Elastin Decreases the Efficiency of Neutrophil Elastase Inhibitors Marc Padrines and Joseph G. Bieth INSERM Unite 237, Universite Louis Pasteur de Strasbourg, Faculte de Pharmacie, Illkirch, France

Elastase inhibitors are potential drugs for the control of lung emphysema. Since neutrophils may release elastase in the lung interstitium, elastin and inhibitors may compete locally for the binding of enzyme. To better evaluate the potential activity of antielastases, we have run experiments that mimic this in vivo competition. Elastase was added to mixtures of human lung elastin and inhibitor, and the solubilization of the fibrous substrate was measured as a function of time. Controls in which a synthetic substrate was used instead of elastin were run under identical conditions. We show that the rate constants for the irreversible inhibition of elastase by methoxysuccinyl-Ala2-Pro-Val-chloromethylketone and L-657,229, a substituted (3 lactam, are 28- and 63-fold lower with elastin than with a synthetic substrate, respectively. The rate constant decreases with increasing concentrations of elastin, indicating that the inhibition is competitive. Elastin also impairs the potency of the following reversible inhibitors: trifluoroacetyl-Lys-Ala-NHC6H.-p-C6HII, trifluoroacety1-Lys-Ala-NH -C 6 H.-pN(C2Hs)z, methoxysuccinyl-Ala- Pro-Boro-Val-OH, and mucus proteinase inhibitor whose K values are 29- to l27-fold higher with elastin than with a synthetic substrate. Again the inhibition is competitive. We conclude that association rate constants of irreversible inhibitors and K values of reversible ones may be measured accurately using elastin as a substrate. The kinetic constants measured with elastin and not those determined with synthetic substrates should be used to decide whether a given inhibitor is potent enough to be a physiologic antielastase or a potential antielastase drug.

Neutrophil elastase is thought to be the primary destructive agent in pulmonary emphysema. It is commonly acknowledged that increased elastolysis may occur as a result of an imbalance between elastase and its major inhibitor, o.-proteinase inhibitor, either because of an increased release of the enzyme in the lung interstitium or because of an acquired or inherited deficiency of the inhibitor (1, 2). Specific elastase inhibitors may therefore be useful therapeutically in the control of emphysema. A wealth of lowmolecular-weight inhibitors have been synthesized (3), and a number of natural polypeptidic antielastases have been studied in vitro. Some of these compounds have also been investigated for their ability to prevent elastase-induced emphysema in laboratory animals (4-6). The potency of most of these inhibitors has been assessed using synthetic elastase substrates. Because the destruction of elastin and the concomitant (Received for publication June 15, 1990) Address correspondence to: Dr. Joseph G. Bieth, INSERM U 237, Faculte de Pharmacie, 74 routedu Rhin, F-67400 Illkirch, France. Abbreviations: aI-proteinase inhibitor, aIPI; MeOSuc-Alaz-Pro-(DL)Boro-Val-OH, Boroval; MeOSuc-A1az-Pro-VaI-chloromethylketone, CMK; 7a-methoxy-8-oxo-3[[(2,5-dihydro-2-methyl-5-oxo-6-hydroxy-I,2,4-triazin3-yl)-thio]-methyl]-5-thia-I-aza-6-R-bicyclo-[4.2.0]-oct-2-ene-5,5-dioxide, LAC; mucus proteinase inhibitor, MPI; methoxysuccinyl, MeOSuc; p-nitroanilide, pNA; succinyl, Sue; CF3CO-Lys-Ala-NH-C6H4-pC6HIl, TFAI; CF3CO-Lys-Ala-NH-C6~-pN(CzHs)2, TFA2. Am. J. Respir. CeU Mol. BioI. Vol. 4. pp. 187-193, 1991

loss of elastic recoil in the emphysematous lung appear to be well established (2), we have decided to measure the kinetic constants of elastase-inhibitor interactions using fibrous elastin as a substrate. We demonstrate that elastin strongly decreases the association rate constant of irreversible inhibitors and increases the K, of reversible ones.

Materials and Methods Materials Human plasma aI-proteinase inhibitor (aIPI) was isolated and tested for purity and activity as described previously (7). Human neutrophil elastase was isolated from purulent sputum (8). The enzyme migrated as a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis and was active site-titrated (9) with acetyl-Ala-Pro-azaAla-p-nitrophenylester (Enzyme System Products, Livermore, CA). Recombinant mucus proteinase inhibitor (10) (MPI) was a gift from Dr. Schnebli (Ciba-Geigy, Basel, Switzerland). It was active site-titrated using neutrophil elastase (11). The synthesis of CF3CO-Lys-Ala-NH-C~-pC~1l (TFAl) and CF3CO-Lys-Ala-NH-C6H4-pN(CzHs)z (TFA2) has been described previously (12). MeOSuc-Alaz-Pro-(DL)-BoroVal-OH (13) (Boroval) and L-657,229, a substituted (3 lactam, 'l-a- methoxy-8-oxo-3-[[(2 ,5-dihydro-2-methy1-5-oxo-6-hydroxy-l,2,4-triazin-3-yl)thio]-methyl]-5-thia-l-aza-6-R-bicyclo[4.2.0]-oct-2-ene-5,5-dioxide (14) (LAC) were kindly supplied by Dr. Kettner (E.!. Du pont de Nemours, Wilmington, DE)



and Dr. Doherty (Merck, Sharp & Dohme, Rahway, NJ), respectively. To hydrolyze the pinacol group of the pinacol derivative of Boroval, we let stand a 2-mM solution of this derivative in 0.1 M sodium phosphate, pH 7.5, for at least 1 h at room temperature. Various dilutions of this solution were prepared in the assay buffer. Human lung elastin, prepared by nondegradative extraction (15), was obtained from Elastin Products Company (Owensville, MO). Succinyl-Ala-p-nitroanilide (Sue-AlapNa) (16), and MeOSuc-Ala2-Pro-Val-chloromethylketone (17) (CMK) came from Bachem (Bubendorf, Switzerland). Stock solutions of Suc-Ala3-pNA, LAC, and CMK were prepared in dimethylformamide. The final concentration of this solvent in the reaction media was less than 2 %. Methods Oxidation of OIIPf. Oxidized OIIPI was prepared by mixing native OIIPI (20 J-tM) with a 20-fold molar excess of N-chlorosuccinimide (Sigma Chemical Co., St. Louis, MO). The mixture was incubated at room temperature for 20 min, and the reaction was stopped by addition of 120 mol methionine/mol OIIPI. The elastase inhibitory capacity of oxidized OIIPI was measured using porcine pancreatic elastase and human neutrophil elastase as described by Padrines and associates (18). The lack of inhibition of the former enzyme evidenced the absence of native aIPI, while the inhibition of the latter elastase showed that the protein was not denatured (18). Kinetics ofelastin solubilization by elastase. The solubilization of elastin was monitored as described by Boudier and colleagues (19). Briefly, samples from continuously stirred elastin + inhibitor + elastase suspensions were removed at appropriate intervals, acidified, centrifuged at 4,000 X g for 15 min and read at 280 urn using a Cary 2200 spectrophotometer with which absorbances as low as 2 X 10-4 U can be measured accurately. This procedure is as sensitive as the method using tritiated elastin (20). Appropriate controls containing either elastase alone, inhibitor alone, or buffer alone were also run. Determination of K with synthetic substrates. The K; of TFAI and TFA2, previously measured at pH 8 and 25° C (200 mM Tris-HCl) (12), was determined again at pH 7.4 and 37° C (50 mM Hepes, 100 mM NaCl). Elastase (0.3 J-tM) was added to a mixture of inhibitor and Sue-AlapNA, and the release of p-nitroaniline was recorded at 410 urn. Two substrate concentrations (0.25 mM and 1.5 mM) and several inhibitor concentrations were used. All progress curves were linear. The K; was estimated by an iterative least-squares fit to the classical equation of competitive inhibition:



The K; of Boroval, previously measured at pH 7.5 and 25° C (100 mM sodium phosphate, 500 mM NaCl) (13), was determined again at pH 7.4 and 37° C (50 mM Hepes, 100 mM NaCl). Elastase (5 nM) was added to mixtures containing variable concentrations of Boroval (0.1 to 5 J-tM) and a fixed concentration of MeOSuc-Ala2-Pro-Val-pNA (0.9 mM). All progress curves were biphasic, diagnosing slow-binding in-

hibition as already shown by Kettner and Shenvi (13). The K; was derived from the progress curves using the equa-

tions for slow-binding reversible competitive inhibition (21, 22). The K; of MPI was determined previously using the same buffer and temperature as those employed here (23). Determination of K with elastin as a substrate. Elastin and inhibitor were stirred for 10 min at pH 7.4 and 37° C (same buffer as above). Elastase (50 nM) was then added, and samples were removed at appropriate intervals to measure the absorbance at 280 nm. The solubilization of elastin was followed for 150 to 300 min. Two elastin concentrations (0.8 mg/ml and 2.4 mg/ml) and several inhibitor concentrations were used. All progress curves were linear. The slopes of these curves were used to calculate the initial rates of elastolysis (v). K; was estimated by an iterative leastsquares fit to an empirical equation of competitive inhibition of elastolysis:



where [So] is the elastin concentration used in the inhibition experiment and S,o, the equivalent of Km , is the elastin concentration for which v = 0.5 Vm,S,o was previously found to be equal to 0.8 mg/ml (24). All other conditions are given in the legends to the figures.

Results We have run competition experiments mimicking in vivo situations by adding elastase to mixtures of elastin and irreversible or reversible elastase inhibitors and measuring the solubilization of elastin as a function of time. Inhibition of Elastolysis by Irreversible Inhibitors Figure 1 shows the kinetics ofthe elastase-catalyzed solubilization of elastin in the presence of synthetic and polypeptidic irreversible elastase inhibitors. Native alPI fully prevented the elastolysis reaction. In contrast, some solubilization of elastin took place in the presence of CMK, LAC, and oxidized alPI. The kinetic curves were concave, suggesting slow-binding inhibition. When the elastase-inhibitor complexes were preformed prior to their addition to elastin (50 nM elastase + 5 J-tM inhibitor, 15 min at 25° C), no elastolysis was detected during the 2-h contact with the substrate. On the other hand, reaction of the inhibitors with elastin for 15 min at 37° C followed by centrifugation and assay of the inhibitory capacity of the supernatants (as indicated in the legend to Figure 2B) showed that the inhibitors were not adsorbed onto elastin. The slow-binding inhibition evidenced in Figure 1 is therefore neither due to a dissociation of the elastase inhibitor complexes during the 2-h incubation nor to a sequestration of the inhibitors by elastin. The concentration of elastin solubilized after a given time in the presence of an inhibitor may be considered as a measure of the efficacy of this inhibitor. The inset of Figure 1 shows that the efficacy of the inhibitors is related to their association rate constants rather than to their chemical structure (e.g., the synthetic inhibitor CMK and the protein inhibitor oxidized aIPI, which exhibit similar association rates, also solubilize similar amounts of elastin).

Padrines and Bieth: Elastase Inhibitors



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Elastin decreases the efficiency of neutrophil elastase inhibitors.

Elastase inhibitors are potential drugs for the control of lung emphysema. Since neutrophils may release elastase in the lung interstitium, elastin an...
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