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A novel inhibitor of mammalian collagenase
Life Sciences, Vol. 37, pp. 575-578 Printed in the U.S.A.
Pergamon Press
A NOVEL INHIBITOR OF MAMMALIAN COLLAGENASE Donald E. Clark, Peter Wel and Norman H. Grant Wyeth Laboratories, Inc. Philadelphia, PA 19101 (Received in final form June 4, 1985) Summary N-[[[(5-chloro-2-benzothlazolyl)thlolphenyllacetyll-~-cysteine (WY-45,368) is a potent inhibitor of human skin flbroblast collagenase. Kinetic data show that the inhibition is competitive, with a Ki of 3.5 BM. WY-45,368 inhibits neither of two other metalloproteinases, thermolysln and angiotensin converting enzyme, nor does it inhibit clostridial collagenase--thus indicating specificity for mammalian collagenase. Enzymatic destruction of collagen occurs in several diseases, including osteoarthrltls (i), rheumatoid arthritis (2), tumor metastasis (3,4), periodontal disease (5), and corneal ulceration (6). Collagenase is found at the sites of tissue damage in these pathological states, while under normal conditions it is either not produced or it is rendered inactive by naturally occurring inhibitors (7). Moreover, since peptidases other than collagenases do not hydrolyze native collagen under physiological conditions, collagenase levels may be rate limiting for irreversible damage to collagenous connective tissue. Inhibitor studies could give further evidence of the pathological role of collagenase, but so far these have been confined to studies with nonspecific inhibitors such as the chelating agents that mitigate corneal ulceration (8). This paper reports the selective competitive inhibition of human skin fibroblast collagenase (HFC) in vitro by a synthetic agent, N-[[[(5-chloro-2-benzothlazolyl thiolphenyllacetyll-_L-cysteine(WY-45,368). Materials and Methods Reagents. Trypsin, soybean trypsin inhibitor, and trizma were obtained from Sigma Chemical ~ (St. Louis, MO), Sepharose 4B from Pharmacla (Piscataway, N.J.), C acetic anhydride from New England Nuclear (Boston, MA), thermolysln and angiotensln converting enzyme from Calbiochem-Behring Corp. (La Jolla, CA), and collagen II from Elastin Products Co., Inc. (Pacific, MO). clostridial collagenase (Clostrldiopeptldase A) was purified by the method of Grant and A l b u m (9). Cell Culture. The KeJo line of normal human skin flbroblasts (WUMS 80567) was received at passage i0 from Dr. Eugene A. Bauer of Washington University in St. Louis MO. It was grown in Dulbeccos modified Eagles Medium with 10% fetal calf serum. Purification of Human Fibroblast Collagenase. The procollagenase was precipitated from the culture media with 50% ammonium sulfate and further purified on a Sepharose 4B collagen affinity column (i0). Before each assay,
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
576
Inhibitor of Mammalian Collagenase
Vol.
37, No. 6, 1985
procollagenase was activated at 37 ° by the addition of trypsin (optimum amount determined for each batch of procollagenase), followed after ten minutes by the addition of a five-fold excessAof soybean trypsin inhibitor. This preparation, HFC, hydrolyzed collagen to TC and TC fragments as evidenced by SDS polyacrylamide gel electrophoresis without evidence of other peptide fragments. ~4 Assay. Rat ta~l tendon was solubilized, acetylated with ~ C acetic anhydride (7.2 x i0 ~ DPM/mg collagen), and assessed for susceptibility to tryptic hydrolysis by the method of Gisslow and McBride (ii); 10-15% was hydrolyzable by trypsin, similar to the amount found by these authors. The assay system consisted of 25 ~i of collagenase diluted with tris buffer (0.05 M trizma pH 7.4, 5 mM CI~) 90 ~l iris buffer, i0 ~i DMS0 or WY-45,368 dissolved in DMS0, and 50 pg C Collagen in 25 ~l 0.01% acetic acid, added last, to give approximately 50% hydrolysis in one to two hours. The samples were incubated o with gentle shaking at 35 in 1.5 ml Eppendorf micro test tubes, and the undigested fibrils were separated by centrifugation for three minutes in a Beckman microfuge. A 25 ~i aliquot of supernatant was used for counting. Product, as measured by solubilized label, varied directly with concentration of added substrate. In the absence of enzyme, fibril formation from freshly prepared substrate reached completion in two to five minutes, with 4-10% of the label remaining in solution. Nonlabeled peptidlc substrates were assayed on a Hewlett Packard I084B HPLC system using a Regis Spherisorb $5C8 column. Results Wy-45,368 was the most potent inhibitor of mammalian collagenase among a series of compounds screened for this purpose. The IC50 under these conditions is 9.2 ~ 2.6 ~M (four runs). Cysteine, by contrast, inhibits this enzyme with an IC50 greater than 2 mM. Figure i, a Lineweaver-Burke double reciprocal plot in the presence and absence of WY-45,368, shows the convergence at I/V typical of competitive inhibition. Determination of Ki, the dissociation constant of the enzyme-inhibitor complex, from the disparate slopes gave the value 3.5 ~M, which reflects a degree of binding uncommonly high for inhibitors of collagenase. Results similar to those of Figure i were also obtained with the synthetic substrate, N-(cyclopentylcarbonyl)GlyProGlnGlylleAlaGlyNH 2 .
1800 ~ ÷ 1350-
1/V
900-
450-
0 0
3~0
6
900
1200
l/IS] FIG. 1 Mode of inhibition of collagenase b~, Wy-45,368. Ordinate: 1/(initial velocity), expressed as 1/cpm X 10°; abscissa: 1/(concentration of collagen), expressed as 1/cpm X 106. 7.9/JM Wy-45,368 . ÷ - ; No inhibitor = -0-
Vol. 37, No. 6, 1985
Inhibitor of Mammalian Collagenase
577
Table I shows that WY-45,368 inhibits the splitting of collagens by human fibroblast collagenase but that it does not inhibit the facile splitting of either type I collagen or AcProGlnTrplleArgNH 2 by the bacterial metalloprotease thermolysin; nor does it inhibit the activity of still another metalloprotease, angiotensin converting enzyme, on angiotensin I or the degradation of collagen by clostridial collagenase.
TABLE I Specificity of WY-45,368 toward Collagenase-related Enzymes Enzyme (collagenase-related)
Substrate (collagen-related)
Human fihroblast collagenase
Rat tail collagen (type I)
Human fibroblast collagenase
Bovine nasal septum collagen (type II)
Thermolysln
Rat tail collagen (type I)
Thermolysin
AcProGlnTrplleArgNH 2
Clostridial collagenase
Rat tail collagen (type I)
Angiotensin converting enzyme (ACE)
Angiotensin I
WY-45,368 ~M
Inhibition (%)
9
50
i0
50
100
0
17
0
I00
0
17
0
Discussion Selectivity for human fibroblast collagenase is shown by the low degree of inhibitory activity of WY-45,368 against thermolysin, angiotensin converting enzyme, and bacterial collagenase. Thermolysin, like mammalian collagenase, is dependent upon zinc for its catalytic activity. Furthermore, thermolysin, which cleaves both type I collagen and the closely associated type III collagen, has been shown to cleave native type III collagen near the collagenase susceptible region (12). Anglotensin converting enzyme, while having no collagenolytic activity, also possesses a zinc-containing catalytic site. Collagenase from Clostridlum histolyticum is capable of hydrolysing native collagen at multiple sites. Table I shows that these enzymes were not affected by concentrations of WY-45,368 that were multiples of the IC50 found against HFC. Cysteine, a potent chelating agent but a less stable thlol than WY-45,368, inhibits mammalian collagenase weakly. The competitive inhibition by WY-45,368, characterized by facile displacement with substrate, points to blocking of the enzyme-substrate binding site, rather than chelation of zinc at the enzyme's catalytic site. Acknowledgements We thank Mrs. Marlene Ronchetti-Blume for expertly coaxing optimal amounts of collagenase from the sometimes reluctant fibroblasts, Dr. Eugene Rosanoff for his help in obtaining and growing the cells, Mr. Jack Kassarlch for competent technical assistance, and Dr. Paul Christner of the University of Pennsylvania School of Dentistry for providing the leukocyte collagenase that helped initiate the study.
578
Inhibitor of Mammalian Collagenase
Vol. 37, No. 6, 1985
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12.
J.P. PELLETIER, J.M. PELLETIER, D.S. HOWELL, L. GHANDUR-MNAYMNEH, J.E. ENIS, J.F. WOESSNER. Arthritis Rheum. 26, 63-68 (1983). E.D. HARRIS JR., S.M. KRANE. Arthritis Rheum. 14, 669-84 (1971). D. TARIN, B.J. HOYT, D.J. EVANS. Br. J. Cancer 46, 266 (1982). L.A. LIOTTA, K. TRYGGVASON, S. GARBISA, I. HART, C.M. FOLTZ, S. SHAFIE. Nature 284, 67 (1980). P.B. ROBERTSON, J. SIMPSON. J. Periodontol 47, 29-33 (1976). M. BERMAN, in Collagenase in Normal and Pathological Connective Tissues eds. D. Woolley and J. Evanson, p.141, John Wiley.(1980). E. HARPER, Ann. Rev. Biochem. 49, 1063-78 (1980). M. BERMAN. Int. Ophthalmol. Clinics 15, 49-66 (1975). N.H. GRANT, H.E. ALBURN. Arch. Biochem Biophys. 82, 245-255 (1959). C. GILLET, Y. EECKHOUT, G. VAES. FEBS Lett. 74, 126-128 (1977). M.T. GISSLOW, B.C. MCBRIDE. Anal. Biochem 68, 70-78 (1975). H.M. WANG, J. CHAN, D.W. PETTIGREW, J. SODEK. Biochem. Biophys. Acta 533, 270-77 (1978).
Pergamon Press
A NOVEL INHIBITOR OF MAMMALIAN COLLAGENASE Donald E. Clark, Peter Wel and Norman H. Grant Wyeth Laboratories, Inc. Philadelphia, PA 19101 (Received in final form June 4, 1985) Summary N-[[[(5-chloro-2-benzothlazolyl)thlolphenyllacetyll-~-cysteine (WY-45,368) is a potent inhibitor of human skin flbroblast collagenase. Kinetic data show that the inhibition is competitive, with a Ki of 3.5 BM. WY-45,368 inhibits neither of two other metalloproteinases, thermolysln and angiotensin converting enzyme, nor does it inhibit clostridial collagenase--thus indicating specificity for mammalian collagenase. Enzymatic destruction of collagen occurs in several diseases, including osteoarthrltls (i), rheumatoid arthritis (2), tumor metastasis (3,4), periodontal disease (5), and corneal ulceration (6). Collagenase is found at the sites of tissue damage in these pathological states, while under normal conditions it is either not produced or it is rendered inactive by naturally occurring inhibitors (7). Moreover, since peptidases other than collagenases do not hydrolyze native collagen under physiological conditions, collagenase levels may be rate limiting for irreversible damage to collagenous connective tissue. Inhibitor studies could give further evidence of the pathological role of collagenase, but so far these have been confined to studies with nonspecific inhibitors such as the chelating agents that mitigate corneal ulceration (8). This paper reports the selective competitive inhibition of human skin fibroblast collagenase (HFC) in vitro by a synthetic agent, N-[[[(5-chloro-2-benzothlazolyl thiolphenyllacetyll-_L-cysteine(WY-45,368). Materials and Methods Reagents. Trypsin, soybean trypsin inhibitor, and trizma were obtained from Sigma Chemical ~ (St. Louis, MO), Sepharose 4B from Pharmacla (Piscataway, N.J.), C acetic anhydride from New England Nuclear (Boston, MA), thermolysln and angiotensln converting enzyme from Calbiochem-Behring Corp. (La Jolla, CA), and collagen II from Elastin Products Co., Inc. (Pacific, MO). clostridial collagenase (Clostrldiopeptldase A) was purified by the method of Grant and A l b u m (9). Cell Culture. The KeJo line of normal human skin flbroblasts (WUMS 80567) was received at passage i0 from Dr. Eugene A. Bauer of Washington University in St. Louis MO. It was grown in Dulbeccos modified Eagles Medium with 10% fetal calf serum. Purification of Human Fibroblast Collagenase. The procollagenase was precipitated from the culture media with 50% ammonium sulfate and further purified on a Sepharose 4B collagen affinity column (i0). Before each assay,
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
576
Inhibitor of Mammalian Collagenase
Vol.
37, No. 6, 1985
procollagenase was activated at 37 ° by the addition of trypsin (optimum amount determined for each batch of procollagenase), followed after ten minutes by the addition of a five-fold excessAof soybean trypsin inhibitor. This preparation, HFC, hydrolyzed collagen to TC and TC fragments as evidenced by SDS polyacrylamide gel electrophoresis without evidence of other peptide fragments. ~4 Assay. Rat ta~l tendon was solubilized, acetylated with ~ C acetic anhydride (7.2 x i0 ~ DPM/mg collagen), and assessed for susceptibility to tryptic hydrolysis by the method of Gisslow and McBride (ii); 10-15% was hydrolyzable by trypsin, similar to the amount found by these authors. The assay system consisted of 25 ~i of collagenase diluted with tris buffer (0.05 M trizma pH 7.4, 5 mM CI~) 90 ~l iris buffer, i0 ~i DMS0 or WY-45,368 dissolved in DMS0, and 50 pg C Collagen in 25 ~l 0.01% acetic acid, added last, to give approximately 50% hydrolysis in one to two hours. The samples were incubated o with gentle shaking at 35 in 1.5 ml Eppendorf micro test tubes, and the undigested fibrils were separated by centrifugation for three minutes in a Beckman microfuge. A 25 ~i aliquot of supernatant was used for counting. Product, as measured by solubilized label, varied directly with concentration of added substrate. In the absence of enzyme, fibril formation from freshly prepared substrate reached completion in two to five minutes, with 4-10% of the label remaining in solution. Nonlabeled peptidlc substrates were assayed on a Hewlett Packard I084B HPLC system using a Regis Spherisorb $5C8 column. Results Wy-45,368 was the most potent inhibitor of mammalian collagenase among a series of compounds screened for this purpose. The IC50 under these conditions is 9.2 ~ 2.6 ~M (four runs). Cysteine, by contrast, inhibits this enzyme with an IC50 greater than 2 mM. Figure i, a Lineweaver-Burke double reciprocal plot in the presence and absence of WY-45,368, shows the convergence at I/V typical of competitive inhibition. Determination of Ki, the dissociation constant of the enzyme-inhibitor complex, from the disparate slopes gave the value 3.5 ~M, which reflects a degree of binding uncommonly high for inhibitors of collagenase. Results similar to those of Figure i were also obtained with the synthetic substrate, N-(cyclopentylcarbonyl)GlyProGlnGlylleAlaGlyNH 2 .
1800 ~ ÷ 1350-
1/V
900-
450-
0 0
3~0
6
900
1200
l/IS] FIG. 1 Mode of inhibition of collagenase b~, Wy-45,368. Ordinate: 1/(initial velocity), expressed as 1/cpm X 10°; abscissa: 1/(concentration of collagen), expressed as 1/cpm X 106. 7.9/JM Wy-45,368 . ÷ - ; No inhibitor = -0-
Vol. 37, No. 6, 1985
Inhibitor of Mammalian Collagenase
577
Table I shows that WY-45,368 inhibits the splitting of collagens by human fibroblast collagenase but that it does not inhibit the facile splitting of either type I collagen or AcProGlnTrplleArgNH 2 by the bacterial metalloprotease thermolysin; nor does it inhibit the activity of still another metalloprotease, angiotensin converting enzyme, on angiotensin I or the degradation of collagen by clostridial collagenase.
TABLE I Specificity of WY-45,368 toward Collagenase-related Enzymes Enzyme (collagenase-related)
Substrate (collagen-related)
Human fihroblast collagenase
Rat tail collagen (type I)
Human fibroblast collagenase
Bovine nasal septum collagen (type II)
Thermolysln
Rat tail collagen (type I)
Thermolysin
AcProGlnTrplleArgNH 2
Clostridial collagenase
Rat tail collagen (type I)
Angiotensin converting enzyme (ACE)
Angiotensin I
WY-45,368 ~M
Inhibition (%)
9
50
i0
50
100
0
17
0
I00
0
17
0
Discussion Selectivity for human fibroblast collagenase is shown by the low degree of inhibitory activity of WY-45,368 against thermolysin, angiotensin converting enzyme, and bacterial collagenase. Thermolysin, like mammalian collagenase, is dependent upon zinc for its catalytic activity. Furthermore, thermolysin, which cleaves both type I collagen and the closely associated type III collagen, has been shown to cleave native type III collagen near the collagenase susceptible region (12). Anglotensin converting enzyme, while having no collagenolytic activity, also possesses a zinc-containing catalytic site. Collagenase from Clostridlum histolyticum is capable of hydrolysing native collagen at multiple sites. Table I shows that these enzymes were not affected by concentrations of WY-45,368 that were multiples of the IC50 found against HFC. Cysteine, a potent chelating agent but a less stable thlol than WY-45,368, inhibits mammalian collagenase weakly. The competitive inhibition by WY-45,368, characterized by facile displacement with substrate, points to blocking of the enzyme-substrate binding site, rather than chelation of zinc at the enzyme's catalytic site. Acknowledgements We thank Mrs. Marlene Ronchetti-Blume for expertly coaxing optimal amounts of collagenase from the sometimes reluctant fibroblasts, Dr. Eugene Rosanoff for his help in obtaining and growing the cells, Mr. Jack Kassarlch for competent technical assistance, and Dr. Paul Christner of the University of Pennsylvania School of Dentistry for providing the leukocyte collagenase that helped initiate the study.
578
Inhibitor of Mammalian Collagenase
Vol. 37, No. 6, 1985
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12.
J.P. PELLETIER, J.M. PELLETIER, D.S. HOWELL, L. GHANDUR-MNAYMNEH, J.E. ENIS, J.F. WOESSNER. Arthritis Rheum. 26, 63-68 (1983). E.D. HARRIS JR., S.M. KRANE. Arthritis Rheum. 14, 669-84 (1971). D. TARIN, B.J. HOYT, D.J. EVANS. Br. J. Cancer 46, 266 (1982). L.A. LIOTTA, K. TRYGGVASON, S. GARBISA, I. HART, C.M. FOLTZ, S. SHAFIE. Nature 284, 67 (1980). P.B. ROBERTSON, J. SIMPSON. J. Periodontol 47, 29-33 (1976). M. BERMAN, in Collagenase in Normal and Pathological Connective Tissues eds. D. Woolley and J. Evanson, p.141, John Wiley.(1980). E. HARPER, Ann. Rev. Biochem. 49, 1063-78 (1980). M. BERMAN. Int. Ophthalmol. Clinics 15, 49-66 (1975). N.H. GRANT, H.E. ALBURN. Arch. Biochem Biophys. 82, 245-255 (1959). C. GILLET, Y. EECKHOUT, G. VAES. FEBS Lett. 74, 126-128 (1977). M.T. GISSLOW, B.C. MCBRIDE. Anal. Biochem 68, 70-78 (1975). H.M. WANG, J. CHAN, D.W. PETTIGREW, J. SODEK. Biochem. Biophys. Acta 533, 270-77 (1978).