.5%, as measured by Trypan blue dye exclusion. Their purity, evaluated using Türk's stain, was 96.2 ± 2.1%.

Effect of Suramin on Platelet Activation

Platelets were reacted with cat G or with formyl-Met-Leu-Phe-treated neutrophils in the presence or absence of suramin, and their activation was measured by their ability to aggregate and to release serotonin. A siliconized cell containing 250 µl of platelet suspension (108 cells), 250 µl of Hank's solution, and 0.7 mg/ml fibrinogen was placed in a Dual Aggro-meter (Chrono-Log Corp., Hevertown, PA) and preincubated at 37 °C for 90 s under continuous stirring (1,100 rpm). Suramin was then mixed with the suspension. Following a further 30 s, 0.25 µM cat G was added, and the resulting aggregation was monitored for 3 min by changes in light transmission. The reactions were terminated by the addition of 125 µl of a "stopping solution" (2.05% formaldehyde, 38.5 mM EDTA, 68.8 mM NaCl) immediately followed by centrifugation (13,000 × g, 3 min). The release of [14C]serotonin by platelets, measured on 400 µl of supernatants, was determined by scintillation counting (Counter 1212 Rackbeta; LKB, Wallac, Stockholm, Sweden). Controls without suramin or cat G were run in an identical way. Aggregation was expressed as a percentage of the maximal light transmission, and serotonin release was expressed as a percentage of the total [14C]serotonin content of the platelets. For the neutrophil-platelet interaction study, the Hanks' solution was replaced by neutrophils (5 × 106 cells/ml final concentration), and cells were preincubated for 5 min at 37 °C in the presence of fibrinogen and 5 µg/ml cytochalasin B. Suramin was then added. Following a further 30 s, aggregation was started with 0.5 µM formyl-Met-Leu-Phe.

Fluorescence Spectroscopy

Fluorescence emission spectra were recorded with a Shimadzu RF-5000 spectrofluorimeter equipped with a thermostated cell holder. The effect of increasing concentrations of NE on the fluorescence intensity of 1 µM suramin was measured at pH 7.4 (50 mM Hepes, 100 mM NaCl) and 25 °C using excitation and emission wavelengths of 315 and 401 nm, respectively.


RESULTS

Inhibition of Neutrophil Proteinases by Suramin

Fig. 1A shows the effect of increasing concentrations of suramin on the kinetics of Suc-Ala3-pNA hydrolysis by NE. While suramin depresses the rate in a concentration-dependent manner, it does not fully inhibit the enzyme, since curves 7 and 8 are barely distinguishable from each other. All progress curves were linear and thus allowed initial rates to be calculated. Linear progress curves were also observed for the four other proteinases. The initial rates derived from these curves were expressed as relative rates so that they could be compared on the same graph. Fig. 1B shows that the activities of NE, cat G, and PR3 sharply decrease with the concentration of suramin and then level off. With large excesses of suramin, the residual activities of PR3, cat G, and NE were about 60, 20, and 10%, respectively. In contrast, trypsin and chymotrypsin were insensitive to suramin when used at nanomolar concentrations. To favor suramin binding, we used 103-fold higher concentrations of trypsin and chymotrypsin. In turn, poor substrates were used to avoid substrate depletion during the rate measurements.2 Despite these conditions, trypsin activity was only slightly affected, and chymotrypsin was not inhibited at all.


Fig. 1. A, progress curves for the hydrolysis of 1 mM Suc-Ala3-pNA by 0.6 µM NE in the absence (curve 1) and the presence of increasing concentrations of suramin (curves 2-8; [suramin] = 0.5, 1.0, 1.5, 2.0, 5.0, 7.5, and 10 µM). B, effect of increasing concentrations of suramin on the activity of constant concentrations of neutrophil and pancreatic proteinases. The enzyme concentrations were as follows: [trypsin] = 1.06 µM (triangle ), [chymotrypsin] = 4 µM (black-triangle), [PR3] = 10 nM (black-square), [cat G] = 0.5 µM (open circle ), [NE] = 0.6 µM (bullet ). The corresponding substrates were benzoyl-Arg-pNA (1.5 mM), Suc-Phe-pNA (1.5 mM), MeOSuc-Ala2-Pro-Val-thiobenzylester (30 µM), Suc-Ala2-Phe-pNA (4 mM), and Suc-Ala3-pNA (1 mM). Ordinates, vi/vo, initial rate in the presence of suramin/initial rate in its absence.
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Incomplete inhibition implies that the enzyme-inhibitor complex EI is able to bind and turn over the substrate S (or that ES is able to bind I). This special case of noncompetitive inhibition may be analyzed using Scheme I (18),
                             E+<UP>S  </UP><LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB>s</SUB></UL></LIM>  E<UP>S  </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>cat</UP></SUB></UL></LIM>  E+<UP>P</UP>
+                    +
n<UP>I</UP>                     n<UP>I</UP>
K<SUB>i</SUB>⥮                 &agr;K<SUB>i</SUB>⥮
                             E<UP>I</UP><SUB>n</SUB>+<UP>S </UP><LIM><OP><ARROW>⇌</ARROW></OP><UL>&agr;K<SUB>s</SUB></UL></LIM> E<UP>SI</UP><SUB>n</SUB> <LIM><OP><ARROW>→</ARROW></OP><UL>&bgr;k<SUB><UP>cat</UP></SUB></UL></LIM> E<UP>I</UP><SUB>n</SUB>+<UP>P</UP>
<UP><SC>Scheme</SC></UP><UP> I</UP>
where n is the number of suramin molecules bound per molecule of free or substrate-bound enzyme and alpha  and beta  are dimensionless numbers representing the change in affinity (alpha ) and the change in the catalytic rate constant (beta ). The conditions under which partial inhibition takes place may be summarized by 0 < beta  <=  1 and 1 <=  alpha  < infinity .

Scheme I leads to a simple (19) or a complex (20) steady-state rate equation depending upon whether the inhibition is "classical" (total I = free I) or "tight binding" (total I = free I + bound I). Classical inhibition does not depend upon the total enzyme concentration, whereas tight binding inhibition does (21). We have therefore measured the inhibition of NE and cat G by suramin using different enzyme concentrations. Fig. 2 shows that the inhibition of NE by suramin decreases with the enzyme concentration; IC50 = 0.5, 0.8, and 1.47 µM for [E]o = 0.1, 0.3, and 0.6, respectively. This diagnoses tight binding inhibition (21). On the other hand, at high enzyme concentration a tight binding inhibitor titrates the enzyme (21). Indeed at [E]o = 2.5 µM, the inhibition was linear up to 80% inhibition, and the extrapolated curve intercepted the abscissa at [I]o = 9.4 µM, indicating that about 4 mol of suramin are required to inhibit 1 mol of NE, i.e. n = 4 (see inset to Fig. 2). Similar experiments demonstrated tight binding inhibition of cat G by suramin and a 3:1 suramin:cat G binding stoichiometry (data not shown).


Fig. 2. Effect of increasing concentrations of suramin on the activity of constant concentrations of NE. The NE concentrations were 0.1 µM (black-triangle), 0.3 µM (open circle ), 0.6 µM (triangle ), and 2.5 µM (bullet , inset). The substrate was 1 mM Suc-Ala3-pNA throughout. vi/vo, see legend to Fig. 1.
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To calculate the equilibrium dissociation constants Ki of the enzyme-suramin complexes we therefore used the complex steady-state rate equation derived by Szedlacsek et al. (20), which takes into account both the tight binding character and the incompleteness of inhibition,
<FR><NU>v<SUB>i</SUB></NU><DE>v<SUB>o</SUB></DE></FR>=<FR><NU>v<SUB>o</SUB>−v<SUB>∞</SUB></NU><DE>2v<SUB>o</SUB></DE></FR><FENCE><FENCE><FENCE>A+<FR><NU>[<UP>I</UP>]<SUB>o</SUB></NU><DE>n[E]<SUB>o</SUB></DE></FR>−1</FENCE><SUP>2</SUP>+4A</FENCE><SUP>1/2</SUP>+<FR><NU>v<SUB>o</SUB>+v<SUB>∞</SUB></NU><DE>v<SUB>o</SUB>−v<SUB>∞</SUB></DE></FR>−A−<FR><NU>[<UP>I</UP>]<SUB>o</SUB></NU><DE>n[E]<SUB>o</SUB></DE></FR></FENCE> (Eq. 1)
with
A=<FR><NU>1+[<UP>S</UP>]<SUB>o</SUB>/K<SUB>m</SUB></NU><DE>&agr;+[<UP>S</UP>]<SUB>o</SUB>/K<SUB>m</SUB></DE></FR> <FR><NU>&agr;K<SUB>i</SUB></NU><DE>n[E]<SUB>o</SUB></DE></FR> (Eq. 2)
and
v<SUB>∞</SUB>=&bgr; <FR><NU>k<SUB><UP>cat</UP></SUB>[E]<SUB>o</SUB>[<UP>S</UP>]<SUB>o</SUB></NU><DE>[<UP>S</UP>]<SUB>o</SUB>+&agr;K<SUB>m</SUB></DE></FR> (Eq. 3)
where vi is the rate of substrate hydrolysis in the presence of suramin, vo is the rate in its absence, vinfinity is the rate extrapolated at infinite suramin concentration, and [I]o and [E]o are the total concentrations of suramin and enzyme, respectively. Separate experiments were run to measure kcat, Km, and beta kcat and alpha Km, the kinetic parameters of the enzyme-substrate reaction in the absence of suramin and in the presence of a saturating concentration of this inhibitor, respectively. The Ki was calculated by nonlinear regression analysis by fitting the (vi, [I]o) pairs to Equation 1, in which Km, kcat, alpha , beta , and n were set as fixed parameters. The Ki for the NE-suramin complex (1.8 × 10-7 M) was determined using 0.1 µM NE and 1 mM Suc-Ala3-pNA, while that of the cat G-suramin interaction (8 × 10-8 M) was measured with 60 nM cat G and 2 mM Suc-Ala2-Pro-Phe-pNA. The fixed parameters used for nonlinear regression analysis are compiled in Table I. The stoichiometry of the PR3-suramin complex (n) could not be determined at high enzyme concentration due to the paucity of material. The Ki for the complex was therefore calculated using 10 nM enzyme, 30 µM MeOSuc-Ala2-Pro-Val-thiobenzylester and arbitrarily chosen values of n and was found to be 5 × 10-7 M in all cases. The low value of the [E]o/Ki ratio indicates that under the present experimental conditions there is no tight binding of inhibitor to enzyme, which explains why Ki does not depend upon the suramin-PR3 binding stoichiometry (21).

Table I.

Parameters of the inhibition by suramin of the hydrolysis of synthetic substrates by neutrophil proteinases

The equilibrium dissociation constants Ki were derived from titration experiments using separately determined values of Km, kcat, alpha , beta , and n. Errors on Km, kcat, alpha , beta , and n were below 15% and are not reported in the table.


Proteinase Substrate Km kcat  alpha  beta Ki n

µM s-1 M
NE Suc-Ala3-pNA 790 0.32 4.0 0.43 (1.8  ± 0.3)  × 10-7 4
cat G Suc-Ala2-Pro-Phe-pNAa 1890 6.6 2.3 0.29 (8.0  ± 0.15)  × 10-8 3
PR3 MeO-Suc-Ala2-Pro-Val-thiobenzylester 9.0 5.0 1.8 0.63 (5.0  ± 1.5)  × 10-7 NDb

a With Suc-Ala2-Phe-pNA, Km = 3800 µM, kcat = 0.49 s-1, alpha  = 1.0, and beta  = 0.12.
b Not determined.

Table I summarizes the kinetic parameters describing the inhibition of leukoproteinases by suramin. It can be seen that partial inhibition occurs because suramin decreases kcat (beta  < 1 but not equal 0). PR3, whose complex with suramin has the highest residual activity (Fig. 1), has also the highest beta  value. On the other hand, suramin has comparable affinities for the free enzymes. The substrates significantly lower these affinities (alpha  > 1).

Effect of NaCl on the Equilibrium Dissociation Constant Ki of the Suramin·NE Complex

The concentration of NaCl was varied from 0.2 to 0.5 M so that we could study the effect of Na+ counterions on the association of suramin to NE. For each NaCl concentration we first determined Km, kcat, alpha , and beta to calculate Ki using Equation 1. Ionic strength was found to affect the inhibition of NE by suramin in several ways. First, alpha  decreases by a factor of about 2, while beta  increases about 5-fold when [NaCl] rises from 0.2 to 0.5 M; ionic strength thus tends to enhance the residual activity of the NE·suramin complex. Second, Ki rises by a factor of 67 when [NaCl] increases from 0.2 to 0.5 M. The latter effect was assumed to be due to the electrostatic effect of Na+ counterions on the ionic interactions involved in the suramin·NE association. If we consider the binding of suramin to NE as the substitution of the Na+ counterions of the polyanion by the positively charged amino acid residues of the protein, we may describe the interaction by the following equilibrium.
<UP>NE</UP><SUP>Z<UP>+</UP></SUP>+<UP>suramin-Na<SUP>+</SUP></UP> <LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB>i</SUB></UL></LIM> <UP>NE · suramin</UP>+<UP>Z&psgr;Na</UP><SUP><UP>+</UP></SUP>
<UP><SC>Reaction</SC></UP><UP> I</UP>
The effect of [NaCl] on Ki may thus be analyzed using the following relation (22, 23),
<UP>log</UP> K<SUB>i</SUB>=<UP>log</UP> K<SUB>i</SUB>(1 <UP><SC>m</SC></UP>)−Z&psgr; <UP>log</UP>[<UP>NaCl</UP>] (Eq. 4)
where Ki (1 M) is the equilibrium dissociation constant extrapolated at [NaCl] = 1 M, Z is the number of discrete ionic interactions involved in the suramin·NE complex, and psi  is the fraction of Na+ bound to suramin per unit charge. Suramin apparently has an extended conformation (24), which suggests that little inner electrostatic screening takes place in the molecule, so that one may assume that psi  = 1. A plot of log Ki versus log [NaCl] yielded a straight line (not shown) whose slope Zpsi , determined by linear regression analysis, gave Z = 4.2 ± 0.7.

Fluorescence Measurement of the Equilibrium Dissociation Constant Ki of the NE·Suramin Complex

Free suramin has a very low intensity of fluorescence when excited at 315 nm. Upon the addition of NE, this intensity increases very strongly (see inset to Fig. 3). NE, however, does not change the maximum emission wavelength of suramin (lambda  = 401 nm). Use was made of this fluorescence enhancement to measure the NE·suramin affinity in a nonenzymatic way. Increasing concentrations of NE were added to constant concentrations of suramin, and the fluorescence intensity of each mixture was measured at lambda ex = 315 nm, and lambda em = 401 nm. Ki was calculated by a nonlinear least squares fit of the data to the following relationship,
&Dgr;F=&Dgr;F<SUB><UP>max</UP></SUB> <FR><NU>(n[E]<SUB>o</SUB>+[<UP>I</UP>]<SUB>o</SUB>+K<SUB>i</SUB>)−<RAD><RCD>(n[E]<SUB>o</SUB>+[<UP>I</UP>]<SUB>o</SUB>+K<SUB>i</SUB>)<SUP>2</SUP>−4n[E]<SUB>o</SUB>[<UP>I</UP>]<SUB>o</SUB></RCD></RAD></NU><DE>2[<UP>I</UP>]<SUB>o</SUB></DE></FR> (Eq. 5)
where n is the suramin:NE binding stoichiometry, Delta F = F - Fo, F being the fluorescence intensity in the presence of NE and Fo the fluorescence intensity in the absence of NE, Delta Fmax = Fmax - Fo, Fmax being the fluorescence intensity in the presence of the saturating concentration of NE.


Fig. 3. Fluorescence titration of suramin by NE. The variation of fluorescence intensity (Delta F) of 1 µM suramin upon the addition of NE was measured at 25 °C, pH 7.4, using a Shimadzu RF-5000 spectrofluorimeter (lambda ex = 315 nm; lambda em = 401 nm). The data were fitted to Equation 5 by nonlinear regression analysis. Inset, emission spectra of free (solid line) and NE-bound (dashed line) suramin excited at lambda = 315 nm (a.u., arbitrary units).
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Ki was found to be (2.1 ± 0.4) × 10-7 M, which, within the limits of experimental error, is identical to the enzymatically determined constant.

Effect of Suramin on the Biological Activities of NE and cat G

Fibrous elastin is the best known biological substrate of NE (14). Fig. 4 shows that suramin is a very potent inhibitor of the NE-catalyzed elastin solubilization. Most of the inhibition curve is linear, confirming the tight binding inhibition diagnosed with the synthetic substrate. Extrapolation of the linear part of this curve to the abscissa indicates that ~10 µM suramin is required to inhibit 2.5 µM NE, thus confirming the 4:1 binding stoichiometry evidenced with the synthetic substrate. Interestingly, the NE·suramin complex has almost no residual activity on elastin in contrast with what has been observed with the synthetic substrate.


Fig. 4. Effect of suramin on the elastolytic activity of NE. Constant concentrations of NE (2.5 µM) were reacted with increasing concentrations of suramin. The residual activities were then measured with elastin as described under "Experimental Procedures."
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Enzymatically active cat G is able to activate platelets through proteolytic cleavage (25, 26). We made use of this property to study the effect of suramin on the biological activity of cat G. Constant amounts of platelets were reacted with increasing amounts of suramin, after which constant amounts of cat G were added. Fig. 5 shows that suramin inhibits both platelet aggregation and serotonin release in a concentration-dependent manner. The concentration of suramin required to get 50% platelet activation (4 µM) is 3.3-fold higher than the IC50 observed with the cat G/Suc-Ala2-Phe-pNA system (Fig. 1) despite the fact that similar enzyme concentrations were used in the two experiments. This might be due to nonspecific binding of part of suramin by fibrinogen and the platelet surface. Alternatively, the peptide cleaved by cat G on platelets might have a much higher affinity for the enzyme than Suc-Ala2-Phe-pNA so that higher suramin concentrations might have been required to overcome this competition effect.


Fig. 5. Inhibition by suramin of the cat G-induced platelet activation. Constant amounts of platelets were reacted with increasing amounts of suramin, after which constant concentrations of cat G were added. Platelet activation was measured by their ability to aggregate (open circle ) or to release serotonin (bullet ). Each point is the mean of three or four experiments conducted with cells from different donors.
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To investigate a more physiologically relevant system, we have also studied the effect of suramin on the platelet activation mediated by formyl-Met-Leu-Phe-stimulated neutrophils (see "Experimental Procedures"). Suramin was again able to prevent platelet aggregation and serotonin release in this biological system (data not shown).


DISCUSSION

Suramin has been shown to inhibit a great number of parasite and human enzymes including trypanosoma glycolytic enzymes (3), human DNA and RNA polymerases (25), human reverse transcriptase (9), ATPase, and protein kinase C (5). It also inhibits human lysosomal enzymes such as beta -glucuronidase (1), but its action on lysosomal proteinases has not heretofore been described. This paper reports its inhibitory activity on NE, cat G, and PR3, the lysosomal proteinases of human neutrophils.

The inhibition of the leukoproteinases by suramin is incomplete, i.e. even with a large excess of suramin there is a substantial rate of substrate hydrolysis (e.g. Fig. 1). This indicates that the proteinase-suramin complexes behave like enzymes that are able to bind substrate and to hydrolyze it. The inhibition data could be satisfactorily fitted to Equation 1, indicating that they are described by Scheme I. Thus, the inhibition of the leukoproteinases by suramin depends upon four parameters: (i) n, the number of binding sites for suramin on the proteinase; (ii) Ki, the equilibrium dissociation constant of the proteinase-suramin complex; (iii) alpha , the factor that affects the binding of substrate to the enzyme-suramin complex or that of suramin to the enzyme-substrate complex; and (iv) beta , the factor that affects kcat. Theoretically, incomplete inhibition may be observed if 0 < beta  <=  1 and 1 <=  alpha  <infinity (see also Equation 3). In our investigation, one of the two limiting cases (alpha  = 1 or beta  = 1) was observed for the cat G/suramin/Suc-Ala2-Phe-pNA system for which alpha  = 1. For all other systems, incomplete inhibition resulted from the combined effects of alpha  > 1 and beta  < 1 (Table I).

The magnitude of alpha  and beta  for the cat G-suramin system depends upon the substrate used (Table I). The same is probably true for the NE-suramin pair for which vinfinity (a function of alpha  and beta ; see Equation 3) is about 10% of vo with the synthetic substrate (Fig. 2), whereas it is virtually 0 with the substrate elastin (Fig. 4). Also, cat G-induced platelet aggregation, a proteolytic event (25, 26), is fully inhibited by suramin (Fig. 5). The observation that full inhibition is observed when large protein substrates are used suggests either that these substrates are unable to bind EI so that the ESI complexes do not form (alpha infinity ) or that the ESI complexes have little or no enzymatic activities (beta  approx  0). With protein substrates, the inhibition of proteolysis by suramin may therefore be simply described by n and Ki. These parameters are substrate-independent at least for NE, since titration of this enzyme with suramin in the presence of a synthetic substrate (Fig. 2) and fluorescence titration of suramin with NE (Fig. 3) yield similar n and Ki values.

The effect of suramin on NE and cat G is reminiscent of that observed with heparin, another polysulfated molecule that is also a partial tight binding inhibitor of these enzymes (27, 28). The two polyanions mainly differ in their ability to depress the enzymatic activities; while the NE·heparin complex has a residual activity of 30 and 20% on synthetic substrates and on elastin, respectively, the NE·suramin complex is only 10% active on a synthetic substrate and is quite inactive on elastin. Similar differences are also observed with cat G. DNA is another polyanion that reacts with NE. At physiological ionic strength, unfractionated DNA yield 25% inhibition of NE activity (29), whereas in vitro selected DNA sequences with high affinity for NE have no significant inhibitory effect on the enzyme (30). Tight enzyme-ligand binding with lack of inhibition might be explained by assuming alpha  = 1 and beta = 1, for instance (see Equation 3). DNA and heparin are therefore less efficient elastase inhibitors than suramin.

The four suramin binding sites of NE are probably equivalent and independent, since the saturation curves show no sign of cooperativity or anticooperativity whether NE is titrated with suramin (Fig. 1 and 2) or suramin is titrated with NE (Fig. 3). The linear titration curve obtained with elastin as a substrate (Fig. 4) also confirms that the binding sites are equivalent and independent.

There is a good relationship between the basicity of the proteinases and their affinity for suramin. (i) PR3 whose pI is 9.1 has a significantly lower affinity for suramin than NE or cat G whose pI is greater than 9.5 (31). (ii) Trypsin and chymotrypsin, which are much less basic proteins, are virtually not inhibited by suramin. This indicates that the enzyme-suramin binding involves electrostatic interactions between the negatively charged sulfonate groups of the polyanion and the positively charged basic amino acid residues of the proteinases. It is worthwhile noticing that the binding of suramin to trypanosoma glycolytic enzymes is also governed by ionic interactions (3, 24). The electrostatic nature of the suramin·NE binding is confirmed by the deleterious effect of ionic strength on Ki, which suggests that an average of four ionic interactions take place per molecule of suramin bound, i.e. that four out of six sulfonate groups participate in binding. NE lacks lysine and has 19 arginine residues, 17 of which are fully exposed to solvent (32). Since four molecules of suramin are bound per molecule of NE, it can be concluded that suramin neutralizes 16 of these guanidino residues.

NE, cat G, and PR3 may cause tissue destruction in lung emphysema, cystic fibrosis, and rheumatoid arthritis because they cleave extracellular matrix proteins (14, 31). Sulfated glycosaminoglycans prevent NE-induced lung emphysema in animal models (31, 33). These ligands are partial inhibitors of the elastolytic activity of NE (27, 34, 35). Suramin, which fully inhibits elastolysis and has already been used in human cancer therapy (5-8) should therefore prove to be an efficient drug in diseases characterized by massive neutrophil recruitment and activation. In addition, as an inhibitor of cat G-induced platelet aggregation, this drug should also be a powerful antithrombotic agent.


FOOTNOTES

*   This work was supported by the Association Française pour la Lutte contre la Mucoviscidose.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Tel.: 333 88 67 69 34; Fax: 333 88 67 92 42; E-mail: jgbieth{at}pharma.u-strasbg.fr.
1   The abbreviations used are: NE, human neutrophil elastase; cat G, human neutrophil cathepsin G; PR3, human neutrophil proteinase 3; Suc, succinyl; MeOSuc, methoxysuccinyl; pNA, p-nitroanilide.
2   In 50 mM Hepes, 100 mM NaCl, 2% (v/v) dimethylformamide, pH 7.4 and 25 °C, the kcat and Km values for the trypsin/benzoyl-Arg-pNA system are 1.8 s-1 and 1.3 mM, those for the chymotrypsin/Suc-Phe-pNA pair being 0.013 s-1 and 0.95 mM, respectively.

ACKNOWLEDGEMENTS

We thank Dr. P. Davies and Dr. J. L. Humes from Merck for the gift of PR3 and Dr. Möller and Dr. Mardin from Bayer for the gift of suramin.


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