The Intermediate S1′ Pocket of the Endometase/Matrilysin-2 Active Site Revealed by Enzyme Inhibition Kinetic Studies, Protein Sequence Analyses, and Homology Modeling
2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês
10.1074/jbc.m310109200
ISSN1083-351X
AutoresHyun I. Park, Yonghao Jin, Douglas R. Hurst, Cyrus A. Monroe, Seakwoo Lee, Martin A. Schwartz, Qing‐Xiang Amy Sang,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoHuman matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1′ pocket in MMPs is a well defined substrate P1′ amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1′ site were selected. The Homephe side chain is designed to probe deep S1′ pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1′ pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1′ pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1′ pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1′ pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1′ pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1′ pockets. Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1′ pocket in MMPs is a well defined substrate P1′ amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1′ site were selected. The Homephe side chain is designed to probe deep S1′ pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1′ pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1′ pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1′ pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1′ pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1′ pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1′ pockets. Matrix metalloproteinases (MMPs, 1The abbreviations used are: MMPmatrix metalloproteinaseBoctert-butoxycarbonylBrij-35polyoxyethylene lauryl etherHomophehomophenylalanineMca(7-methoxycoumarin-4-yl)acetylDpaN-3-(2,4-dinitrophenyl)-2,3-diaminopropionylMMPImatrix metalloproteinase inhibitor. matrixins) are believed to participate in angiogenesis, embryonic development, morphogenesis, reproduction, tissue resorption and remodeling, and tumor growth, progression, invasion, and metastasis through breakdown of the extracellular matrix, cell surface proteins, and processing growth factors, cytokines, and chemokines (1Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 207-214Crossref PubMed Scopus (970) Google Scholar, 2Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 163-175Crossref Scopus (5169) Google Scholar, 3Overall C.M. Mol. Biotech. 2002; 22: 51-86Crossref PubMed Google Scholar). Recently, human MMP-26 (endometase/matrilysin 2) was identified and its mRNA expression was detected in normal tissues of the human uterus and placenta, and in many types of malignant tumors (4Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 5Uría J.A. López-Otín C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar, 6De Coginac A.B. Elson G. Delneste Y. Magistrelli G. Jeannin P. Aubry J.-P. Berthier O. Schmitt D. Bonnefoy J.-Y. Gauchat J.-F. Eur. J. Biochem. 2000; 267: 3323-3329Crossref PubMed Scopus (107) Google Scholar, 7Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 359: 705-718Crossref Google Scholar). Characterization of the MMP-26 promoter suggests that this proteinase may be expressed in cancer cells of epithelial origin (8Marchenko G.N. Marchenko N.D. Leng J. Strongin A.Y. Biochem. J. 2002; 363: 253-262Crossref PubMed Scopus (88) Google Scholar). MMP-26 may play an important role in human prostate and breast cancer invasion (9Zhao Y.-G. Xiao A.-Z. Newcomer R.G. Park H.I. Kang T. Chung L.W. Swanson M.G. Zhau H.E. Kurhanewicz J. Sang Q.-X. J. Biol. Chem. 2003; 278: 15056-15064Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 10Zhao Y.-G. Xiao A.-Z. Park H.I. Newcomer R.G. Yan M. Man Y.G. Heffelfinger S.C. Sang Q.-X. Cancer Res. 2003; (in press)Google Scholar). matrix metalloproteinase tert-butoxycarbonyl polyoxyethylene lauryl ether homophenylalanine (7-methoxycoumarin-4-yl)acetyl N-3-(2,4-dinitrophenyl)-2,3-diaminopropionyl matrix metalloproteinase inhibitor. MMP-26 cleaves type I gelatin, α1-proteinase inhibitor, fibrinogen, fibronectin, vitronectin, type IV collagen, and insulin-like growth factor binding protein-1 (4Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 7Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 359: 705-718Crossref Google Scholar, 11Park H.I. Turk B.E. Gerkema F.E. Cantley L.C. Sang Q.-X. J. Biol. Chem. 2002; 277: 35168-35175Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Studies of MMP-26 indicate that it has substrate specificity similar to other MMPs, with the exception of a preference for Ile at the P2 and P2′ positions, for small residues at the P3′ and P4′ positions, and Lys at the P4 position (11Park H.I. Turk B.E. Gerkema F.E. Cantley L.C. Sang Q.-X. J. Biol. Chem. 2002; 277: 35168-35175Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). MMP-26 also hydrolyzes several synthetic fluorogenic peptide substrates designed for stromelysin-1, gelatinases, collagenases, and tumor necrosis factor-α converting enzyme (4Park H.I. Ni J. 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Fernandez-Catalan C. Tschesche H. Grams F. Nagase H. Maskos K. Cell. Mol. Life Sci. 1999; 55: 639-652Crossref PubMed Scopus (300) Google Scholar, 20Nagase H. Clendeninn N.J. Appelt K. Matrix Metalloproteinase Inhibitors in Cancer Therapy. Humana Press, Totowa, NJ2001: 39-66Google Scholar). One type is a shallow pocket, as found in MMP-1 (human fibroblast collagenase; 13) and MMP-7 (matrilysin; 16), where the pockets are limited by the side chains of Arg and Tyr, respectively, crossing the pockets. Many of the structurally known MMPs possess Leu at the corresponding site, and its side chain forms the top of the pocket rather than crossing the pocket. These Leu-containing MMPs may be further classified as deep and intermediate S1′ pocket MMPs. A deep, tunnel-like pocket is found in MMP-3 (stromelysin-1; 12), MMP-12 (metalloelastase; 17), and MMP-14 (MT1-MMP; 21), whereas MMP-2 (gelatinase A; 22), MMP-8 (human neutrophil collagenase; 15), and MMP-9 (gelatinase B; 23) possess an intermediate-sized pocket, which is neither deep nor shallow. An enzyme with a shallow pocket prefers large, aliphatic residues in the P1′ position, such as Leu and Met (24Netzel-Arnett S. Sang Q.-X. Moore W.G.I. Narve M. Birkedal-Hansen H. Van Wart H.E. Biochemistry. 1993; 32: 6427-6432Crossref PubMed Scopus (162) Google Scholar, 25Nagase H. Fields C.G. Fields G.B. J. Biol. Chem. 1994; 269: 20952-20957Abstract Full Text PDF PubMed Google Scholar). The remainder of the MMPs can accommodate larger amino acid derivatives, such as homophenylalanine, in the P1′ position (26Mucha A. Cuniasse P. Kannan R. Beau F. Yiotakis A. Basset P. Dive V. J. Biol. Chem. 1998; 273: 2763-2768Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). MMP-26, composed of 261 amino acid residues and lacking a hemopexin-like domain, represents the smallest member of the MMP family. The primary structure of MMP-26 can be divided into three regions that include a signal peptide, a propeptide domain, and a catalytic domain. MMP-26 identification, expression, and substrate specificity have been explored by several groups (4Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 5Uría J.A. López-Otín C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar, 6De Coginac A.B. Elson G. Delneste Y. Magistrelli G. Jeannin P. Aubry J.-P. Berthier O. Schmitt D. Bonnefoy J.-Y. Gauchat J.-F. Eur. J. Biochem. 2000; 267: 3323-3329Crossref PubMed Scopus (107) Google Scholar, 7Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 359: 705-718Crossref Google Scholar, 8Marchenko G.N. Marchenko N.D. Leng J. Strongin A.Y. Biochem. J. 2002; 363: 253-262Crossref PubMed Scopus (88) Google Scholar, 9Zhao Y.-G. Xiao A.-Z. Newcomer R.G. Park H.I. Kang T. Chung L.W. Swanson M.G. Zhau H.E. Kurhanewicz J. Sang Q.-X. J. Biol. Chem. 2003; 278: 15056-15064Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 10Zhao Y.-G. Xiao A.-Z. Park H.I. Newcomer R.G. Yan M. Man Y.G. Heffelfinger S.C. Sang Q.-X. Cancer Res. 2003; (in press)Google Scholar, 11Park H.I. Turk B.E. Gerkema F.E. Cantley L.C. Sang Q.-X. J. Biol. Chem. 2002; 277: 35168-35175Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, the S1′ pocket characteristics of MMP-26 are unknown because of the absence of an MMP-26 x-ray crystallographic structure. Therefore, in this study we have utilized previously characterized and newly developed mercaptosulfide MMPIs (27Schwartz, M. A., and Van Wart, H. E. (October 3, 1995) U. S. Patent 5455262Google Scholar, 28Sang Q.-X. Jia M.C. Schwartz M.A. Jaye M.C. Kleinman H.K. Ghaffari M.A. Luo Y.L. Biochem. Biophys. Res. Commun. 2000; 274: 780-786Crossref PubMed Scopus (16) Google Scholar, 29Jin Y. Ghaffari M.A. Schwartz M.A. Tetrahedron Lett. 2002; 43: 7319-7321Crossref Scopus (13) Google Scholar) together with protein sequence analyses and molecular modeling to understand the S1′ pocket characteristics of MMP-26. Materials—The fluorescent peptide substrates for MMPs used in this study were purchased from Bachem Chemical Co. The metal salts and Brij-35 were purchased from Fisher Scientific Inc. The hydroxamate MMPIs 444237, 444238, 444225, and GM6001 were purchased from Calbiochem. All other chemicals were purchased from Sigma. The mercaptosulfide inhibitors were prepared and characterized as previously described (27Schwartz, M. A., and Van Wart, H. E. (October 3, 1995) U. S. Patent 5455262Google Scholar, 28Sang Q.-X. Jia M.C. Schwartz M.A. Jaye M.C. Kleinman H.K. Ghaffari M.A. Luo Y.L. Biochem. Biophys. Res. Commun. 2000; 274: 780-786Crossref PubMed Scopus (16) Google Scholar, 29Jin Y. Ghaffari M.A. Schwartz M.A. Tetrahedron Lett. 2002; 43: 7319-7321Crossref Scopus (13) Google Scholar). cis-1-Acetylthio-2-tert-butoxycarbonylthiocyclopentane and cis-3-acetylthio-4-tert-butoxycarbonylthio-N-tert-butoxycarbonylpyrrolidine were synthesized (29Jin Y. Ghaffari M.A. Schwartz M.A. Tetrahedron Lett. 2002; 43: 7319-7321Crossref Scopus (13) Google Scholar) and S-alkylated with (2S)-2-bromo-4-methylpentanoic acid or (2S)-2-bromo-4-phenylbutanoic acid; the latter bromoacids were derived from l-leucine and l-homophenylalanine, respectively (27Schwartz, M. A., and Van Wart, H. E. (October 3, 1995) U. S. Patent 5455262Google Scholar). Subsequent coupling with l-PheNHMe or l-leucine-p-methoxyanilide (27Schwartz, M. A., and Van Wart, H. E. (October 3, 1995) U. S. Patent 5455262Google Scholar) afforded the S-Boc and N-Boc protected inhibitors as mixtures of two diastereomers. The N-Boc group was selectively removed and replaced by the other acyl groups (29Jin Y. Ghaffari M.A. Schwartz M.A. Tetrahedron Lett. 2002; 43: 7319-7321Crossref Scopus (13) Google Scholar). The diastereomers were separated by flash chromatography on silica gel or by reverse-phase preparative high performance liquid chromatography on a C18 column. The slower-eluting S-Boc protected diastereomer exhibited the more potent MMP inhibition in each case. Its stereochemistry was assigned by 1H NMR NOE analysis (MAG-182), x-ray crystallography (YHJ-294-2) (29Jin Y. Ghaffari M.A. Schwartz M.A. Tetrahedron Lett. 2002; 43: 7319-7321Crossref Scopus (13) Google Scholar), or by analogy. Finally, the S-Boc protecting groups were removed by brief treatment with 2 N HCl in acetic acid and the mercaptosulfide inhibitors were isolated by lyophilization of the reaction mixture. MAG-181: m.p. 174–176 °C; [α]25D + 11.2° (c = 0.4, MeOH); analysis (CHNS). S-Boc derivative: m.p. 118–119 °C; [α]25D + 33.5° (c = 0.49, MeOH); analysis (CHNS). MAG-182: m.p. 173–174 °C; [α]25D + 98.6° (c = 0.45, MeOH); analysis (CHNS). S-Boc derivative: m.p. 159–160 °C; [α]25D + 63.4 (c = 0.52, MeOH); analysis (CHNS). YHJ-72: m.p. 136–137 °C; [α]20D – 67.9° (c = 0.14, CHCl3); analysis (CHNS). S-Boc derivative: m.p. 94–95 °C; [α]25D +0.4° (c = 0.24, CHCl3); analysis (CHNS). YHJ-73: m.p. 145–146 °C; [α]20D – 0.7° (c = 0.14, CHCl3); analysis (CHNS). S-Boc derivative: m.p. 126–127 °C; [α]25D – 8.8° (c = 0.25, CHCl3); HRMS. YHJ-294-1: m.p. 98–100 °C; [α]20D + 54.4° (c = 0.50, MeOH); analysis (CHNS). S-Boc derivative: m.p. 123–124 °C; [α]20D + 11.5° (c = 0.55, MeOH); analysis (CHNS). YHJ-294-2: m.p. 128–130 °C; [α]20D + 38.5° (c = 0.40, MeOH); analysis (CHNS). S-Boc derivative: m.p. 173–175 °C; [α]20D + 82.6° (c = 0.50, MeOH); analysis (CHNS). YHJ-74: m.p. 174–175 °C; [α]20D + 2.4° (c = 0.50, CDCl3); HRMS. S-Boc derivative: m.p. 112–113 °C; [α]20D – 42.1° (c = 0.24, CHCl3); analysis (CHNS). YHJ-75: m.p. 105–106 °C; [α]20D – 35.4° (c = 0.24, CHCl3); analysis (CHNS). S-Boc derivative: m.p. 171–172 °C; [α]20D + 17.2° (c = 0.25, CHCl3); HRMS. Enzyme Preparation and Folding of the Denatured Protein—MMP-7/matrilysin, MMP-3/stromelysin-1 (30Sang Q.-X. Birkedal-Hansen H. Van Wart H.E. Biochim. Biophys. Acta. 1995; 1251: 99-108Crossref PubMed Scopus (96) Google Scholar), and MMP-12/metalloelastase (4Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) were kindly provided by Dr. Harold E. van Wart (Roche Diagnostics), Professor L. Jack Windsor (Indiana University), and Dr. C. Bruun Schiødt (OsteoPro A/S), respectively. MMP-1/human fibroblast collagenase, MMP-2/human fibroblast gelatinase, MMP-8/human neutrophil collagenase, and MMP-9/human neutrophil gelatinase were described previously (30Sang Q.-X. Birkedal-Hansen H. Van Wart H.E. Biochim. Biophys. Acta. 1995; 1251: 99-108Crossref PubMed Scopus (96) Google Scholar, 31Sang Q.A. Bodden M.K. Windsor L.J. J. Prot. Chem. 1996; 15: 243-253Crossref PubMed Scopus (41) Google Scholar). The catalytic domain of MT1-MMP/MMP-14 was provided by Professor Harald Tschesche (Bielefeld University) (32Hurst D.R. Schwartz M.A. Ghaffari M.A. Jin Y. Tschesche H. Fields G.B. Sang Q.-X. Biochem. J. October 8, 2003; (10.1042/BJ20031067)PubMed Google Scholar). MMP-26 was prepared as described previously (4Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 11Park H.I. Turk B.E. Gerkema F.E. Cantley L.C. Sang Q.-X. J. Biol. Chem. 2002; 277: 35168-35175Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, MMP-26 was expressed as inclusion bodies from a transformed BL-21 DE3 strain. After bacterial insoluble body preparation with B-Per™ reagent, the isolated insoluble protein was folded by following the procedures previously outlined (4Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 5Uría J.A. López-Otín C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar, 6De Coginac A.B. Elson G. Delneste Y. Magistrelli G. Jeannin P. Aubry J.-P. Berthier O. Schmitt D. Bonnefoy J.-Y. Gauchat J.-F. Eur. J. Biochem. 2000; 267: 3323-3329Crossref PubMed Scopus (107) Google Scholar, 7Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 359: 705-718Crossref Google Scholar, 8Marchenko G.N. Marchenko N.D. Leng J. Strongin A.Y. Biochem. 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For inhibition assays, 10 μl of inhibitor stock solution, 176 μl of assay buffer, and 10 μl of enzyme stock solution were mixed and incubated for 30 to 60 min prior to initiation of the assay, which was accomplished by adding and mixing 4 μl of the substrate stock solution. Enzyme concentrations ranged from 0.2 to 7 nm during the assay. Apparent inhibition constant Kiapp values were calculated by fitting the kinetic data to the Morrison equation for tight-binding inhibitors (34Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (736) Google Scholar, 35Copeland R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. 2nd Ed. Wiley-VCH, Inc., New York2000Crossref Google Scholar), where νi and ν0 are the initial rates with and without inhibitor, respectively, and [E]o and [I]o are the initial (total) enzyme and inhibitor concentrations, respectively.vivo=[E]o-[I]o-Kiapp+([I]o+Kiapp-[E]o)2+4[E]oKiapp2[E]o(Eq. 1) Determination of Mercaptosulfide Inhibitor Concentration—The active inhibitor concentrations were estimated by titrating the mercapto group with 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) as described previously (36Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar, 37Riddles P.W. Blakeley R.L. Zerner B. Anal. Biochem. 1979; 94: 75-81Crossref PubMed Scopus (929) Google Scholar). Briefly, the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) with the mercapto group produces 2-nitro-5-thiobenzoic acid. The concentration of 2-nitro-5-thiobenzoic acid is then measured by monitoring the absorbance at 412 nm. Cysteine was used to generate the standard curve with a molar extinction coefficient of 14,000 ± 500 m–1 cm–1, which is close to the value in the literature (37Riddles P.W. Blakeley R.L. Zerner B. Anal. Biochem. 1979; 94: 75-81Crossref PubMed Scopus (929) Google Scholar). Computational Protein Sequence Analyses and Homology Modeling Structure of MMP-26 —The sequence alignment of MMP catalytic domains was performed by the PILEUP program in Genetics Computer Group (GCG) software (Wisconsin Package version 10), with a default gap weight of 8 and gap length weight of 2. To align MMP-2 and -9, the 183-residue inserts of fibronectin type II-like modules were deleted before the alignment. The homology modeling structure of the MMP-26 catalytic domain was constructed using the Swiss Model program (38Peitsch M.C. Bio/Technology. 1995; 13: 658-660Crossref Scopus (116) Google Scholar, 39Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (900) Google Scholar, 40Guex N. 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Soc. 1989; 111: 4379-4386Crossref Scopus (1209) Google Scholar) with the Amber force field modified to include parameters for zinc and calcium. Residues within 7 Å of the inhibitor were included in the minimizations. All modeling was performed using the continuum solvent model. The crystallographic structures of MMP-1 (Protein Data Bank number 1HFC) (44Spurlino J.C. Smallwood A.M. Carlton D.D. Banks T.M. Vavra K.J. Johnson J.S. Cook E.R. Falvo J. Wahl R.C. Pulvino T.A. Wendoloski J.J. Smith D.L. Proteins. 1994; 19: 98-109Crossref PubMed Scopus (196) Google Scholar), MMP-7 (Protein Data Bank number 1MMQ) (16Browner M.F. Smith W.W. Castelhano A.L. Biochemistry. 1995; 34: 6602-6610Crossref PubMed Scopus (252) Google Scholar), MMP-8 (Protein Data Bank number 1BZS) (45Matter H. Schwab W. Barber D. Billen G. Hasse B. Neises B. Schudok M. Thorwart W. Schreuder H. Brachvogel V. Lönze P. Weithmann K.U. J. Med. Chem. 1999; 42: 1908-1920Crossref PubMed Scopus (90) Google Scholar), MMP-12 (Protein Data Bank number 1JK3) (17Lang R. Kocourek A. Braun M. Tschesche H. Huber R. Bode W. Maskos K. J. Mol. Biol. 2001; 312: 731-742Crossref PubMed Scopus (96) Google Scholar), and MMP-14 (Protein Data Bank number 1BUV) (21Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (315) Google Scholar) were used for comparison of the S1′ pocket. Inhibition of MMPs with Mercaptosulfide MMPIs—An inhibitor set consisting of eight mercaptosulfide inhibitors was chosen to evaluate the S1′ pocket of MMP-26 (Fig. 1). These inhibitors contain P1′ and P2′ residues and have a mercapto and a sulfide group as a possible bidentate metal-binding moiety. The inhibitors contain a Leu side chain (MAG-181 and -182 and YHJ-294-1 and -2) or a Homophe side chain (YHJ-72, -73, -74, and -75) at the P1′ site. These inhibitors were tested against MMPs with known pocket characteristics (MMP-1–3, -7–9, -12, and -14). The inhibition potency of this class of inhibitors for the MMPs is significantly enhanced with a β-H configuration at the five-membered ring containing the mercapto and sulfide groups. The inhibitors with a Leu side chain are more potent against the shallow pocket MMPs, MMP-1/human fibroblast collagenase, and MMP-7/matrilysin than those with a Homophe side chain. Inhibitors with a Homophe side chain (YHJ-72, -73, 74, and -75) were more potent against the known deep-pocket MMPs such as MMP-3, -12, and -14 than those with Leu side chain. The inhibitors with the Leu side chain at the P1′ site (MAG-182 and YHJ-294-2) inhibit MMP-7 (40 and 26 nm, respectively) and MMP-12 (130 and 93 nm, respectively) without significant differences in Kiapp values. However, the presence of Homophe at the P1′ site dramatically distinguishes MMP-12 from MMP-7. YHJ-73 efficiently inhibits MMP-12 (13 nm), however, the potency is decreased to 1 μm against MMP-7. This trend is also displayed by YHJ-75, which has a high nmKiapp value against MMP-7 (300 nm) but retains potency against MMP-12 (5.6 nm). This dramatic change of potency because of changes in the P1′ site of the inhibitors is consistently observed with the remaining shallow- and deep-pocket MMPs. MMPs with an intermediate pocket can also accommodate the Homophe at the P1′ residue. However, the difference in inhibitor potency observed with Leu or Homophe at the P1′ residue is not as remarkable as that in the shallow- and deep-pocket MMPs. Inhibitors containing Leu at the P1′ site (MAG-182 and YHJ-294-2) are only slightly more potent against MMP-2 and MMP-9 than inhibitors with Homophe (YHJ-73 and -75). These Homophe inhibitors are still potent against MMP-8 with Kiapp values in the low nanomolar range. In general, these results indicate that mercaptosulfide inhibitors are suitable for characterizing the S1′ pocket of MMPs. Characteristics of the S1′ Pocket of MMP-26 as Probed by Mercaptosulfide MMPIs—Inhibition constants for the inhibitors in Fig. 1 were measured with MMP-26 (Table I). YHJ-294-2 is the most potent inhibitor of MMP-26 among the mercaptosulfide inhibitors tested, with a Kiapp value of 2.8 nm. MMP-26 also favors the β-H configuration at the cyclopentyl or pyrrolidine ring moiety in the inhibitor. Addition of the urea-substituted pyrrolidine ring in place of the cyclopentyl ring (YHJ-294-1 and -2; YHJ-74 and -75) enhances the stereoselectivity for the β-H configuration. Importantly, MMP-26 prefers Leu over Homophe at the S1′ site, similar to the intermediate pocket MMPs, MMP-2, -8, and -9.Table IInhibition of human MMPs by mercaptosulfide MMP inhibitorsInhibitorKiappShallowDeepIntermediateMMP-1MMP-7MMP-3MMP-12MMP-14MMP-2MMP-8MMP-9MMP-26nmnmnmMAG-18168071025001,300259854.14481MAG-1824940470130241.10.890.5717YHJ-72>12 × 1035,500150130380930530180160YHJ-73>12 × 1031,000100131620708.628YHJ-294-15,2003,500>40 × 10316,0003,000430130550450YHJ-294-21002636093136.11.21.22.8YHJ-743600270117208830022082YHJ-752400300215.63.76.9443.08.6 Open table in a new tab Characterization of MMP-26 S1′ Pock
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