Low Molecular Weight, Sequence Based, Collagenase Inhibitors Selectively Block the Interaction Between Collagenase and TIMP (Tissue Inhibitor of Metalloproteinases)

Matrix Vol. 10/1990, pp. 292-299 © 1990 by Gustav Fischer Verlag, Stuttgart

Low Molecular Weight, Sequence Based, Collagenase Inhibitors Selectively Block the Interaction Between Collagenase and TIMP (Tissue Inhibitor of Metalloprotei nases) YVES LELIEVRE, ROMAINE BOUBOUTOU, JANINE BOIZIAU, DIDIER FAUCHER, DANIEL ACHARD and TERENCE CARTWRIGHT Institut de Biotechnologie de Vitry et Departement de Chimie, Centre de Recherches de Vitry-Alfortville, France.

Abstract Sequence-based inhibitors of collagenase bearing an hydroxamate group capable of chelating the active site zinc atom were synthesized and tested. The effect of one of these molecules (RP 59 794; Ki about 10- 8M) on the formation of the TIMP: collagenase complex was also tested. RP 59794 blocks complex formation and can partially dissociate established TIMP: collagenase complexes. It exhibits the same stereospecificity in this activity as in its inhibition of collagenase suggesting that TIMP and RP 59 794 both interact with the active site region of collagenase. Key words: collagenase inhibitors, TIMP.

Introduction Degradation of interstitial collagen occurs in several connective tissue diseases including the arthritides (Harris et al., 1970), periodontal disease (Birkedal-Hansen, 1980), and corneal ulceration (Berman, 1980); it is also probably involved in the mechanism of tumour invasion (Liotta et al., 1980). In higher animals, collagen degradation is mediated by collagenase (EC 3.4.24), a metalloproteinase which specifically cleaves triple helical collagen molecules at a single site between gly 775 - leu 776 or gly 775 - ile 776 depending on the type of collagen. The possible role of collagenase in these pathologies has stimulated considerable study of specific collagenase inhibitors. Several synthetic inhibitors have been described that are based on chelation of the active site zinc atom of collagenase by moities carried on peptide mimetics that respect the substrate specifity of collagenase (Dickens et al., 1986; Shaw et al., 1987; Johnson et al., 1987; Cartwright et al., 1987). A natural low molecular weight inhibitor of collagenase which apparently functions by the same mechanism

has also been described (Faucher et al., 1987; Lelievre et al., 1989). Natural macromolecular inhibitors of collagenase also exist. The best known of these, TIMP, (Tissue Inhibitor of Metalloproteinase) is a specific tight-binding inhibitor of collagenase and related metalloproteinases which blocks collagenase activity by an 1: 1 stoichiometric interaction with an apparent Kd of 1.41O- IOM (Cawston et al., 1983). TIMP activity was initially demonstrated in rabbit bone (Sellers et al., 1977) and in culture supernatants derived from bovine aorta (Nolan et al., 1980). Later it was shown to be widely distributed in tissues. TIMP is a 23-kDa glycoprotein, and has now been cloned in E. coli and yeast (Docherty et al., 1985; Kaczorek et al., 1987). Efforts to produce active fragments of TIMP have not succeeded (Faucher et al., 1989), and little is known about its mechanism of action. It was thus of interest to test whether some of the more powerful small molecule inhibitors could influence the interaction between collagenase and TIMP, and whether this might provide clues to the mechanism of this interaction. By coupling a zinc-chelating hydroxamate

Synthetic Collagenase Inhibitors and TIMP residue to a peptide sequence analogous to that immediately C-terminal to the cleaved bond, we have designed molecules that specifically inhibit collagenase. We investigated the effect of one these inhibitors (RP 59 794 or Molecule 9, Table I: Ki 10- 8 M) on complex formation between TIMP and collagenase. RP 59 794 selectively blocked complex formation with the same stereospecificity as that observed for its own, direct, inhibition of collagenase, suggesting that TIMP and RP 59 794 may compete for the same site on the enzyme.

Materials and Methods Collagenase Collagenase was prepared from the culture supernatant of porcine synovial cells (Dayer et aI., 1976). Purification was as described by Cawston et aI. (1979) but with an additional immunopurification step using anti-collagenase monoclonal antibody (Crespo et aI., 1988). Briefly antibody was immobilized on Sepharose 4B and the collagenase was fixed in 50mM Tris/HCl, pH7.6 containing 5mM CaCh, 300 mM NaCI and 0.05% Brij 35. Collagenase was eluted with 10 mM acetic acid pH 4,5 mM CaCI 2 , 150 mM NaCl, 0.05% Brij 35 and fractions were immediately neutralized by collection into tubes containing 50 mM Tris/ HCl,pH7.6.

Collagenase and Inhibitor Assays [14C]-labelled rat skin collagen was used in a diffuse fibril assay similar to that described (Cawston et aI., 1979). Collagenase activity was also estimated using a synthetic octapeptide substrate (Masui et aI., 1977). For the assay of collagenase inhibitors, 30 f-tl of [14C]-collagen (200,000 dpm/mg, 5 mg/ml) in CH3COOH 1 mM, was diluted to 130 f-tl with 60 mM Tris, 15 mM CaCI2> pH 7.6, then 30 f-tl of H 2 0 and 20 f-tl of an aqueous solution of the inhibitor to be tested were added. The enzyme reaction was initiated by addition of 20111 of collagenase (3.2 U/ml in the same buffer) and the mixture was incubated for 18 h at 36°C and then centrifuged for 15 min at 10,000 g. 160 f-tl of supernatant was transferred to scintillation vials containing 3 ml of Ready -Solv HP/b Beckman and counted using a Beckman LS 3801 scintillation counter. ICso was determined directly using a Sord micro-computer and Ki was then determined from this value (Cheng et aI., 1976).

Preparation of Synthetic Collagenase Inhibitors Peptide hydroxamate derivatives were synthesized as already described (Cartwright et aI., 1987). Briefly the synthesis of N-[3-(hydroxy amino) carbonyl -2-isobutyl -1-oxo propyll amino acid derivatives started from ethyl 2-isobutylene 3-carboxypropanoate (C 2 Hs-

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OOC-CHrC[ =CH-CH(CH3hl-COOH), obtained according to Cohen and Milovanovic (1968). This starting material was coupled with appropriate C-protected amino acids. After the ethyl ester was hydrolyzed in alkaline medium, a second coupling step with a-benzyl hydroxylamine in the presence of dicyclohexylcarbodiimide was used to add the protected hydroxamate group. Finally catalytic hydrogenation resulted in both deprotection of the hydroxamate group and saturation of the isobutylene double bond giving the synthetic inhibitors as diastereoisomeric mixtures. Diastereoisomers were separated by reverse phase HPLC. Precise conditions varied for different inhibitors, but for RP 59 794, 50 f-tl of the mixture at 1O- 3M in 10% dimethyl sulphoxide in water were applied to a C18 column (10 f-t, 0.46 x 25 cm) equilibrated with 0.07% trifluoracetic acid (TFA) in 10% acetonitrile. The products were eluted using a linear gradient (40 ml, 1 mVmin) to 0.07% TFA in 75% acetonitrile. For inhibition studies, synthetic inhibitors were dissolved in dimethyl sulphoxide at 10- 2 M, then diluted in water to give the required concentration.

Other enzyme assays Aminopeptidase M activity was measured by the method of Appel (1974), angiotensin converting enzyme by that of Hayakari et aI. (1978), enkephalinase according to Florentin et aI. (1984) and thermolysin as described by Fritz et aI. (1974).

Preparation afTIMP and study afTIMP-Collagenase complexes Pure TIMP was produced from bovine aortic fibroblasts as described by Nolan et aI. (1980). For study of complex formation, solutions of pure TIMP and of pure collagenase wre prepared at about 10- 6 M in 50 mM cacodylate buffer, pH 7.4, 5 mM CaCl2 • The solutions were mixed to give equimolar concentration of enzyme and inhibitor and incubated together (5 min at 36°C) for complex formation. To study antagonism of complex formation, potential inhibitors in aqueous solution were incubated at the desired concentration (5 min 36°C) with the collagenase prior to mixing with TIMP. To study dissociation of a preformed TIMP: collagenase complex, the inhibitors were added after formation of the complex as described above and incubated for a further 3 h at 36°C. After reaction, the mixture was centrifuged (2 min, 10,000 g) and 175 f-tl was injected into an HPLC gel-filtration column (TSK SW 2000 + TSK SW 3000 in tandem, each column 2 X 30 em) and eluted with a buffer containing 200 mM sodium sulphate, 10 mM sodium cacodylate, 5 mM CaCh, pH 7.4. The column was calibrated using bovine albumin (67 kDa), ovalbumin (43 kDa), chymo-

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trypsin (25 kDa) and ribonuclease (13.7 kDa) as molecular weight standards.

Results Synthetic inhibitors of collagenase

Several potential collagenase inhibitors were synthesized with a structure based on the amino acid sequence Cterminal to the collagenase cleavage site and carrying an

hydroxamate group at their N-terminal end. The best inhibitors of this series had apparent Ki values of 0.4 - 3 x 1O- 8 M (Tablel, molecules 9, 11, 12, 14, 15). One of these molecules, RP 59 794, N-[3-(hydroxyamino) carbonyl-2isobutyl-1-oxo propyl) valine benzylamide (molecule 9 in Tablel), was choosen for more detailed analysis of collagenase inhibition in this study. Previous studies (Lelievre et aI., 1989) have shown the importance of the presence of a methylene group between the hydroxamate moiety and the P'1 site, and that the P'1 site itself needs to be hydrophobic

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1.3 10- 8M

10

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13

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VAL

14

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0.3 10-~ 0.7 1O-4M 0.4 10- 5M

10-7 M

1. 8 10- 7M 0.3 10-7M 2

10- 7M

0.7 la- 8M 0.4l0- 8M >0.41O-4M

Table!. Inhibition of collagenase by hydroxamate derivatives of pep tides analogous to the sequence C-terminal to the scissile bond. P'2 represents the lateral chain of amino acids labelled as G2.

Synthetic Collagenase Inhibit?rs and TIMP although its precise structure is not very critical (Dickens et aI., 1986; Cartwright et aI., 1987). The P'2 site appears also to require a hydrophobic residue since introduction of groups of lower hydrophobicity results in dramatic loss of activity (TableI, molecules 1,2,4,5,8). Substitution of Dvaline for L-valine at this point also results in activity loss (Table I, molecule 3). Adding an aromatic group to the C-terminus improves activity, presumably be increasing overall hydrophobicity in this region. Using less hydrophobic groups progressively lowers activity (Table I, molecules 10, 11, and 13). The effect of the aromatic C-terminal can also be achieved by addition of o-methyl tyrosine in the P'2 position (molecules 14 and 15 and Dickens et a1. (1986).

Table n. Inhibition specificity: comparison of the activity of the two diastereoisomers of RP 59794 on several metalloproteinases. Inhibition by TIMP is included in the table for comparison. Enzyme

Specificity of inhibition In order to test the specificity of action of this class of inhibitor, both stereo isomers of RP 59 794 were tested against collagenase using either [14C]-collagen or a synthetic octapeptide as substrate, and also against a variety of other metalloproteases. Results of these experiments are summarized in Table II. Stereoisomer A, which is the most active on collagenase is also the most active on aminopeptidase M, thermolysin and angiotensin converting enzyme, although the difference between the two forms is much less marked than for collagenase. For enkephalinase however, the stereospecificity is reversed and form B behaves as a potent enkephalinase inhibitor (Ki measured in other experiments 10- 8 M), (Lelievre et aI., unpublished data). RP 59794 shows considerable specificity towards collagenase inhibition and, importantly, is effectively inactive with respect to angiotension converting enzyme (Table II).

Enzyme inhibition by TIMP The activity of TIMP was also measured in the same enzyme assays and was shown to be specific for the matrix metaUoproteinases (Table II). Inhibition of collagenase by TIMP was examined using

Molecule 9 FormB FormA Ki Ki

T.I.M.P.

Ki

Collagenase Substrate: collagen Substrate: peptide

1.3 1O- 8 M 4.51O- 8 M

0.61O- 5 M 0.31O- 9 M 0.3 1O- 5 M 2.11O- 8 M

Angiotensin converting Enzyme (A.C.E.)

>10- 4 M

inactive

Aminopeptidase M

1.61O- 6 M

4.61O- 6 M inactive at 1O- 6 M

Enkephalinase

3.

Thermolysin

l.21O- 7 M

Collagenase inhibition is stereospecific As synthesized, the synthetic inhibitors were racemic mixtures from which the diastereoisomers were resolved by reverse phase HPLC. In each case, one diastereoisomer, designated "form A", was about 500-fold more active than the other form (data for RP 59 794 shown in Table 11). The same stereospecificity was observed in a test of bone resorption from mouse calvaria stimulated by PTH, a system in which metalloproteinases are thought to be involved (Delaisse et aI., 1985). RP 59 794 form A, the form most active in collagenase inhibition, was also active in this model while the other diastereoisomer was essentially inactive (Vaes, personal communication).

295

1O- 7 M

0.31O- 4M

",,11O- 8 M N.D.

llO- 7 M inactive at 1O- 7 M

N. D.: not determined

both collagen and an octapeptide as substrate. In each case, collagenase activity was fully inhibited by TIMP at relative concentrations which suggest the formation of a 1: 1 molar ratio enzyme: inhibitor complex.

TIMP - Collagenase complex formation Mixtures of TIMP and procollagenase or active collagenase were pre-incubated together and then analysed by HPLC gel filtration. In the HPLC system used, pure pro collagenase was eluted at 37.2 min, active collagenase at 41.3 min, and TIMP at 37.3 min (Fig. 1, data for procollagenase not shown). When TIMP and collagenase were preincubated at a 1: 1 molecular ratio, a new molecular species was observed which eluted at 35.1 min, corresponding to an apparent molecular weight of 55 kDa. The same complex was seen if either enzyme or substrate were present in excess; no higher molecular weight complexes were seen under any conditions (data not shown). When procollagenase was incubated with TIMP, no complex formation occurred. Although the complex and its individual components were incompletely resolved in this system, the resolution obtained permitted easy identification of peaks corresponding to the complex, to free collagenase and to TIMP (Fig. 2). As previously reported (Faucher et aI., 1989; Murphy et aI., 1989), the complex could be dissociated by SDS-PAGE. The same pattern was observed in either reducing or nonreducing conditions and the complex dissociated into two components that were electrophoretically identical to the free active enzyme and the free inhibitor. No other bands were observed.

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When RP 59 794 was added in lOOO-fold molar excess (10- 3 M) to a preformed TIMP-collagenase complex that showed no free TIMP or enzyme, the complex was partially dissociated and free TIMP and collagenase appeared. However dissociation was limited to 20-30% (data not shown). Higher concentrations could not be used due to the limited solubility of the inhibitor in aqueous medium.

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Pre-incubation of collagenase (0.8 X lO- 6M) with increasing concentrations of unresolved RP 59 794 before incubation with TIMP (0.8 x 10- 6 M) resulted in a dose dependent inhibition of TIMP-collagenase complex formation as shown by the progressive appearance of HPLC peaks corresponding to free enzyme and free inhibitor (Fig. 2). When the resolved diasteroisomers of RP 59 794 were tested separately in another experiment for their ability to block complex formation, RP 59 794 form A showed potent inhibition, blocking complex formation completely at 10- 3 M, while the B form reduced complex formation by only about 10% at the same concentration (data not shown). Thus, RP 59 794 displays the same stereospecificity in inhibition of TIMP-collagenase complex formation as it does in inhibition of collagenase activity and in the bone resorption model.

o

10

20 30 40 50 60 TIME (minutes)

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Fig. 1. Demonstration of the formati on of a TIMP: collagenase complex. Pure collagenase (Panel a) , pure TIMP (Panel b) or an equimolar mixture of the two (panel c) were chromatographed by HPLC gel permeation. The mixture showed a single peak at a retention time (35.1 min.) distinct from either collagenase (39.5 min.) or TIMP (37.3 min.) corresponding to the enzyme: inhibitor complex. Column calibration: bovine albumin (67 kDa) : 34.7 min., ovalbumin (43 kDa ): 37.4 min., chymotrypsin (25 kDa ): 41.4 min., ribonuclease (13.7 kDa): 42.6 min., Flow rate 0,5 ml/min.

The series of collagenase inhibitors reported in this study and those described elsewhere (Dickens et al., 1986; Shaw et al., 1987; Johnson et al., 1987; Lelievre et al., 1989) indicate that, with this type of inhibitor, the collagen sequence around the collagen cleavage site must be mimicked in order to achieve effective inhibition. It is thus supposed that these molecules act by being recognized as substrate analogues and by subsequently chelating the enzyme's active site zinc atom (Johnson et al., 1987). The observation reported here that inhibitors such as RP 59 794 can block the formation of the TIMP-collagenase complex, and can, at high molar excess, partially dissociate preformed complexes, suggests that these inhibitors and TIMP may compete for the same region of collagenase. By analogy, this region should include the active site of the enzyme, although the possibility cannot be excluded that RP 59 794 induces a conformational change in the enzyme, distant from the active site, that hinders complex formation with TIMP. The view that the active site of collagenase is directly

297

Synthetic Collagenase Inhibitors and TIMP 40

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TIME (minute.) Fig. 2. Inhibition of collagenase: TIMP complex formation by increasing concentrations of RP 59 794: HPLC gel filtration of constant amount of an equimolar mixture of TIMP and collagenase incubated with RP 59 794 at a: 23, b: 230 or c: 2300-fold molar excess. (EI = TIMP-collagenase complex, 1= TIMP, Cse = collagenase).

involved in the interaction with TIMP is supported by the fact that only the stereoisomer of RP 59794 which is itself active as a collagenase inhibitor blocks the TIMP-enzyme complex. This argues against a non-specific conformation effect of RP 59 794, as does the fact that the active stereoisomer of RP 59 794 inhibits collagenase much more strongly than it inhibits other metalloproteases (Table II). That TIMP does not form a complex with procollagenase, in which the active site is masked, also supports this view. Since the peptide inhibitors were modelled on substrate, it is possible that "collagen-like" regions might exist in TIMP which are recognized by active collagenase. Analysis of the sequence of TIMP shows two glycine-leucine bonds (G92 - L93 and GI72 - L173) which might resemble the collagen cleavage site, but no other regions are identifiable as being significantly "collagen-like". We have synthesized a peptide corresponding to the sequence flanking the GI72-L173 bond but this peptide showed no anti-collagenase activity (data not shown). Presentation to the enzyme of a substrate-like "bait" region and subsequent effectively irreversible binding to the enzyme is a strategy used by several well known classes of endogenous protease inhibitors. In some cases, such as the aI-proteinase inhibitors, this results in cleavage of the bait region, and subsequent dissociation of the enzyme inhibitor complex yields inactive fragments of inhibitor (Johnson et a1. 1976; 1978). However, TIMP dissociated from the complex appears to be unaltered in this and other studies, and

has been reported to retain biological activity (Murphy et aI., 1989). In inhibition by a2-macroglobulin type inhibitors, the inhibitor binds, often covalently, to the enzyme in such a way as to prevent access of protein substrates to the enzyme by steric hindrance. The active site, however, remains functional and capable of hydrolysis of small peptide substrates (Borth, 1984). TIMP inhibited collagenase action equally using either collagen or an octapeptide as substrate (T able II) suggesting that an a2-macroglobulin-like steric hindrance mechanism is not involved in this case. There was no formation of covalent bonds between collagenase and TIMP. Thus collagenase inhibition by TIMP does not exactly fit either of the two "classical" protease inhibitor strategies although recognition via a substrate-like region and subsequent blocking of the active site remains the most likely mechanism of action. The Ki values for TIMP and for RP 59794 have been determined as lO-lOM and 1O- 8 M respectively using collagen as substrate. This result for TIMP agrees with Kd value of 1.4 10- 10 M found by Cawston et a1. (1983). A 100-fold excess of RP 59 794 should therefore be able to inhibit the TIMP-collagenase complex formation by 50%. In fact, lOOO-fold excess of RP 59 794 is needed to significantly antagonize complex formation indicating that, even though TIMP and RP 59 794 may recognize the same, probably active site, region in collagenase, the TIMP-collagenase

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interaction is reinforced by other inter-molecular contacts giving a "fine tuning" of the interaction between the two molecules (Colman et aI., 1987). Identification of these contacts must await the availability of direct structural information on the TIMP-collagenase complex. An important corollary to the data discussed here is that collagenase inhibitors of the RP 59 794 family might be useful as drugs potentially capable of neutralizing free, active collagenase in diseases where the level of endogenous inhibition by TIMP is decreased, but would not be capable of liberating collagenase already inactivated by complex formation with TIMP. Acknowledgements We acknowledge the contribution of Laurence Barillet, JeanClaude Wallet, Nadine Fromage, Jean-Dominique Guitton, Sylvie M eaux, Andre Crespo and Jean-Michel Cherel. References Appel, W.: Amino acid arylamidases. In: Methods of Enzymatic Analysis, Vol. 2, ed. by Bergmeyer, H.A., Verlag Chemie, Weinheim Academic Press, Inc., 2nd Ed., 1974, pp. 958-974. Berman, M. B.: Collagenase and Corneal Ulceration. In: Collagenase in Normal and Pathological Tissues, ed. by Woolley D. and Evanson,J., Wiley and Sons, New York, 1980, pp.141-174. Birkdael-Hansen, H.: Collagenase in Periodontal diseases. In: Collagenase in Normal and Pathological Tissues, ed. by Woolley, D. and Evanson, J., Wiley and Sons, New York, 1980, pp. 127 -140. Borth, W.:a2-macroglobulin in connective tissue matrix metabolism. Collagen Rei. Res. 4: 83-95, 1984. Cartwright, T., Bouboutou-Tello, R., Lelievre, Y. and FournieZaluski, M. c.: Nouveaux composes aactivite d'inhibition de la collagenase. (Laboratoire Roger Bellon) French Patent 87 00 053 , 1987. Caws ton, T.E. and Barret, A.J.: A rapid and reproducible assay for collagenase using 14C acetylated collagen. Anal. Biochem. 99: 340-345, 1979. Cawston, E., Murphy, G., Mercer, E., Galloway, W.A., Hazleman, B. L. and Reynolds, J.J.: The interaction of purified rabbit bone collagenase with purified rabbit bone metalloproteinase inhibitor. Biochem.J. 211: 313-318, 1983. Cawston, T. E. and Tyler, T.A.: Purification of pig synovial collagenase to high specificity activity. Biochem. J. 183: 647-656, 1979. Cheng, Y. C. and Prusoff, W. H.: Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (ICsol of an enzymatic reaction. Proc. Nat. Acad. Sci. USA 22: 3099-3198, 1976. Cohen, S. G. and Milanovic, A.: Absolute steric course ofhydrolysis by a-chymotrypsin. Esters of a-benzylsuccinic, a methyl-~­ phenylpropionic and a-Methylsuccinic acids. J. Am. Chem. Soc. 90: 3495-3502, 1968. Colman, P.M., Laver, W.G., Varghese, J.N., Baker, A.I., Tulloch, P.A., Air, G.M. and Webster, R.G.: Three dimensional structure of a complex of antibody with influenza virus rieurarninidase. Nature 325: 358- 363, 1987. Crespo, A., Duchesne, M., Cartwright, T., Pernelle, C. and Cherel,

J. M.: Monoclonal antibodies against synovial collagenase: use for immunopurification and characterization of the latent active enzyme. Collagen Rei. Res. 1: 1-10, 1988. Dayer, M., Krane, S. M., Russer, G. and Robinson, D.: Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. Proc. Nat. Acad. Sci. USA 73: 945-949,1976. Delaisse, J. M., Eeckhout, Y., Sear, c., Galloway, A., McCullagh, K. and Vaes, G.: A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture. Biochem. Biophys. Res. Comm. 133: 483-490, 1985. Dickens, J.P., Donald, D. K., Kneen, G. and McKay, W.R. (G.D. SEARLE): Hydroxamic acid based collagenase inhibitors. U.S. PATENT 4, 599, 361, 1986. Docherty, A.J. P., Lyons, A., Smith, B.J., Wright, E.M., Stephens, P. E.and Harris, T.J. R.: Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid potentiating activity. Nature 318: 66-69, 1985. Faucher, D., Lelievre, Y., Boiziau, J., Cornet, P. and Cartwright, T.: Inhibiteur naturel des metalloproteinases: etude structurale et fonctionnelle. Pathologie Biologie 37: 199-205,1989. Faucher, D., Lelievre, Y. and Cartwright, T. : An inhibitor of mammalian collagenase active at micromolar concentration from an Actinomycete culture broth. J. Antibiotics 40: 1757-1761,1987. Fritz, H., Trautschold, I. and Werle, E. : Protease inhibitors. In: Methods in Enzymatic Analysis, ed by Bergmeyer, H. A., Verlag Chemie, Weinheim, Academic Press, Inc., New York, 2nd Ed., 1974, pp. 1071-1074. Florentin, D., Sassi, A. and Roques, P.: A highly sensitive f1uorometric assay for "Enkephalinase", a neutral aminopeptidase that releases tyrosine-glycine-glycine from enkephalins. Anal. Biochem.141: 62-69, 1984. Harris, E.D., Evanson, J.M. Dibona, D.R. and Krane, S. M.: Collagenase and rheumatoid arthritis. Arthr. Rheum. 13: 83-94,1970. Hayakari, M., Kondo, Y. and Izumi, H.: A rapid and simple spectrometric assay of angiotensin-converting enzyme. Anal. Biochem. 84.361-369,1978 . Johnson, W.H., Roberts, N.A. and Borkakoti, N.: Collagenase inhibitors: their design and potential therapeutic use. J. Enzyme Inhibition 2: 1-22,1987. Johnson, D. and Travis, J. : Human aI-proteinase inhibitor mechanism of action: evidence for activation by limited proteolysis. Biochem. Biophys. Res. Comm. 72: 33-39, 1976. Johnson, D. and Travis, J.: Structural evidence for methionine at the reactive site of human aI-proteinase inhibitor. J. BioI. Chern . 253: 7142- 7 144, 1978. Kaczorek, M., Honore, N ., Ribes, V., Dehoux, P., Cornet, P., Cartwright, T. and Streeck, R. E. : Molecular cloning and synthesis of biologically active human tissue inhibitor of metalloproteinases in yeast. Biotechnology 5: 595 - 598, 1987. Lelievre, Y., Bouboutou, R., Boiziau, J. and Cartwright, T.: Inhibition de la collagenase synovia Ie par I'actinonine. Etude de relation structure-activite. Pathologie Biologie 3 7: 43-46, 1989. Liotta, L. A., Tryggvason, K., Garbisa, S., Hart, I., Foltz, C. M. and Shafie, S.: Metastatic potential correlate with enzymatic degradation of basement membrane collagen. Nature 284 : 67-68, 1980. Masui, Y., Takemoto, T., Sakakibara, Ibori, H. and Nagai, Y.: Synthetic substrate for vertebrate collagenase. Biochem. Med. 17: 215-221, 1977. Murphy, G., Koklitis, P. and Carne, F.: Dissociation of tissue

Synthetic Collagenase Inhibitors and TIMP inhibitor of metalloproteinase (TIMP) from enzyme complexes yields fully active inhibitor. Biochern. J. 261: 1031-1034, 1989. Nolan, ].C, Ridge, S.C, Oronsky, A. L. and Kerwar, 5.5.: Purification and properties of collagenase inhibitor from culture of bovine aorta. Atherosclerosis 35: 93 -102, 1980. Sellers, A. and Reynolds,].].: Identification and partial characterization of an inhibitor of collagenase from rabbit bone. J. Bioi. Chern. 167: 353-360, 1977.

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Shaw, A. and Wolanin, D.]., (I.CI): New peptides containing hydroxamic acids useful as inhibitors of metalloproteinases and especially endopeptidases for treating osteoartritis. Eur. Patent Application No. 87300366.9., 1987. Dr. Y. Lelievre, Departement de Biochimie des Macromolecules, Institut de Biotechnologie de Vitry, CR.V.A., 13, quai Jules Guesde, 94403 Vitry Sur Seine Cedex, France.