Normal and abnormal processes of cellular invasion often are initiated by degradation of basement membranes. The process of corneal ulceration might operate via similar mechanisms; degradation of the corneal stroma is not seen until after the basement membrane underlying the corneal epithelium in the preulcerative lesion is lost. Recent data implicate a member of the matrix metalloproteinase (MMP) family of enzymes, 92 kD gelatinase/type IV collagenase (MMP-9) in both cellular invasion processes and degradation of epithelial basement membrane before corneal ulceration. This suggests that use of nontoxic substances that block activity of MMP-9 might be useful in preventing or inhibiting pathologic invasion processes in vivo. An agent that fits these criteria is N-[D,L-2-isobutyl-3(N'-hydroxycarbonylamido)propanoyl]-O-methyl-L-tyrosine methylamide, which previously has been characterized as an inhibitor of tumor cell collagenases. In this study, the authors show that the inhibitor can efficiently block activity of MMP-9 purified from cultures of rabbit corneal epithelial cells. Results suggest that the recently reported efficacy of a closely related inhibitor in blocking progression of alkali burns to ulceration might be attributable to its action against MMP-9. Invest Ophthalmol Vis Sci 32:2997-3001,1991

NMR spectroscopy techniques have been used to determine the conformation of DG35-VIII in DMSO, acetone, and methanol. COSY and Heteronuclear Correlation experiments were used to confirm the proton spectral assignments. NOESY experiments were used to identify proton internuclear distances which were used to determine the 3D structure. The NOESY data identified a single "U-shaped" conformer of DG35-VIII in acetone, and an alternate "extended" conformer in methanol and two possible conformations in DMSO. Restrained molecular minimization methods using the Molecular Mechanics Program "DISCOVER" and "DYANA" were used to determine a low energy structure consistent with the NMR data. The extended structure of DG35-VIII was compared with closely related HIV protease inhibitors (VX-478 and ABT-538) and showed similar backbone structures, with the functional isostere groups superimposed on each other. The binding energy of DG35-VIII with HIV protease was examined and found to be comparable with VX-478 and ABT-538.

Modifications around the dipeptide-mimetic core of a hydroxamic acid based matrix metalloproteinase inhibitor were studied. These variations incorporated a variety of natural, unnatural, and synthetic amino acids in addition to modifications of the P1' and P3' substituents. The results of this study indicate the following structural requirements: (1) Two key hydrogen bonds must be present between the enzyme and potent substrates. (2) Potent inhibitors must possess strong zinc-binding functionalities. (3) The potential importance of the hydrophobic group at position R3 as illustrated by its ability to impart greater relative potency against stromelysin when larger hydrophobic groups are used. (4) Requirements surrounding the nature of the amino acid appear to be more restrictive for stromelysin than for neutrophil collagenase, 72 kDa gelatinase, and 92 kDa gelatinase. These requirements may involve planar fused-ring aryl systems and possibly hydrogen-bonding capabilities.

The protease of human immunodeficiency virus (HIV) is an important target for antiretroviral drug therapy. The synthesis and in vitro antiviral activity of a novel protease inhibitor, DG-35-VIII, which contains an hydroxyethylhydrazide core unit, is described. DG-35-VIII had potent activity against HIV-1 and related viruses (HIV-2 and simian immunodeficiency virus) in an acutely infected T lymphocyte line (MT-2) and was also active in cells chronically infected with HIV-1, where it inhibited processing of the Pr55(gag) and Pr160(gag-pal) precursor proteins.

The inhibitor N-[2R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]-L- tryptophan methylamide specifically blocks several matrix metalloproteases, enzymes which are thought to be involved in angiogenesis. An extract of Walker 256 carcinoma in Hydron pellets implanted in the corneas of Sprague-Dawley rats was used to stimulate angiogenesis from the vessels of the limbus. Angiogenesis was graded visually as the distance penetrated into the cornea and the number of vessels generated. The vessel area was also measured by image analysis using Image 1 software. Continuous i.v. administration of N-[2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]- L-tryptophan methylamide at 32 mg/kg/day (n = 17) via syringe pump reduced vessel number [25.06 +/- 5.9 (SEM) compared to 65.33 +/- 9.0] and vessel area (26.14 +/- 3.2 mm2 compared with 40.96 +/- 4.6 mm2), but not distance penetrated, compared to vehicle-treated control eyes after 6 days. These results confirm the suspected role for matrix metalloproteases in angiogenesis and suggest that inhibitors of these enzymes may be angiostatic agents.

Annals of the New York Academy of SciencesVolume 732, Issue 1 p. 315-323 Low Molecular Weight Inhibitors in Corneal Ulcerationa RICHARD E. GALARDY, Corresponding Author RICHARD E. GALARDYCorresponding author.Search for more papers by this authorMARIE E. CASSABONNE, MARIE E. CASSABONNE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorCARLANNE GIESE, CARLANNE GIESE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorJAMES H. GILBERT, JAMES H. GILBERT Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorFRANCE LAPIERRE, FRANCE LAPIERRE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorHENRY LOPEZ, HENRY LOPEZ Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorMARY E. SCHAEFER, MARY E. SCHAEFER Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorROBERT STACK, ROBERT STACK Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorMICHAEL SULLIVAN, MICHAEL SULLIVAN Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorBRENT SUMMERS, BRENT SUMMERS Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorROB TRESSLER, ROB TRESSLER Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorDAVE TYRRELL, DAVE TYRRELL Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorJENNIFER WEE, JENNIFER WEE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorSCOTT D. ALLEN, SCOTT D. ALLEN Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorJOHN J. CASTELLOT, JOHN J. CASTELLOT Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.Search for more papers by this authorJOHN P. BARLETTA, JOHN P. BARLETTA Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorGREGORY S. SCHULTZ, GREGORY S. SCHULTZ University of Florida, Gainesville, Florida 32610.Search for more papers by this authorLEONARDO A. FERNANDEZ, LEONARDO A. FERNANDEZ Department of Diagnostic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.Search for more papers by this authorSUSAN FISHER, SUSAN FISHER Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorTIAN-YI CUI, TIAN-YI CUI Department of Stomatology, University of California, San Francisco, California 94143.Search for more papers by this authorHARALD G. FOELLMER, HARALD G. FOELLMER Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06437.Search for more papers by this authorDAMIAN GROBELNY, DAMIAN GROBELNY 43 Brassey Avenue, Rosanna 3084, Victoria, Australia.Search for more papers by this authorWALTER M. HOLLERAN, WALTER M. HOLLERAN Department of Dermatology, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121.Search for more papers by this author RICHARD E. GALARDY, Corresponding Author RICHARD E. GALARDYCorresponding author.Search for more papers by this authorMARIE E. CASSABONNE, MARIE E. CASSABONNE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorCARLANNE GIESE, CARLANNE GIESE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorJAMES H. GILBERT, JAMES H. GILBERT Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorFRANCE LAPIERRE, FRANCE LAPIERRE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorHENRY LOPEZ, HENRY LOPEZ Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorMARY E. SCHAEFER, MARY E. SCHAEFER Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorROBERT STACK, ROBERT STACK Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorMICHAEL SULLIVAN, MICHAEL SULLIVAN Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorBRENT SUMMERS, BRENT SUMMERS Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorROB TRESSLER, ROB TRESSLER Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorDAVE TYRRELL, DAVE TYRRELL Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorJENNIFER WEE, JENNIFER WEE Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorSCOTT D. ALLEN, SCOTT D. ALLEN Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorJOHN J. CASTELLOT, JOHN J. CASTELLOT Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston, Massachusetts 02111.Search for more papers by this authorJOHN P. BARLETTA, JOHN P. BARLETTA Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorGREGORY S. SCHULTZ, GREGORY S. SCHULTZ University of Florida, Gainesville, Florida 32610.Search for more papers by this authorLEONARDO A. FERNANDEZ, LEONARDO A. FERNANDEZ Department of Diagnostic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.Search for more papers by this authorSUSAN FISHER, SUSAN FISHER Glycomed Incorporated, 860 Atlantic Avenue, Alameda, California 94501.Search for more papers by this authorTIAN-YI CUI, TIAN-YI CUI Department of Stomatology, University of California, San Francisco, California 94143.Search for more papers by this authorHARALD G. FOELLMER, HARALD G. FOELLMER Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06437.Search for more papers by this authorDAMIAN GROBELNY, DAMIAN GROBELNY 43 Brassey Avenue, Rosanna 3084, Victoria, Australia.Search for more papers by this authorWALTER M. HOLLERAN, WALTER M. HOLLERAN Department of Dermatology, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121.Search for more papers by this author First published: September 1994 https://doi.org/10.1111/j.1749-6632.1994.tb24746.xCitations: 120 a This work was supported in part by National Institutes of Health grant EY05587. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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The function of proteases in brain tumor invasion is currently not well established. For tumors of epithelial and fibromatous origin collagenase production can enhance the invasive capacity of cells to penetrate basement membranes. We showed previously that a c-Ha-ras transformed glial cell line (CxT24neo3) invaded hamster brain tissuein vivo. These cells were also capable of invading reconstituted basement membrane and embryonic chick heartsin vitro. Since the histopathology of CxT24neo3 tumors mimics that of glioblastoma multiforme in humans, CxT24neo3 was used as the modelin vitro for this type of brain tumor. Presently, we detected by zymogram analysis a gelatinase that was secreted by CxT24neo3 and that had an apparent molecular mass of 62 kD.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

The hydroxamic acid HONHCOCH2CH(i-Bu)CO-L-Trp-NHMe, isomer 6A (GM 6001), inhibits human skin fibroblast collagenase with Ki of 0.4 nM using the synthetic thiol ester substrate Ac-Pro-Leu-Gly-SCH(i-Bu)CO-Leu-Gly-OEt at pH 6.5. The other isomer, 6B, which has the opposite configuration at the CH2CH(i-Bu)CO alpha-carbon atom, has a Ki of 200 nM for this enzyme. GM 6001 is one of the most potent inhibitors of human skin fibroblast collagenase yet reported. GM 6001 has a Ki of 20 nM against thermolysin and Pseudomonas aeruginosa elastase. Isomer 6B has a Ki of 7 nM against thermolysin and 2 nM against the elastase. 6A and 6B are the most potent hydroxamate inhibitors reported for these bacterial enzymes. The pattern of inhibition for all three enzymes suggests that isomer 6A is the (R,S) compound, stereochemically analogous to the L,L-dipeptide, and isomer 6B is the (S,S) compound, analogous to the DL-dipeptide. The tolerance of the D configuration by thermolysin and the elastase allows these inhibitors to discriminate between the human and bacterial enzymes simply by inversion of configuration at the CH2CH(i-Bu)CO alpha-carbon atom. Substitution of the potential metal liganding groups carboxylate and hydrazide for the hydroxamate group yields much weaker inhibitors for all three enzymes.

Substitution of the phosphonamidate linkage (PO2-NH) for the peptide bond (CO-NH) in substrate-like sequences produces inhibitors of human skin fibroblast collagenase with Ki's far below Km for the native collagen substrate. Using a thiol ester substrate at pH 6.5, phthaloyl-GlyP-Ile-Trp-(S)NHCH-(Me)Ph, the phosphonamidate analog of phthaloyl-Gly-Ile-Trp-(S)NHCH(Me)Ph, has a Ki of 20 nM. Peptide phosphonamidates with amino acid sequences extended further to the right or the left of the Gly-Ile-Trp sequence had higher Ki's. Substitution of the phosphinate linkage (PO2-CH2) for the peptide bond also gives potent inhibitors such as napthoyl-GlyP-C-Leu-Trp-NHBzl, the phosphinate analog of naphtholyl-Gly-Leu-Trp-NHBzl, which has a Ki of 10 nM. Some of the phosphonamidates and phosphinates are also excellent inhibitors of the bacterial zinc metalloproteases thermolysin and Pseudomonas aeruginosa elastase.

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.

Normal and abnormal processes of cellular invasion often are initiated by degradation of basement membranes. The process of corneal ulceration might operate via similar mechanisms; degradation of the corneal stroma is not seen until after the basement membrane underlying the corneal epithelium in the preulcerative lesion is lost. Recent data implicate a member of the matrix metalloproteinase (MMP) family of enzymes, 92 kD gelatinase/type IV collagenase (MMP-9) in both cellular invasion processes and degradation of epithelial basement membrane before corneal ulceration. This suggests that use of nontoxic substances that block activity of MMP-9 might be useful in preventing or inhibiting pathologic invasion processes in vivo. An agent that fits these criteria is N-[D,L-2-isobutyl-3(N'-hydroxycarbonylamido)-propanoyl]-O-methyl-L-tyrosine methylamide, which previously has been characterized as an inhibitor of tumor cell collagenases. In this study, the authors show that the inhibitor can efficiently block activity of MMP-9 purified from cultures of rabbit corneal epithelial cells. Results suggest that the recently reported efficacy of a closely related inhibitor in blocking progression of alkali burns to ulceration might be attributable to its action against MMP-9.

The phosphinic acid isosteres of di-, tetra- and hexapeptides containing a hydrophobic amino acid side chains at the P1-P′1 positions are powerful inhibitors of Human Immunodeficiency Virus protease. Ki's ranged from 0.4 nM to 26 μM at pH 6.5 and were lower at pH 4.5. The compounds showed no activity against trypsin, weak activity against renin at pH 6.5, moderate activity against pepsin at pH 2.0 (Ki values in the μM range) and substantial activity against cathepsin D at pH 3.5 (Ki values from 9 to 300 nM).

The importance of a specific hydrogen bond between thermolysin and a phosphonamidate inhibitor, Z-NHCH2-PO(O-)-Leu-Leu (1) [Bartlett, P. A., & Marlowe, C. K. (1987) Science (Washington D.C.) 235, 569-571], has been reevaluated. We have determined the inhibition constants (binding free energies) for thermolysin of phosphonamidate n-hexyl-P(O)(O-)-Leu-Trp-NHMe (4), phosphonate n-hexyl-P-(O)(O-)OCH(iBu)CO-Trp-NHMe (5), and phosphinates n-hexyl-P(O)(O-)CH2CH(iBu)CO-Trp-NHMe (6) and Z-NHCH2PO(O-)CH2CH(iBu)CO-Leu (3). Replacement of the P-NH group by P-CH2 (1----3 and 4----6) weakens the overall binding free energy by about 1.5 kcal/mol. A negligible difference in solvation energy has been measured for these pairs, and the basicity of the P-O- ligand for zinc in each pair remains nearly unchanged as determined by pH titration of their 31P NMR resonances. Therefore, this value of 1.5 kcal/mol can be assigned to the specific hydrogen bond known to exist between the P-NH of 1 and thermolysin [Tronrud, D. E., Holden, H. M., & Matthews, B. W. (1987) Science (Washington, D.C.) 235, 871-574] and inferred to exist between 4 and the enzyme. Substitution of P-O for P-NH (1----2 [Bartlett, P. A., & Marlowe, C. K. (1987) Science (Washington, D.C.) 235, 569-571] and 4----5) weakens the overall binding free energy by 4.1 kcal/mol for each pair as the basicity of the P-O- ligand decreases by about 1.6 pH units. The measured solvation energy difference between 4 and 5 (and by inference between 1 and 2) is negligible.(ABSTRACT TRUNCATED AT 250 WORDS)

The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro [(4-13C-5-cinnamido-4-oxo-6-phenylhexanoyl)-Pro-Pro 2] competitively inhibits a mixture of collagenases from Clostridium histolyticum with a Ki of 40 +/- 6 nM. 13C-nmr spectroscopy of the ketone in the presence of this collagenase shows a bound 13C resonance at 102.6 ppm and the resonance of the free ketone at 212 ppm. Ketone alone shows no trace (less than 0.5%) of a resonance in the region around 100 ppm. The bound resonance is displaceable by another competitive inhibitor. This ketone is thus a transition state analog which is rehybridized from trigonal planar to tetrahedral upon binding to collagenase.