European Journal of Pharmacology, 193 (1991) 153-158 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991001383
153
EJP 51704
Inhibition of human neutrophil elastase by ICI 200,355 Christian P. S o m m e r h o f f 1, R o b e r t D. Krell, J o s e p h L. Williams, B r u c e C. G o m e s , A n n e M. S t r i m p l e r and J a y A. N a d e l Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, CA 94143, U.S.A. and Department of Pharmacology, ICI Americas, Inc., Wilmington, DE 19897, U.S.A. Received 13 August 1990, revised MS received 31 October 1990, accepted 13 November 1990
To examine the pathogenetic role of neutrophil elastase in airway hypersecretion, we have studied the novel inhibitor of this enzyme, [4-(4-bromophenylsulfonylcarbamoyl)benzoyl-L-valyl-L-proline1 (RS)-(1-trifluroacetyl-2-methylprolyl)amide] (ICI 200, 355). This compound was a potent (K i = 0.6 _+0.22 riM) inhibitor of human neutrophil elastase and a much weaker inhibitor ot other hydrolases. ICI 200,355 also inhibited the ongoing destruction of insoluble elastin by human neutrophil elastase. ICI 200,355 produced a concentration-dependent inhibition of the secretory response induced by human neutrophil elastase (10-s M), with an ICs0 of 1.6 × 10 -8 M. ICI 200,355 had no effect on baseline secretion or on the secretory response to chymase, cathepsin G oi Pseudomonas aeruginosa elastase. Thus, ICI 200,355 appears to be a useful tool for investigating the role of human neutrophi] elastase in inflammatory disorders associated with hypersecretion, such as cystic fibrosis, chronic bronchitis, and asthma. Airway; Submucosal gland; Mucus; Serine protease; Elastin (insoluble); Neutrophil elastase (EC 3.4.21.11)(human); Proteinase inhibitor; Hydrolase; (Bovine)
1. Introduction
Human neutrophil elastase is a neutral serine proteinase with broad substrate specificity which is released from neutrophils during phagocytosis and upon cell death (Weissmann et al., 1972; Ohlsson and Olsson, 1977). This enzyme has been implicated in the pathogenesis of a variety of inflammatory diseases such as emphysema, acute respiratory distress syndrome, and rheumatoid arthritis (Janoff, 1985a; Bonney and Smith, 1986; Janoff, 1985b). Evidence has also been presented that it may be involved in the pathogenesis of increased and abnormal airway secretions commonly associated with airway inflammatory diseases. Thus, increased numbers of neutrophils are found in the airways of subjects with abnormal airway secretions such as smokers (Reynolds and Merrill, 1981; Hunninghake et al., 1979) and in patients with chronic bronchitis and cystic fibrosis (Esterly and Oppenheimer, 1968; Gibson
a Present address: Department of Clinical Chemistry and Clinical Biochemistry, Surgical Clinic City, University of Munich, Munich, F.R.G. Correspondence to: J.A. Nadel, Cardiovascular Research Institute, Box 0130, University of California, San Francisco, CA 94143-0130, U.S.A.
et al., 1989). Continuous mucus hypersecretion in exsmokers has also been associated with ongoing inflammation of airway submucosal glands (Mullen et al.. 1987). Also, levels of free neutrophil elastase in the airways may be greatly increased in smokers and in patients with chronic bronchitis and cystic fibrosk, (Goldstein and Doring, 1986; Stockley and Burnett. 1979; Weitz et al., 1987; Janoff et al., 1983; Tetley el al., 1987). Finally, neutrophil elastase has been shown to release cell-surface glycoconjugates from trachea] surface epithelial cells in vitro (Varsano et al., 1987'. Kim et al., 1987; Breuer et al., 1989) and to inducc goblet cell discharge and hypertrophy in hamsters in vivo (Breuer et al., 1987). In addition, among othel proteinases such as human neutrophil cathepsin G, masl cell chymase and 'classic' mediators such as histamine neutrophil elastase is the most potent secretagogue for cultured serous cells of airway submucosal glands (Sommerhoff et al., 1989; Sommerhoff et al., 1990), th~ structures that are considered to be the major source ol airway secretions in humans (Reid, 1960). The pathogenetic role of human neutrophil elastas~ in conditions such as airway hypersecretion can only b~ validated by the use of potent and selective inhibitors oJ the enzyme in vivo. To aid these investigations, we hav~ studied the novel inhibitor of human neutrophil elastase, [4-(4-bromophenylsulfonylcarbamoyl)benzoyl-L.
154
0
mostatically controlled, water-jacketed holder in the cell compartment of a Cary B210 spectrophotometer and was allowed to reach thermal equilibrium. The temperature was maintained at 25 + 0.1°C. The reaction was initiated by the addition of 5 #1 enzyme solution (0.14 m g / m l ) . Absorbance was continuously monitored and stored in a D E C PDP 11/32 minicomputer. Initial and steady state velocities were calculated by a fit of the experimental data to a linear dependence on time by linear least-squares analysis. Triplicate determinations were conducted for each inhibitor concentration.
cF,
Fig. 1. Structure of ICI 200,355 ([4-(4-bromophenylsulsulfonylcarbamoyl)benzoyl-L-valyl-L-proline l(RS)-(1-trifluroacetyl-2-methylprolyl)amidel). valyl-L-proline 1 (RS)-(1-trifluroacetyl-2-methylprolyl) amide] (ICI 200,355) (fig. 1). Our results demonstrate that this compound is a potent and selective inhibitor of human neutrophil elastase that blocks not only the amidolytic activity and the ongoing destruction of insoluble elastin but also the secretion from cultured airway gland serous cells induced by the enzyme. ICI 200,355 therefore appears promising as a tool for the in vivo investigation of the role of human neutrophil elastase in inflammatory disorders and, subsequently, perhaps for a rational therapy of diseases involving airway hypersecretion, such as cystic fibrosis, chronic bronchitis, and asthma.
2. Materials and methods
2.1. Determination of kinetics of inhibition of human neutrophil elastase The substrate methoxysuccinyl-Ala-Ala-Pro-Valp N A was hydrolyzed by human neutrophil elastase releasing p-nitroanalide which was continuously measured spectrophotometrically by monitoring absorbance changes at 410 nm. Both substrate and inhibitor were dissolved in DMSO. Fifty microliters of both substrate and inhibitor or D M S O were added to a cuvette containing 2.895 ml butter I (10 m M N a phosphate, 500 m M NaC1, p H 7.6), The cuvette was placed in a ther-
2.2. Determination of protease selectivity The protease selectivity of ICI 200,355 was determined by using minor modifications of the protocol above. All substrates were dissolved in D M S O and were combined with inhibitor (dissolved in the same solvent). Equal volumes (50 #1) of inhibitor and substrate were added to 2.895 ml buffer I in a cuvette, and the cuvettes were placed in the spectrophotometer described above. When the reaction mixtures had reached thermal equilibrium (25°C), the reactions were initiated by the addition of 5 /~1 enzyme, and absorbance was continuously monitored at a wavelength determined by the substrate-leaving group. Assay conditions for the various enzymes are illustrated in table 1. 2.3. Inhibition of human neutrophil elastase-catalyzed hydrolysis of insoluble elastin
Bovine neck elastin was suspended at a concentration of 20 m g / m l in 0.1 M Tricine, 0.75 M NaC1 (pH 8.0) buffer containing 250 ~1 triton X-100/250 ml. The suspension was stirred at room temperature for 18 h, then it was washed on a glass-fritted funnel with buffer, and resuspended in fresh buffer. The elastin solution was diluted with an equal volume of buffer to give a
TABLE 1 Assay conditions for the various enzymes. Enzyme Porcine pancreatic elastase Bovine pancreatic chymotrypsin Human plasma thrombin Acetylcholinesterase Human leukocyte cathepsin G
Substrate (concentration) Succinyl-Ala-Ala-Ala-pNA(0.5 mM) Succinyl-Ala-Ala-Pro-Phe-pNA(155 ttM) Bz-Phe-Val-Arg-pNA(51 ~M) Acetylthiocholine (20 ~ M) Succinyl-Ala-Ala-Pro-Phe-pNA(1 raM)
Bovine pancreatic trypsin Papain
Bz-Phe-Val-Arg-pNA(51 ~M) N-CBZ-GIy-oNP(83 ~M)
Pseudomonas aeruginosa elastase
Bovine ligament elastin (10 mg/ml)
Buffer
Enzyme concentration
Wavelength(nm)
Buffer I Buffer I Buffer 1 Buffer I 0.1 M Tris, 0.7 mM NaN 2 pH 8.3 Buffer I 0.2 M NaPi 0.005 M EDTA pH 6.0 1.65% DMSO 0.8% MeCN Buffer I
24 nM 3.3 nM 5 units/ml 14 nM 25 nM
410 410 410 420 410
3.3 nM 87 nM
410 347.5
0.25/LM
280
155 final volume of 10 mg/ml. Aliquots of substrate (8 ml) were added to 25-mi flasks, and human neutrophil elastase was added to a final concentration of 0.2 /~M. The flasks were stirred continuously at 25°C. Ten minutes after the initiation of the reaction, inhibitor was added to final concentrations of 0.2, 0.4 or 0.8/~M. At various times, 0.75-ml samples were removed from the reaction flasks and were added to centrifuge tubes containing 0.75 ml 0.1 M acetic acid, 4.0 M NaC1 (pH 4.7). After centrifugation, the absorbance of the supernatant at 276 nm was determined on a Cary B210 spectrophotometer.
2.4. Culture of bovine tracheal serous cells A line of bovine trachea gland serous cells was cultured as described previously (Finkbeiner et al., 1986; Sommerhoff et al., 1989). Cells were grown in tissue culture plastic flasks (surface area, 25 cm 2) coated with human placental collagen in medium containing 40% Dulbecco's modified Eagles H21 medium, 40% Ham's F12 medium, 20% fetal calf serum, and 50 /~g/ml gentamicin. Flasks were maintained at 3 7 ° C in 5% CO2-95% air. Cells were studied between passages 10 and 25.
2.5. Release of 35S-labeled macromolecules On day 9, confluent monolayers were incubated with medium containing Na35SO4 (7.5 /~Ci/ml). After 24 h, the medium containing the radiolabel was removed, and the cells were washed 3 times with phosphate-buffered saline. Serum-free medium (50% Dulbecco's modified Eagles H21 medium, and 50% Ham's F12 medium) containing 100 / t g / m l streptomycin and 100 U / m l penicillin was then added and was renewed every 30 min for 210 rain. At 210 min, medium containing proteases was added to the cells. To determine the inhibition of protease-induced secretion by ICI 200,355, proteases were preincubated with ICI 200,355 prior to addition to the cells. In preliminary studies, we compared preincubation for 5 and 30 min and found no differences in the degree of inhibition. Therefore, we performed all studies after 5 min of preincubation. After 30 rain, the medium was collected again, and the culture flasks were examined using a phase microscope ( I T M - 2, Olympus, Cherry Hill, N J) to verify the integrity of the cell monolayer. The spent medium from the 180- to 210-rain and the 210- to 240-rain incubation periods was dialyzed (Spectrapor tubing; MW cutoff, 12 000-14 000 Da), against distilled water containing 10 mg/1 sodium azide to remove unincorporated 35SO4. After addition of scintillation fluid (Hydrofluor, National Diagnostics, Somerville, N J), non-dialyzable 3sS-labeled macromolecules were counted by scintillation spectroscopy to an accu-
racy of 2% (beta counter model LS7500, Beckman Instruments, Inc., Irvine, CA). Secretion was expressed as percent increase of release of 3sS-labeled macromolecules during incubation with the agonists over the release during the immediately preceding time period. This calculation was corrected for the declining baseline, which was determined in controls incubated with medium alone.
2.6. Statistics All values were expressed as means + S.E.M. Statistical analysis was performed using unpaired Student's t-test or analysis of variance and Dunetts multiple comparison test, where appropriate (Zar, 1984). A P value of less than 0.05 was considered significant.
2.7. Materials Media, fetal calf serum, and antibiotics were obtained from the Cell Culture Facility of the University of California, San Francisco. Human placental collagen (type IV), bovine pancreatic trypsin and chymotrypsin, porcine pancreatic elastase, human plasma thrombin, acetylcholinesterase, papain and synthetic enzyme substrates were obtained from Sigma (St. Louis, MO). Carrier-free Na35SO4 (specific activity, 43 C i / m g ) was purchased from ICN Radiochemicals, Inc. (Irvine, CA). Human neutrophil elastase and cathepsin G isolated from human purulent sputum and bovine neck elastin were purchased from Elastin Products (Pacific, MO). Pseudomonas aeruginosa elastase was obtained from Nagase Biochemicals (Kyoto, Japan). Chymase was purified from canine mastocytoma cells as described previously (Caughey et al., 1988). ICI 200,355 was synthesized by the Department of Medicinal Chemistry, ICI Americas, Wilmington, DE.
3. Results
3.1. Potency and selectivity of 1C1 200,355 against several hydrolases ICI 200,355 is a slow, tight, reversible inhibitor of human neutrophil elastase with a K i = 0.60 ___0.22 nM (mean + S.E.M., n = 5). Reaction progress curves for compounds of this type are typically exponential followed by positive linear slope, the reversible nature of the inhibitor being demonstrated by the positive linear, as opposed to a zero, slope. The slow nature of the inhibition of human neutrophil elastase by ICI 200,355 derives from kon = 87 000 _+ 54 000 M -1 s -1 (mean _+ S.E.M., n = 5) compared with more traditional inhibitors with konvalues > 107 M -1 s -1. Pharmacologically,
156 TABLE 2 Potency and selectivity of ICI 200,355 against several hydrolases.
I O0
/
80
Enzyme
K i (nM)
H u m a n leukocyte elastase Porcine pancreatic elastase Bovine pancreatic chymotrypsin Bovine pancreatic trypsin Hunztan plasma thrombin Acetylcholinesterase Papain H u m a n leukocyte cathepsin G Pseudomonas aeruginosa elastase
0.6 + 0.22 ~ 46 + 5 150000 + 2 000 NI b NI c NI ~ 60 000 _+ 8 000 81 000 +_20 000 140 000 d
z 6O o F-
~= 4 0 bq
ij
IIj
i l l
20
a Values are m e a n s + S . E M , of n > 3; b no inhibition at concentrations of 0.1 m M ; ~ no inhibition at concentrations of 1.0 # M ; d iCs0"
10
-10
10
-9
10
-8
10
-7
6
10
ICI 200,355 [M]
differences between traditional and slow binding inhibitors are imperceptible in our hands. In contrast, ICI 200,355 was only a weak inhibitor of several other hydrolases (table 2 and fig. 5). The single exception was porcine pancreatic elastase, where ICI 200,355 demonstrated only a 77-fold lower affinity than for human neutrophil elastase.
3.2. Inhibition of human neutrophil elastase-catalyzed hydrolysis of insoluble elastin The ability of ICI 200,355 to inhibit human neutrophil elastase hydrolysis of insoluble elastin is illustrated in fig. 2. In these experiments, the reaction was initiated by combining human neutrophil elastase with insoluble bovine ligament elastin followed 10 min later by the addition of ICI 200,355. When inhibitor concentration was equal to enzyme concentration (0.2 #M), approximately 33% inhibition of elastinolysis was observed. When inhibitor was in 4-fold excess, approximately 83% inhibition was obtained. 0.6 _/~
0.5
Fig. 3. Concentration-dependent inhibition of h u m a n neutrophil elastase-induced (10 - s M) secretion of macromolecules from serous cells by ICI 200,355. Data are reported as means_+ S.E.M, (n = 5).
3. 3. Inhibition of serous cell secretion by ICI 200, 355 At the highest concentration used in these studies (1 /~M), ICI 200,355 had no effect on baseline secretion of sulfated macromolecules from cultured serous cells. However, ICI 200,355 did inhibit the secretory response induced by 10 -8 M h u m a n neutrophil elastase in a concentration-dependent fashion (fig. 3). A significant inhibition of the elastase-induced response was achieved at concentrations equal to or greater than 10 8 M (P < 0.01). The interpolated ICs0 from these data is 1 . 6 x 1 0 - 8 M. In further studies, the inhibitor ICI 200,355 (3 x 10 8 M) produced a rightward, parallel shift of the concentration-response curve to human neutrophil elastase. The shift was slightly more than one log (fig. 4). ICI 200,355 had no effect on the secretory response ot serous cells to chymase, cathepsin G or Pseudomonas aeruginosa elastase (each, 10-8 M) at a concentration (1
6
O
0.4
0
r
2000
,/•
0.3
z a m o
0.2
O/•/z~/~'
I000
01 o. o " - 2" ~ ' : t z -•: ~ - - ° : - L - :
Ol
I I
60
120
'u . . . .
180
u, . . . .
240
9.~W
300
V
,u.
360
INCUBATION TIME (MIN)
ADDITION OF ICI 200,355
Fig. 2. Inhibition of h u m a n neutrophil elastase-catalyzed elastinolysis by ICI 200,355. ( o ) Control; ( I ) 0.2 # M ICI 200,355; (zx) 0.4 # M ICI 200,355; (A) 0.8 ktM ICI 200,355; (D) 0 # M , h u m a n neutrophil elastase. The inhibitor was added 10 rain after h u m a n neutrophil elastase and substrate had been combined. Symbols with vertical lines represent means:t: S.E.M. (n = 3).
0 -~
...... ~=~0~" ..............
-10
10
-9
10
8
10
-7
10
H u m a n N e u t r o p h i l Elastase [ M ]
Fig. 4. Competitive inhibition of secretion of h u m a n leukocyte elastase-induced secretion of macromolecules by ICI 200,355. (11) H u m a n neutrophil elastase alone; (D) h u m a n neutrophil elastase plus ICI 200,355 (3 × 10 -8 M). Data represent m e a n s + S.E.M. (n = 5).
157
2000
i--~x~
,,-4,
--,w-
x> >(> x> x>
X> X~ X> X>
X~
x> x> x> x>
1000
i
x~ x> x~ X> ×A
~>
K> b b
153
EJP 51704
Inhibition of human neutrophil elastase by ICI 200,355 Christian P. S o m m e r h o f f 1, R o b e r t D. Krell, J o s e p h L. Williams, B r u c e C. G o m e s , A n n e M. S t r i m p l e r and J a y A. N a d e l Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, CA 94143, U.S.A. and Department of Pharmacology, ICI Americas, Inc., Wilmington, DE 19897, U.S.A. Received 13 August 1990, revised MS received 31 October 1990, accepted 13 November 1990
To examine the pathogenetic role of neutrophil elastase in airway hypersecretion, we have studied the novel inhibitor of this enzyme, [4-(4-bromophenylsulfonylcarbamoyl)benzoyl-L-valyl-L-proline1 (RS)-(1-trifluroacetyl-2-methylprolyl)amide] (ICI 200, 355). This compound was a potent (K i = 0.6 _+0.22 riM) inhibitor of human neutrophil elastase and a much weaker inhibitor ot other hydrolases. ICI 200,355 also inhibited the ongoing destruction of insoluble elastin by human neutrophil elastase. ICI 200,355 produced a concentration-dependent inhibition of the secretory response induced by human neutrophil elastase (10-s M), with an ICs0 of 1.6 × 10 -8 M. ICI 200,355 had no effect on baseline secretion or on the secretory response to chymase, cathepsin G oi Pseudomonas aeruginosa elastase. Thus, ICI 200,355 appears to be a useful tool for investigating the role of human neutrophi] elastase in inflammatory disorders associated with hypersecretion, such as cystic fibrosis, chronic bronchitis, and asthma. Airway; Submucosal gland; Mucus; Serine protease; Elastin (insoluble); Neutrophil elastase (EC 3.4.21.11)(human); Proteinase inhibitor; Hydrolase; (Bovine)
1. Introduction
Human neutrophil elastase is a neutral serine proteinase with broad substrate specificity which is released from neutrophils during phagocytosis and upon cell death (Weissmann et al., 1972; Ohlsson and Olsson, 1977). This enzyme has been implicated in the pathogenesis of a variety of inflammatory diseases such as emphysema, acute respiratory distress syndrome, and rheumatoid arthritis (Janoff, 1985a; Bonney and Smith, 1986; Janoff, 1985b). Evidence has also been presented that it may be involved in the pathogenesis of increased and abnormal airway secretions commonly associated with airway inflammatory diseases. Thus, increased numbers of neutrophils are found in the airways of subjects with abnormal airway secretions such as smokers (Reynolds and Merrill, 1981; Hunninghake et al., 1979) and in patients with chronic bronchitis and cystic fibrosis (Esterly and Oppenheimer, 1968; Gibson
a Present address: Department of Clinical Chemistry and Clinical Biochemistry, Surgical Clinic City, University of Munich, Munich, F.R.G. Correspondence to: J.A. Nadel, Cardiovascular Research Institute, Box 0130, University of California, San Francisco, CA 94143-0130, U.S.A.
et al., 1989). Continuous mucus hypersecretion in exsmokers has also been associated with ongoing inflammation of airway submucosal glands (Mullen et al.. 1987). Also, levels of free neutrophil elastase in the airways may be greatly increased in smokers and in patients with chronic bronchitis and cystic fibrosk, (Goldstein and Doring, 1986; Stockley and Burnett. 1979; Weitz et al., 1987; Janoff et al., 1983; Tetley el al., 1987). Finally, neutrophil elastase has been shown to release cell-surface glycoconjugates from trachea] surface epithelial cells in vitro (Varsano et al., 1987'. Kim et al., 1987; Breuer et al., 1989) and to inducc goblet cell discharge and hypertrophy in hamsters in vivo (Breuer et al., 1987). In addition, among othel proteinases such as human neutrophil cathepsin G, masl cell chymase and 'classic' mediators such as histamine neutrophil elastase is the most potent secretagogue for cultured serous cells of airway submucosal glands (Sommerhoff et al., 1989; Sommerhoff et al., 1990), th~ structures that are considered to be the major source ol airway secretions in humans (Reid, 1960). The pathogenetic role of human neutrophil elastas~ in conditions such as airway hypersecretion can only b~ validated by the use of potent and selective inhibitors oJ the enzyme in vivo. To aid these investigations, we hav~ studied the novel inhibitor of human neutrophil elastase, [4-(4-bromophenylsulfonylcarbamoyl)benzoyl-L.
154
0
mostatically controlled, water-jacketed holder in the cell compartment of a Cary B210 spectrophotometer and was allowed to reach thermal equilibrium. The temperature was maintained at 25 + 0.1°C. The reaction was initiated by the addition of 5 #1 enzyme solution (0.14 m g / m l ) . Absorbance was continuously monitored and stored in a D E C PDP 11/32 minicomputer. Initial and steady state velocities were calculated by a fit of the experimental data to a linear dependence on time by linear least-squares analysis. Triplicate determinations were conducted for each inhibitor concentration.
cF,
Fig. 1. Structure of ICI 200,355 ([4-(4-bromophenylsulsulfonylcarbamoyl)benzoyl-L-valyl-L-proline l(RS)-(1-trifluroacetyl-2-methylprolyl)amidel). valyl-L-proline 1 (RS)-(1-trifluroacetyl-2-methylprolyl) amide] (ICI 200,355) (fig. 1). Our results demonstrate that this compound is a potent and selective inhibitor of human neutrophil elastase that blocks not only the amidolytic activity and the ongoing destruction of insoluble elastin but also the secretion from cultured airway gland serous cells induced by the enzyme. ICI 200,355 therefore appears promising as a tool for the in vivo investigation of the role of human neutrophil elastase in inflammatory disorders and, subsequently, perhaps for a rational therapy of diseases involving airway hypersecretion, such as cystic fibrosis, chronic bronchitis, and asthma.
2. Materials and methods
2.1. Determination of kinetics of inhibition of human neutrophil elastase The substrate methoxysuccinyl-Ala-Ala-Pro-Valp N A was hydrolyzed by human neutrophil elastase releasing p-nitroanalide which was continuously measured spectrophotometrically by monitoring absorbance changes at 410 nm. Both substrate and inhibitor were dissolved in DMSO. Fifty microliters of both substrate and inhibitor or D M S O were added to a cuvette containing 2.895 ml butter I (10 m M N a phosphate, 500 m M NaC1, p H 7.6), The cuvette was placed in a ther-
2.2. Determination of protease selectivity The protease selectivity of ICI 200,355 was determined by using minor modifications of the protocol above. All substrates were dissolved in D M S O and were combined with inhibitor (dissolved in the same solvent). Equal volumes (50 #1) of inhibitor and substrate were added to 2.895 ml buffer I in a cuvette, and the cuvettes were placed in the spectrophotometer described above. When the reaction mixtures had reached thermal equilibrium (25°C), the reactions were initiated by the addition of 5 /~1 enzyme, and absorbance was continuously monitored at a wavelength determined by the substrate-leaving group. Assay conditions for the various enzymes are illustrated in table 1. 2.3. Inhibition of human neutrophil elastase-catalyzed hydrolysis of insoluble elastin
Bovine neck elastin was suspended at a concentration of 20 m g / m l in 0.1 M Tricine, 0.75 M NaC1 (pH 8.0) buffer containing 250 ~1 triton X-100/250 ml. The suspension was stirred at room temperature for 18 h, then it was washed on a glass-fritted funnel with buffer, and resuspended in fresh buffer. The elastin solution was diluted with an equal volume of buffer to give a
TABLE 1 Assay conditions for the various enzymes. Enzyme Porcine pancreatic elastase Bovine pancreatic chymotrypsin Human plasma thrombin Acetylcholinesterase Human leukocyte cathepsin G
Substrate (concentration) Succinyl-Ala-Ala-Ala-pNA(0.5 mM) Succinyl-Ala-Ala-Pro-Phe-pNA(155 ttM) Bz-Phe-Val-Arg-pNA(51 ~M) Acetylthiocholine (20 ~ M) Succinyl-Ala-Ala-Pro-Phe-pNA(1 raM)
Bovine pancreatic trypsin Papain
Bz-Phe-Val-Arg-pNA(51 ~M) N-CBZ-GIy-oNP(83 ~M)
Pseudomonas aeruginosa elastase
Bovine ligament elastin (10 mg/ml)
Buffer
Enzyme concentration
Wavelength(nm)
Buffer I Buffer I Buffer 1 Buffer I 0.1 M Tris, 0.7 mM NaN 2 pH 8.3 Buffer I 0.2 M NaPi 0.005 M EDTA pH 6.0 1.65% DMSO 0.8% MeCN Buffer I
24 nM 3.3 nM 5 units/ml 14 nM 25 nM
410 410 410 420 410
3.3 nM 87 nM
410 347.5
0.25/LM
280
155 final volume of 10 mg/ml. Aliquots of substrate (8 ml) were added to 25-mi flasks, and human neutrophil elastase was added to a final concentration of 0.2 /~M. The flasks were stirred continuously at 25°C. Ten minutes after the initiation of the reaction, inhibitor was added to final concentrations of 0.2, 0.4 or 0.8/~M. At various times, 0.75-ml samples were removed from the reaction flasks and were added to centrifuge tubes containing 0.75 ml 0.1 M acetic acid, 4.0 M NaC1 (pH 4.7). After centrifugation, the absorbance of the supernatant at 276 nm was determined on a Cary B210 spectrophotometer.
2.4. Culture of bovine tracheal serous cells A line of bovine trachea gland serous cells was cultured as described previously (Finkbeiner et al., 1986; Sommerhoff et al., 1989). Cells were grown in tissue culture plastic flasks (surface area, 25 cm 2) coated with human placental collagen in medium containing 40% Dulbecco's modified Eagles H21 medium, 40% Ham's F12 medium, 20% fetal calf serum, and 50 /~g/ml gentamicin. Flasks were maintained at 3 7 ° C in 5% CO2-95% air. Cells were studied between passages 10 and 25.
2.5. Release of 35S-labeled macromolecules On day 9, confluent monolayers were incubated with medium containing Na35SO4 (7.5 /~Ci/ml). After 24 h, the medium containing the radiolabel was removed, and the cells were washed 3 times with phosphate-buffered saline. Serum-free medium (50% Dulbecco's modified Eagles H21 medium, and 50% Ham's F12 medium) containing 100 / t g / m l streptomycin and 100 U / m l penicillin was then added and was renewed every 30 min for 210 rain. At 210 min, medium containing proteases was added to the cells. To determine the inhibition of protease-induced secretion by ICI 200,355, proteases were preincubated with ICI 200,355 prior to addition to the cells. In preliminary studies, we compared preincubation for 5 and 30 min and found no differences in the degree of inhibition. Therefore, we performed all studies after 5 min of preincubation. After 30 rain, the medium was collected again, and the culture flasks were examined using a phase microscope ( I T M - 2, Olympus, Cherry Hill, N J) to verify the integrity of the cell monolayer. The spent medium from the 180- to 210-rain and the 210- to 240-rain incubation periods was dialyzed (Spectrapor tubing; MW cutoff, 12 000-14 000 Da), against distilled water containing 10 mg/1 sodium azide to remove unincorporated 35SO4. After addition of scintillation fluid (Hydrofluor, National Diagnostics, Somerville, N J), non-dialyzable 3sS-labeled macromolecules were counted by scintillation spectroscopy to an accu-
racy of 2% (beta counter model LS7500, Beckman Instruments, Inc., Irvine, CA). Secretion was expressed as percent increase of release of 3sS-labeled macromolecules during incubation with the agonists over the release during the immediately preceding time period. This calculation was corrected for the declining baseline, which was determined in controls incubated with medium alone.
2.6. Statistics All values were expressed as means + S.E.M. Statistical analysis was performed using unpaired Student's t-test or analysis of variance and Dunetts multiple comparison test, where appropriate (Zar, 1984). A P value of less than 0.05 was considered significant.
2.7. Materials Media, fetal calf serum, and antibiotics were obtained from the Cell Culture Facility of the University of California, San Francisco. Human placental collagen (type IV), bovine pancreatic trypsin and chymotrypsin, porcine pancreatic elastase, human plasma thrombin, acetylcholinesterase, papain and synthetic enzyme substrates were obtained from Sigma (St. Louis, MO). Carrier-free Na35SO4 (specific activity, 43 C i / m g ) was purchased from ICN Radiochemicals, Inc. (Irvine, CA). Human neutrophil elastase and cathepsin G isolated from human purulent sputum and bovine neck elastin were purchased from Elastin Products (Pacific, MO). Pseudomonas aeruginosa elastase was obtained from Nagase Biochemicals (Kyoto, Japan). Chymase was purified from canine mastocytoma cells as described previously (Caughey et al., 1988). ICI 200,355 was synthesized by the Department of Medicinal Chemistry, ICI Americas, Wilmington, DE.
3. Results
3.1. Potency and selectivity of 1C1 200,355 against several hydrolases ICI 200,355 is a slow, tight, reversible inhibitor of human neutrophil elastase with a K i = 0.60 ___0.22 nM (mean + S.E.M., n = 5). Reaction progress curves for compounds of this type are typically exponential followed by positive linear slope, the reversible nature of the inhibitor being demonstrated by the positive linear, as opposed to a zero, slope. The slow nature of the inhibition of human neutrophil elastase by ICI 200,355 derives from kon = 87 000 _+ 54 000 M -1 s -1 (mean _+ S.E.M., n = 5) compared with more traditional inhibitors with konvalues > 107 M -1 s -1. Pharmacologically,
156 TABLE 2 Potency and selectivity of ICI 200,355 against several hydrolases.
I O0
/
80
Enzyme
K i (nM)
H u m a n leukocyte elastase Porcine pancreatic elastase Bovine pancreatic chymotrypsin Bovine pancreatic trypsin Hunztan plasma thrombin Acetylcholinesterase Papain H u m a n leukocyte cathepsin G Pseudomonas aeruginosa elastase
0.6 + 0.22 ~ 46 + 5 150000 + 2 000 NI b NI c NI ~ 60 000 _+ 8 000 81 000 +_20 000 140 000 d
z 6O o F-
~= 4 0 bq
ij
IIj
i l l
20
a Values are m e a n s + S . E M , of n > 3; b no inhibition at concentrations of 0.1 m M ; ~ no inhibition at concentrations of 1.0 # M ; d iCs0"
10
-10
10
-9
10
-8
10
-7
6
10
ICI 200,355 [M]
differences between traditional and slow binding inhibitors are imperceptible in our hands. In contrast, ICI 200,355 was only a weak inhibitor of several other hydrolases (table 2 and fig. 5). The single exception was porcine pancreatic elastase, where ICI 200,355 demonstrated only a 77-fold lower affinity than for human neutrophil elastase.
3.2. Inhibition of human neutrophil elastase-catalyzed hydrolysis of insoluble elastin The ability of ICI 200,355 to inhibit human neutrophil elastase hydrolysis of insoluble elastin is illustrated in fig. 2. In these experiments, the reaction was initiated by combining human neutrophil elastase with insoluble bovine ligament elastin followed 10 min later by the addition of ICI 200,355. When inhibitor concentration was equal to enzyme concentration (0.2 #M), approximately 33% inhibition of elastinolysis was observed. When inhibitor was in 4-fold excess, approximately 83% inhibition was obtained. 0.6 _/~
0.5
Fig. 3. Concentration-dependent inhibition of h u m a n neutrophil elastase-induced (10 - s M) secretion of macromolecules from serous cells by ICI 200,355. Data are reported as means_+ S.E.M, (n = 5).
3. 3. Inhibition of serous cell secretion by ICI 200, 355 At the highest concentration used in these studies (1 /~M), ICI 200,355 had no effect on baseline secretion of sulfated macromolecules from cultured serous cells. However, ICI 200,355 did inhibit the secretory response induced by 10 -8 M h u m a n neutrophil elastase in a concentration-dependent fashion (fig. 3). A significant inhibition of the elastase-induced response was achieved at concentrations equal to or greater than 10 8 M (P < 0.01). The interpolated ICs0 from these data is 1 . 6 x 1 0 - 8 M. In further studies, the inhibitor ICI 200,355 (3 x 10 8 M) produced a rightward, parallel shift of the concentration-response curve to human neutrophil elastase. The shift was slightly more than one log (fig. 4). ICI 200,355 had no effect on the secretory response ot serous cells to chymase, cathepsin G or Pseudomonas aeruginosa elastase (each, 10-8 M) at a concentration (1
6
O
0.4
0
r
2000
,/•
0.3
z a m o
0.2
O/•/z~/~'
I000
01 o. o " - 2" ~ ' : t z -•: ~ - - ° : - L - :
Ol
I I
60
120
'u . . . .
180
u, . . . .
240
9.~W
300
V
,u.
360
INCUBATION TIME (MIN)
ADDITION OF ICI 200,355
Fig. 2. Inhibition of h u m a n neutrophil elastase-catalyzed elastinolysis by ICI 200,355. ( o ) Control; ( I ) 0.2 # M ICI 200,355; (zx) 0.4 # M ICI 200,355; (A) 0.8 ktM ICI 200,355; (D) 0 # M , h u m a n neutrophil elastase. The inhibitor was added 10 rain after h u m a n neutrophil elastase and substrate had been combined. Symbols with vertical lines represent means:t: S.E.M. (n = 3).
0 -~
...... ~=~0~" ..............
-10
10
-9
10
8
10
-7
10
H u m a n N e u t r o p h i l Elastase [ M ]
Fig. 4. Competitive inhibition of secretion of h u m a n leukocyte elastase-induced secretion of macromolecules by ICI 200,355. (11) H u m a n neutrophil elastase alone; (D) h u m a n neutrophil elastase plus ICI 200,355 (3 × 10 -8 M). Data represent m e a n s + S.E.M. (n = 5).
157
2000
i--~x~
,,-4,
--,w-
x> >(> x> x>
X> X~ X> X>
X~
x> x> x> x>
1000
i
x~ x> x~ X> ×A
~>
K> b b
