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• W2022172589 abstract "Gelatinases have been shown to play a key role in angiogenesis and tumor metastasis. Small molecular weight synthetic inhibitors for these enzymes are highly sought for potential use as anti-metastatic agents. Virtually all of the known inhibitors of matrix metalloproteinases (MMPs) are broad spectrum. We report herein the synthesis and kinetic characterization of two compounds, 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol (compound 1) and 5-(4-phenoxyphenylsulfonyl)pentane-1,2-dithiol (compound 2), that are potent and selective gelatinase inhibitors. These compounds are slow, tight-binding inhibitors of gelatinases (MMP-2 and MMP-9) with Ki values in the nanomolar range. In contrast, competitive inhibition of the catalytic domain of membrane-type 1 metalloproteinase (MMP-14cat) with comparable Ki values (Ki ∼200 nm) was observed. Binding to stromelysin (MMP-3) was substantially weaker, with Ki values in the micromolar range (Ki ∼10 μm). No binding to matrilysin (MMP-7) and collagenase 1 (MMP-1) was detected at inhibitor concentrations up to 60 μm. We have previously shown that synthetic MMP inhibitors work synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP in a process that depends on the affinity of the inhibitor toward MT1-MMP. It is shown herein that the dithiols are significantly less efficient (>100-fold) than marimastat, a broad-spectrum MMP inhibitor, in enhancing pro-MMP-2 activation in cells infected to express MT1-MMP, consistent with the lower affinity of the dithiols toward MT1-MMP. Thus, in contrast to broad-spectrum MMP inhibitors, the dithiols are less likely to promote MT1-MMP-dependent pro-MMP-2 activation in the presence of TIMP-2, while maintaining their ability to inhibit active MMP-2 effectively. Gelatinases have been shown to play a key role in angiogenesis and tumor metastasis. Small molecular weight synthetic inhibitors for these enzymes are highly sought for potential use as anti-metastatic agents. Virtually all of the known inhibitors of matrix metalloproteinases (MMPs) are broad spectrum. We report herein the synthesis and kinetic characterization of two compounds, 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol (compound 1) and 5-(4-phenoxyphenylsulfonyl)pentane-1,2-dithiol (compound 2), that are potent and selective gelatinase inhibitors. These compounds are slow, tight-binding inhibitors of gelatinases (MMP-2 and MMP-9) with Ki values in the nanomolar range. In contrast, competitive inhibition of the catalytic domain of membrane-type 1 metalloproteinase (MMP-14cat) with comparable Ki values (Ki ∼200 nm) was observed. Binding to stromelysin (MMP-3) was substantially weaker, with Ki values in the micromolar range (Ki ∼10 μm). No binding to matrilysin (MMP-7) and collagenase 1 (MMP-1) was detected at inhibitor concentrations up to 60 μm. We have previously shown that synthetic MMP inhibitors work synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP in a process that depends on the affinity of the inhibitor toward MT1-MMP. It is shown herein that the dithiols are significantly less efficient (>100-fold) than marimastat, a broad-spectrum MMP inhibitor, in enhancing pro-MMP-2 activation in cells infected to express MT1-MMP, consistent with the lower affinity of the dithiols toward MT1-MMP. Thus, in contrast to broad-spectrum MMP inhibitors, the dithiols are less likely to promote MT1-MMP-dependent pro-MMP-2 activation in the presence of TIMP-2, while maintaining their ability to inhibit active MMP-2 effectively. Matrix metalloproteinases (MMPs) 1The abbreviations used are: MMPmatrix metalloproteinaseDMEMDulbecco's modified Eagle's mediumMe2SOdimethyl sulfoxideMT-MMPmembrane-type MMPTIMPtissue inhibitor of metalloproteinase are zinc-dependent endopeptidases known to play key roles in normal and pathological conditions involving remodeling and turnover of extracellular matrix, such as embryonic development, wound healing, angiogenesis, arthritis, cardiovascular diseases, and cancer. In cancer, MMPs are known to be required at all stages of tumorigenesis, including tumor establishment and growth, neovascularization, intravasation, extravasation, and metastasis (1.Kleiner D.E. Stetler-Stevenson W.G. Cancer Chemother. Pharmacol. 1999; 43: S42-S51Crossref PubMed Scopus (645) Google Scholar,2.McCawley L.J. Matrisian L.M. Mol. Med. Today. 2000; 6: 149-156Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). MMPs are expressed as zymogenic latent enzymes. The zymogenic form has a propeptide that achieves coordination to the catalytic zinc ion by a strictly conserved cysteine residue (for a comparative review of MMP structures see Ref. 3.Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (703) Google Scholar). MMP activation occurs when the cysteine thiolate-zinc ion coordination is broken, usually as a consequence of two proteolytic cleavages of the propeptide, either by autolysis or by hydrolytic action of other proteinases (4.Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar, 5.Murphy G. Stanton H. Cowell S. Butler G. Knauper V. Atkinson S. Gavrilovic J. Acta Pathol. Microbiol. Immunol. Scand. 1999; 107: 38-44Crossref PubMed Scopus (391) Google Scholar, 6.Kotra L.P. Cross J.B. Shimura Y. Fridman R. Schlegel H.B. Mobashery S. J. Am. Chem. Soc. 2001; 123: 3108-3113Crossref PubMed Scopus (26) Google Scholar). MMPs are inhibited by the tissue inhibitors of metalloproteinases (TIMPs), a family of four proteins that are the natural inhibitors of MMPs. matrix metalloproteinase Dulbecco's modified Eagle's medium dimethyl sulfoxide membrane-type MMP tissue inhibitor of metalloproteinase A subgroup of the MMP family, gelatinases A and B (MMP-2 and MMP-9, respectively), has been shown to play a key role in angiogenesis and tumor metastasis (7.Moses M.A. Stem Cells. 1997; 15: 180-189Crossref PubMed Scopus (263) Google Scholar, 8.Nguyen M. Arkell J. Jackson C.J. Int. J. Biochem. Cell Biol. 2001; 33: 960-970Crossref PubMed Scopus (264) Google Scholar). MMP-2 has been the subject of intense investigation since the observation was made that its activation correlates with tumor spread and poor prognosis (9.Seiki M. Acta Pathol. Microbiol. Immunol. Scand. 1999; 107: 137-143Crossref PubMed Scopus (274) Google Scholar). Latent MMP-2 (pro-MMP-2) is activated by membrane-type MMPs (MT-MMPs) (10.Polette M. Birembaut P. Int. J. Biochem. Cell Biol. 1998; 30: 1195-1202Crossref PubMed Scopus (126) Google Scholar, 11.Ellerbroek S.M. Stack M.S. Bioessays. 1999; 21: 940-949Crossref PubMed Scopus (134) Google Scholar), a unique MMP subfamily currently comprising six members (MT1- to MT6-MMP). Contrary to the other MMPs, which are secreted into the extracellular milieu, the MT-MMPs are tethered to the plasma membrane either by a transmembrane domain (MT1-, MT2-, MT-3 and MT5-MMP) or by a glycosylphosphatidylinositol anchor (MT4- and MT6-MMP). MT1-MMP (MMP-14) (12.Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2377) Google Scholar) is known to be the primary pro-MMP-2 physiological activator through a highly regulated mechanism (13.Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar) involving TIMP-2 (14.Brew K. Dinakarpandian D. Nagase H. Biochim. Biophys. Acta. 2000; 1477: 267-283Crossref PubMed Scopus (1608) Google Scholar). It has been shown that TIMP-2 binds active MT1-MMP through the N-terminal cysteine residue (15.Fernandez-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, 16.Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. van Westrum S.S. Crabbe T. Clements J. d'Ortho M.P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar), whereas the C-terminal domain of TIMP-2 interacts with the hemopexin-like domain of pro-MMP-2 (17.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), forming a ternary complex (18.Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar) that efficiently concentrates pro-MMP-2 on the cell surface, thereby promoting its interaction with a neighboring TIMP-2-free MT1-MMP. MT1-MMP cleaves pro-MMP-2 at the Asn37–Leu38 peptide bond, generating an inactive 64-kDa intermediate. Subsequently, a second autolytic cleavage at Asn80–Tyr81 bond achieves the full activation of pro-MMP-2 (19.Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). This process requires protonation of the coordinated cysteine thiolate before its dissociation from the active site zinc ion (6.Kotra L.P. Cross J.B. Shimura Y. Fridman R. Schlegel H.B. Mobashery S. J. Am. Chem. Soc. 2001; 123: 3108-3113Crossref PubMed Scopus (26) Google Scholar). The key role that MMPs play in neovascularization and metastasis has made these enzymes major targets for therapeutic intervention, and therefore synthetic small molecular weight MMP inhibitors are highly sought. Several orally active MMP inhibitors were synthesized and tested in animal models yielding encouraging results (20.Shalinsky D.R. Brekken J. Zou H. Bloom L.A. McDermott C.D. Zook S. Varki N.M. Appelt K. Clin. Cancer Res. 1999; 5: 1905-1917PubMed Google Scholar, 21.Price A. Shi Q. Morris D. Wilcox M.E. Brasher P.M. Rewcastle N.B. Shalinsky D. Zou H. Appelt K. Johnston R.N. Yong V.W. Edwards D. Forsyth P. Clin. Cancer Res. 1999; 5: 845-854PubMed Google Scholar). Some of these MMP inhibitors, including the broad-spectrum hydroxamate-based batimastat and marimastat (22.Rasmussen H.S. McCann P.P. Pharmacol. Ther. 1997; 75: 69-75Crossref PubMed Scopus (361) Google Scholar, 23.Brown P.D. Breast Cancer Res. Treat. 1998; 52: 125-136Crossref PubMed Scopus (91) Google Scholar), reached phase 3 of clinical trials. However, administration of these inhibitors, either alone or in combination with standard chemotherapeutic agents, to cancer patients produced severe side effects and showed no clinical efficacy. The current view is that these trials were misguided because the subjects had pre-existing metastasis and improved survival rates should not have been anticipated (24.Zucker S. Cao J. Chen W.T. Oncogene. 2000; 19: 6642-6650Crossref PubMed Scopus (501) Google Scholar). On the other hand, a recent study (25.Kruger A. Soeltl R. Sopov I. Kopitz C. Arlt M. Magdolen V. Harbeck N. Gansbacher B. Schmitt M. Cancer Res. 2001; 61: 1272-1275PubMed Google Scholar) demonstrated that batimastat administration led to liver metastasis in mice inoculated with human breast carcinoma cells. Results from our laboratories (26.Toth M. Bernardo M.M. Gervasi D.C. Soloway P.D. Wang Z. Bigg H.F. Overall C.M. DeClerck Y.A. Tschesche H. Cher M.L. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 41415-41423Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) showed that both batimastat and marimastat, in concert with TIMP-2, enhanced MT1-MMP-dependent pro-MMP-2 activation when compared with the activation observed with TIMP-2 alone. On the cell membrane, active MT1-MMP (57 kDa) undergoes autolysis and shedding of the catalytic domain, yielding a major membrane-bound species of 44 kDa, which is devoid of proteolytic activity (27.Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Binding of TIMP-2 or of a synthetic MMP inhibitor to active MT1-MMP prevents autolysis, resulting in accumulation of enzyme on the cell surface. We have shown that in the presence of TIMP-2 accumulation of active MT1-MMP by synthetic MMP inhibitors can generate MT1-MMP·TIMP-2 complexes on the cell surface, which then promote pro-MMP-2 activation (26.Toth M. Bernardo M.M. Gervasi D.C. Soloway P.D. Wang Z. Bigg H.F. Overall C.M. DeClerck Y.A. Tschesche H. Cher M.L. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 41415-41423Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Thus, under certain conditions, synthetic MMP inhibitors with high affinity toward MT1-MMP can enhance the effects of TIMP-2 in pro-MMP-2 activation by MT1-MMP. We have recently reported the first mechanism-based inhibitor (“suicide substrate”) for MMPs, SB-3CT (28.Brown S. Bernardo M.M. Li Z.-H. Kotra L.P. Tanaka Y. Fridman R. Mobashery S. J. Am. Chem. Soc. 2000; 122: 6799-6800Crossref Scopus (184) Google Scholar, 29.Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), which exhibits high selectivity for the gelatinases but poor affinity for MT1-MMP. Consistently, SB-3CT does not enhance the MT1-MMP-mediated activation of pro-MMP-2 in the presence of TIMP-2 (26.Toth M. Bernardo M.M. Gervasi D.C. Soloway P.D. Wang Z. Bigg H.F. Overall C.M. DeClerck Y.A. Tschesche H. Cher M.L. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 41415-41423Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Thus, selective inhibitors for gelatinases may have clinical advantages by reducing side effects and improving efficacy. We report herein the synthesis and kinetic characterization of two novel inhibitors, 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol (1) and 5-(4-phenoxyphenylsulfonyl)pentane-1,2-dithiol (2), which also exhibit selectivity toward gelatinases. However, their mechanisms of inhibition are different. A comparative study of these compounds on the activation of pro-MMP-2 in cells expressing MT1-MMP shows that, when compared with marimastat, significantly higher dithiol concentrations are required for active MT1-MMP binding and enhancement of pro-MMP-2 activation in the presence of TIMP-2. Moreover, at higher concentrations both marimastat and the dithiols inhibit MMP-2, leading to accumulation of the 64-kDa MMP-2 inactive intermediate and preventing further MMP-2 generation. 1H and 13C NMR spectra were recorded on either a Varian Gemini-300, a Varian Mercury-400, or a Varian Unity-500 spectrometer. Chemical shifts are reported in ppm from tetramethylsilane on the δ scale. Infrared spectra were recorded on a Nicolet 680 DSP spectrophotometer. Mass spectra were recorded on a Kratos MS 80RFT spectrometer. Melting points were taken on an electrothermal melting point apparatus and are uncorrected. Thin-layer chromatography was performed with Whatman 0.25-mm silica gel 60-F plates. All other reagents were purchased from either Aldrich or Across Organics. 2-(4-Phenoxyphenylsulfonyl)ethylthiirane and 3-(4-phenoxyphenylsulfonyl)propylthiirane were prepared according to the method reported previously (28.Brown S. Bernardo M.M. Li Z.-H. Kotra L.P. Tanaka Y. Fridman R. Mobashery S. J. Am. Chem. Soc. 2000; 122: 6799-6800Crossref Scopus (184) Google Scholar). A solution of sodium hydrosulfide (13 mg, 0.18 mmol) in methanol (0.5 ml) was cooled to 0 °C. Hydrogen sulfide gas was then bubbled slowly through the solution for 5 min. To this solution was added 2-(4-phenoxyphenylsulfonyl)ethylthiirane (39 mg, 0.12 mmol) in chloroform/methanol (1:2, 0.8 ml) over a period of 10 min, with continuous bubbling of hydrogen sulfide. This mixture was stirred at 0 °C for a further 30 min and then at room temperature for 2 h. The reaction mixture was poured into water (20 ml) and then acidified with a few drops of sulfuric acid (1 m) before extraction with ether (3 × 10 ml). The combined organic extracts were dried over magnesium sulfate and concentrated to give the desired product as a waxy, white solid (42 mg, 99%). 1H (400 MHz, CDCl3) δ7.86–7.83 (m, 2H), 7.44–7.40 (m, 2H), 7.26–7.22 (m, 1H), 7.10–7.06 (m, 5H), 3.41–3.34 (m, 1H), 3.25–3.18 (m, 1H), 3.02–2.93 (m, 1H), 2.84–2.69 (m, 2H), 2.36–2.26 (m, 1H), 1.90–1.80 (m, 1H), 1.66 (d, J6 Hz, 1H), 1.64 (t, J8.8 Hz, 1H); 13C (100 MHz, CDCl3) δ162.94, 155.02, 132.54, 130.55, 130.51, 125.48, 120.70, 117.94, 54.57, 42.44, 34.13, 29.91; m/z (EI) 320 ([M − SH2]+, 30%), 235 (40.Holmquist B. Vallee B.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6216-6220Crossref PubMed Scopus (104) Google Scholar), 217 (60), 170 (100); HRMS calculated for C16H18O3S3–H2S 320.0541, found 320.0540. This material was prepared in the same manner as 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol with the exception that 3-(4-phenoxyphenylsulfonyl)propylthiirane was used in place of 2-(4-phenoxyphenylsulfonyl)ethylthiirane. The desired product was obtained as a waxy white solid (99%). 1H (400 MHz, CDCl3) δ7.86–7.83 (m, 2H), 7.44–7.40 (m, 2H), 7.26–7.22 (m, 1H), 7.09–7.07 (m, 4H), 3.11–3.08 (m, 2H), 2.84–2.82 (m, 1H), 2.75–2.71 (m, 1H), 2.04–1.47 (m, 4H), 1.66 (d, J7.2 Hz, 1H), 1.63 (t, J8 Hz, 1H);13C (100 MHz, CDCl3) δ162.85, 155.08, 132.60, 130.58, 130.50, 125.43, 120.68, 117.93, 56.16, 43.34, 35.19, 34.07, 20.99; m/z (ESI) 391 ([M + Na]+, 100%); HRMS calculated for C17H20O3S3–H2S 334.0697, found 334.0695. HeLa S3 cells were obtained from American Type Culture Collection (ATTC, Manassas, VA) (CCL-2.2) and grown in suspension in MEM Spinner (Quality Biologicals, Inc., Gaithersburg, MD) supplemented with 5% horse serum. Nonmalignant monkey kidney epithelial BS-C-1 (CCL-26) cells were obtained from the ATTC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. All tissue culture reagents were purchased from Invitrogen. Recombinant vaccinia viruses expressing either bacteriophage T7 RNA polymerase (vTF7–3) or MT1-MMP (vTF-MT1) under the T7 promoter were produced by homologous recombination as described previously (27.Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 30.Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (334) Google Scholar, 31.Fridman R. Fuerst T.R. Bird R.E. Hoyhtya M. Oelkuct M. Kraus S. Komarek D. Liotta L.A. Berman M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1992; 267: 15398-15405Abstract Full Text PDF PubMed Google Scholar, 32.Fridman R. Bird R.E. Hoyhtya M. Oelkuct M. Komarek D. Liang C.M. Berman M.L. Liotta L.A. Stetler-Stevenson W.G. Fuerst T.R. Biochem. J. 1993; 289: 411-416Crossref PubMed Scopus (79) Google Scholar). Human recombinant pro-MMP-2, pro-MMP-9, TIMP-1, and TIMP-2 were expressed in HeLa S3 cells infected with the corresponding recombinant vaccinia viruses and purified to homogeneity as previously described (17.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Heat-activated human stromelysin 1 (MMP-3) was generously provided by Dr. Paul Cannon (Center for Bone and Joint Research, Palo Alto, CA). Pro-MMP-2 and pro-MMP-9 were activated by incubation with p-aminophenylmercuric acetate and heat-activated stromelysin 1, respectively, as described previously (28.Brown S. Bernardo M.M. Li Z.-H. Kotra L.P. Tanaka Y. Fridman R. Mobashery S. J. Am. Chem. Soc. 2000; 122: 6799-6800Crossref Scopus (184) Google Scholar). The recombinant catalytic domain of human MT1-MMP (MMP-14cat) encompassing residues Ile114–Ile318 expressed in Escherichia coli (33.Lichte A. Kolkenbrock H. Tschesche H. FEBS Lett. 1996; 397: 277-282Crossref PubMed Scopus (62) Google Scholar), was a generous gift from Dr. Harald Tschesche (University of Bielefeld, Bielefeld, Germany). Recombinant human active matrilysin (MMP-7) was obtained from Chemicon (Temecula, CA). Human recombinant interstitial collagenase (pro-MMP-1) was a generous gift from Dr. William Parks (Washington University, St. Louis, MO). Pro-MMP-1 was activated by incubation with p-aminophenylmercuric acetate (1 mm) in a buffer consisting of 0.1 m Tris, 10 mmCaCl2, pH 7.5. The concentrations of heat-activated stromelysin 1 and MMP-9 were determined by active site titration with recombinant TIMP-1, whereas MMP-2, MMP-7, and MT1-MMPcatconcentrations were obtained by titration with recombinant TIMP-2. The hydroxamate-based inhibitors marimastat (BB-2516) and batimastat (BB-94) were obtained from British Biotech (Annapolis, MD). Stock solutions of marimastat, batimastat, and the dithiol compounds were prepared in Me2SO in the millimolar concentration range. Fluorescence was measured with a PTI spectrofluorometer (Photon Technology International) interfaced to a Pentium computer equipped with RadioMasterTM and FeliXTM hardware and software, respectively. The assays were carried out at 25.0 °C, and the cuvette holder was thermostatted at the same temperature. Excitation and emission band passes of 1 and 3 nm, respectively, were used. Fluorescence measurements were taken every 4 s. MMP-2, MMP-9, MMP-7, and MT1-MMP activities were monitored with the fluorescence-quenched substrate MOCAcPLGLA2pr(Dnp)AR-NH2 at excitation and emission wavelengths of 328 and 393 nm, respectively. MOCAcRPKPVE(Nva)WRK(Dnp)NH2 was the fluorogenic substrate used to monitor MMP-3 enzymatic activity at 325 and 393 nm. MMP-1 activity was assayed with the fluorogenic synthetic substrate (Dnp)P(Cha)GC(Me)HAK(NMa)NH2 at 340 and 440 nm. All fluorogenic substrates were obtained from Peptides International, Inc. (Louisville, KY). Less than 10% hydrolysis of the substrates was monitored as described by Knight (34.Knight C.G. Methods Enzymol. 1995; 248: 85-101Crossref PubMed Scopus (75) Google Scholar). The assays were performed as described previously (35.Olson M.W. Bernardo M.M. Pietila M. Gervasi D.C. Toth M. Kotra L.P. Massova I. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 2661-2668Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Progress curves for slow-binding inhibition analysis were obtained by adding enzyme (0.5–1 nm) to a mixture of MOCAcPLGLA2pr(Dnp)AR-NH2(7 μm) and varying concentrations of the dithiol inhibitors in buffer R (50 mm HEPES (pH 7.5), 150 mm NaCl, 5 mm CaCl2, 0.01% Brij-35, and 5% Me2SO; final volume 2 ml) in acrylic cuvettes with stirring. Hydrolysis of the fluorogenic substrate was monitored for 15 to 30 min. The progress curves were nonlinear least squares fitted to Equation 1 (36.Muller-Steffner H.M. Malver O. Hosie L. Oppenheimer N.J. Schuber F. J. Biol. Chem. 1992; 267: 9606-9611Abstract Full Text PDF PubMed Google Scholar), F=vst+(vo−vs)(1−exp(−kt))/k+FoEquation 1 where vo represents the initial rate, vs the steady-state rate, k the apparent first-order rate constant characterizing the formation of the steady-state enzyme-inhibitor complex, and Fo the initial fluorescence, using the program SCIENTIST (MicroMath Scientific Software, Salt Lake City, UT). The obtained k values, vo and vs, were further analyzed according to Equations 2 and 3 for a one-step association mechanism. k=koff+kon[I]/(1+[S]/Km)Equation 2 (vo−vs)/vs=[I]/(Ki(1+[S]/Km))Equation 3 The Km values used for the reaction of MMP-2 and MMP-9 with the fluorogenic substrate were 2.46 ± 0.34 and 3.06 ± 0.74 μm, respectively (17.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Intercept and slope values, obtained by linear regression of the k versusinhibitor concentration plot (Eq. 2), yielded the association and dissociation rate constants kon and koff, respectively, and the inhibition constant Ki(koff/kon). Alternatively, Ki was determined from the slope of the (vo − vs)/vsversus [I] plot according to Equation 3. The dissociation rate constants were determined independently from the enzyme activity recovered after dilution of a preformed enzyme-inhibitor complex. Thus, 200–400 nm enzyme was incubated with ∼100 μm inhibitor for at least 45 min at 25.0 °C. The complex was diluted 400-fold into 2 ml of buffer R containing fluorogenic substrate (10 μm final concentration). Recovery of enzyme activity was monitored for 60 min. The data were analyzed as described elsewhere (26.Toth M. Bernardo M.M. Gervasi D.C. Soloway P.D. Wang Z. Bigg H.F. Overall C.M. DeClerck Y.A. Tschesche H. Cher M.L. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 41415-41423Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 28.Brown S. Bernardo M.M. Li Z.-H. Kotra L.P. Tanaka Y. Fridman R. Mobashery S. J. Am. Chem. Soc. 2000; 122: 6799-6800Crossref Scopus (184) Google Scholar). For competitive inhibition, the initial rates were obtained by adding enzyme (0.5 nm) to a mixture of fluorogenic substrate (7 μm) and varying concentrations of inhibitor in buffer R (final volume 1 ml) in quartz semi-micro cuvettes and monitoring the increase in fluorescence with time for 5–10 min. The initial velocities were determined by linear regression analysis of the fluorescence versus time traces using FeliXTM. The initial rates were fitted to Equation 4(37.Segel I.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1975Google Scholar), where vi and vo represent the initial velocity in the presence and absence of inhibitor, respectively, using the program SCIENTIST. vi/vo=(Km+[S])/(Km(1+[I]/Ki)+[S])Equation 4 To express MT1-MMP, ∼90% confluent BS-C-1 cells cultured in 6-well plates were co-infected with 5 plaque-forming units/cell each of vTF7–3 and vTF-MT1 viruses for 45 min in infection medium (DMEM supplemented with 2.5% fetal bovine serum and antibiotics) at 37 °C. After infection the cells were rinsed with serum-free DMEM supplemented with l-glutamine and antibiotics and incubated in the same medium containing varying concentrations of synthetic inhibitors. After ∼16 h the inhibitor-containing media were aspirated, and the cells were rinsed twice with phosphate-buffered saline and incubated with Opti-MEM, without phenol red, supplemented with pro-MMP-2 (25 nm). At varying times, media aliquots were removed and MMP-2 activity was assayed with MOCAcPLGLA2pr(Dnp)AR-NH2, as described (35.Olson M.W. Bernardo M.M. Pietila M. Gervasi D.C. Toth M. Kotra L.P. Massova I. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 2661-2668Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). After 4–6 h the remaining media were collected, and the cells were rinsed twice with cold phosphate-buffered saline and lysed with cold lysis buffer (25 mm Tris-HCl (pH 7.5), 1% IGEPAL CA-630, 100 mm NaCl) containing protease inhibitors (1 pellet of Complete Mini, EDTA-free protease inhibitor mixture from Roche Diagnostics/10 ml of buffer). The media and lysate samples were analyzed for pro-MMP-2 cleavage by gelatin zymography. The lysates were also subjected to immunoblot analysis to ascertain the level of MT1-MMP expression and processing as described below. Gelatin zymography was performed using 8% Tris-glycine SDS-polyacrylamide gels containing 0.1% gelatin under nonreducing conditions as described previously (38.Toth, M., Gervasi, D. C., and Fridman, R. (1997) p. 105, @@[email protected]@Cancer [email protected]@/[email protected]@ 57, 3159–3167Google Scholar). The samples for immunoblot analysis were subjected to reducing SDS-PAGE followed by transfer to nitrocellulose membranes. The transferred proteins were developed using rabbit polyclonal antibody 437 to MT1-MMP (39.Gervasi D.C. Raz A. Dehem M. Yang M. Kurkinen M. Fridman R. Biochem. Biophys. Res. Commun. 1996; 228: 530-538Crossref PubMed Scopus (38) Google Scholar). Horseradish peroxidase-labeled anti-rabbit IgG (ImmunoPureR, Pierce) was the secondary antibody used. Detection was performed using SuperSignalR enhanced chemiluminescence substrate for horseradish peroxidase, with West pico or femto sensitivity, according to the manufacturer's (Pierce) instructions. Thiolates are the best ligands for zinc ions, a factor that has been used in designing enzyme inhibitors (40.Holmquist B. Vallee B.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6216-6220Crossref PubMed Scopus (104) Google Scholar, 41.Gomez-Monterrey I. Beaumont A. Nemecek P. Roques B.P. Fournie-Zaluski M.-C. J. Med. Chem. 1994; 37: 1865-1873Crossref PubMed Scopus (23) Google Scholar, 42.Fink C.A. Qiao Y. Berry C.J. Sakane Y. Ghai R.D. Trapani A.J. J. Med. Chem. 1995; 38: 5023-5030Crossref PubMed Scopus (24) Google Scholar, 43.De Bohacek R. Lombaert S. McMartin C. Priestle J. Gruetter M. J. Am. Chem. Soc. 1996; 118: 8231-8249Crossref Scopus (63) Google Scholar, 44.Babine R.E. Bender S.L. Chem. Rev. 1997; 97: 1359-1472Crossref PubMed Scopus (913) Google Scholar, 45.Freskos J.N. Mischke B.V. DeCrescenzo G.A. Heintz R. Getman D.P. Howard S.C. Kishore N.N. McDonald J.J. Munie G.E. Rangwala S. Swearingen C.A. Voliva C. Welsch D.J. Bioorg. Med. Chem. Lett. 1999; 9: 943-948Crossref PubMed Scopus (34) Google Scholar, 46.Gaucher J.F. Selkti M. Tiraboschi G. Prange T. Roques B.P. Tomas A. Fournie-Zaluski M.C. Biochemistry. 1999; 38: 12569-12576Crossref PubMed Scopus (48) Google Scholar, 47.Paulvannan, K., and Chen, T. (1999) @@[email protected]@[email protected]@/[email protected]@ 1371–1374Google Scholar, 48.Sang Q.-X. A. 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). A report for a dithiol inhibitor of VanX, a bacterial zinc-dependentd,d-dipeptidase, was published recently (49.Wu Z. Walsh C. J. Am. Chem. Soc. 1996; 118: 1785-1786Crossref Scopus (33) Google Scholar), which prompted us" @default.
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