No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Orally active inhibitors of stromelysin-1 (MMP-3)
Abstract
Further development of N-carboxyalkyl dipeptide inhibitors of stromelysin-1 (MMP-3) led to the discovery of C-carboxyalkyl dipeptide analogs with improved oral bioavailability. An in vivo assay of human MMP-3 mediated degradation of a macromolecular substrate in an extravascular space is described and inhibition studies are reported.
... An excellent alternative wasreduction under mild conditions (slight positiveH 2 pressure at ambient temperature) with Pd(OH) 2 in acetic acid. [31] The desired 18 was obtained in 73-75% yield. Direct conversion of naphthalene (15)i nto acid 18 by the Friedel-Crafts reaction with butyrolactone, [32,33] either neat, in n-decane, or in nitrobenzene,d id not give satisfactory results. ...
A convergent synthesis of racemic 5-amino[6]hexahelicene is described. Cross-coupling reactions are used to assemble a pentacyclic framework, and a metal-catalyzed ring-closure comprises the final step. The enantiomers were separated by means of chromatography and the absolute configurations were assigned by comparison of the CD spectra with hexahelicene. The t1/2 value for racemization at 210 °C was approximately 1 hour. Scanning tunneling microscopy (STM) measurements were carried out on enantiopure and racemic samples of aminohelicene on Au(111) under ultrahigh vacuum (UHV) conditions.
... An excellent alternative wasreduction under mild conditions (slight positiveH 2 pressure at ambient temperature) with Pd(OH) 2 in acetic acid. [31] The desired 18 was obtained in 73-75% yield. Direct conversion of naphthalene (15)i nto acid 18 by the Friedel-Crafts reaction with butyrolactone, [32,33] either neat, in n-decane, or in nitrobenzene,d id not give satisfactory results. ...
A combination of Scanning Tunnelling Microscopy and Density Functional Theory simulations highlights the role of van der Waals interactions in the self-assembly of an aminohelicene on Cu(100) and Au(111).
Tomi K Sawyer received a BSc degree in Chemistry at Moorhead State University (now Minnesota State University –Moorhead) and PhD in Organic Chemistry at the University of Arizona. His research has integrated synthetic chemistry, drug design, structural biology, chemoinformatics, biochemistry, cell biology, and in vivo disease models with a focus on cancer. Tomi's drug discovery track record includes contributions to clinical candidates and/or noteworthy molecular tools for several therapeutic targets, including GPCRs (melanocortin), aspartyl proteases (renin and HIV protease), and protein kinases (Src and Abl). He has published more than 200 scientific articles, reviews, commentaries, monographs, and books. Tomi is an inventor of more than 50 issued patents and patent filings. He worked at Upjohn Company and Parke-Davis/Warner-Lambert (now both Pfizer Global Research & Development), and is currently Senior-Vice President, Drug Discovery, at ARIAD Pharmaceuticals. He is concurrently adjunct professor, Chemistry as well as Biochemistry & Molecular Biology, University of Massachusetts, and also adjunct professor, Cancer Biology, at University of Massachusetts School of Medicine. Tomi has served on the highlights advisory panel of Nature Reviews Drug Discovery and the editorial advisory boards of Trends in the Pharmacological Sciences, Expert Reviews in Molecular Medicine, Expert Opinion on Investigational Drugs, Journal of Medicinal Chemistry, Chemistry and Biology, Current Medicinal Chemistry (Anti-Cancer Agents), Current Organic Synthesis, Expert Reviews in Molecular Medicine, Expert Opinion on Therapeutic Patents (Oncology), Drug Design and Discovery, Pharmaceutical Research, Molecular Biotechnology, and Biopolymers (Peptide Science). Most recently, Tomi was appointed Editor-in-Chief, Chemical Biology & Drug Design.
This chapter covers primarily the conjugate addition of carbon nucleophiles to auxiliary-containing α,β-unsaturated substrates derived from carbonyl (aldehydes and ketones) and acyl (aliphatic, aromatic, and heteroaromatic acids) compounds. Conjugate addition has relatively recently benefitted from enantiomeric approaches through great strides in catalyst design. This change is reflected in the range of research reference material.
Key Chiral Auxiliary Applications, Second Edition is a detailed compilation of chiral auxiliary applications organized by type of transformation. Continuing from the most important and relevant auxiliaries described in its predecessor, the three-volume set Compendium of Chiral Auxiliary Applications (2001), as well as advances in the field, the book provides a vital and timely resource for chemists in the field. Each reaction class includes a series of tables and graphical abstracts of real reactions from the literature and patents to enable easy review and comparison of results. This anticipated edition is based on a screening of nearly 40,000 auxiliary reaction applications, with details supplied for the more than 13,000 selected representative entries: synthetic route, reagents, yields, diastereomeric/enantiomeric excesses, and characterization data. Updated and streamlined with more than 60% new material, Key Chiral Auxiliary Applications provides valuable guidance and reliable content for selecting the best auxiliary for a specific asymmetric synthetic transformation.
A novel model to assess the in vivo bioactivity of matrix metalloproteinase (MMP) inhibitors is described. A solution containing the target enzyme, either stromelysin (MMP-3) or collagenase (MMP-1), was put inside dialysis tubing which was then implanted either intraperitoneally or subcutaneously in the back of a rat. The rat was dosed with inhibitor, the dialysis tubing was recovered after a set period of time, and its contents were analyzed for inhibition of enzyme activity. The MMP inhibitors tested were each able to significantly block enzyme activity after either intravenous or oral administration. In vitro, a minimum of 30 min was required for the drugs to maximally equilibrate across the dialysis membrane and inhibit enzyme activity. In the rat, significant inhibition of stromelysin activity was observed at 45 min after intravenous drug administration but was maximal at 60 min. Dialysis tubing of various molecular weight cutoffs from 3,500 to 14,000 did not affect the amount of inhibition exerted by the compounds. The enzyme activity recovered from the dialysis tubing was quantitated by either spectrophotometric or fluorometric assays using specific substrates. This model determined the bioactivity of different chemical classes of known inhibitors of stromelysin and collagenase in a rapid and convenient manner. The dialysis tubing implant model can be expanded to assess the pharmacodynamic bioactivity of a diverse group of drugs using various targets inside the dialysis tubing. Drug Dev. Res. 43:200–205, 1998. © 1998 Wiley-Liss, Inc.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Matrix metalloproteinases (MMPs) are involved in the normal processes of tissue remodelling and repair, but have increasingly been implicated in a range of disease states where they are over-expressed. This review summarises developments in the design and clinical evaluation of synthetic MMP inhibitors since the subject was last covered in this journal in 1995.
Enantioselective alkylation of chiral amide enolates derived from L-prolinol with β-branched chiral iodides afforded good yields of hydroxy amide adducts, which were elaborated in four steps to give C-carboxyalkyl dipeptide inhibitors of stromelysin-1 (MMP-3).
This chapter briefly describes the metalloproteinase (MMP) family of enzymes and the putative role it plays in both physiology and pathology, the structures of these enzymes and the current status of MMP inhibitor development for the treatment of this wide range of diseases. The MMP family currently includes fourteen members encoded by unique genes, 10 of which are secreted from cells in a soluble form and 4 new members that are bound to the cell membrane. The MMPs are zinc dependent, calcium requiring enzymes, which are expressed as inactive zymogens. These enzymes are also inhibited by one or more members of the tissue inhibitor of metalloproteinase (TIMP) family, of which three have been cloned and sequenced. The physiological activators of many of these enzymes remain unknown; however, some of the members of the MMP family have the capacity to activate other family members. Therefore, inhibition of the enzyme(s), ultimately responsible for this activation cascade, could result in blocking the activity of multiple enzymes without directly inhibiting their activity. Recently four membrane type MMPs (MT-MMPs) have been cloned and sequenced. Two of these enzymes have the capacity to activate MMP-2 suggesting that they may be a target to consider for the intervention of tumor metastasis. These enzymes are unique among the MMPs because they appear to be associated with the cell membrane and not released into the extracellular space. Using MMP inhibitors, shedding of a number of cell associated proteins, including TNFα, IL-6 and tumor necrosis factors (TNF) receptors, FAS ligand, ACE, TSH receptor ectodomain, and CD-23, may be mediated by membrane associated MMPs.
A new method of synthesis of phosphoramidite of 2′-deoxycytydine carrying a protected spermine moiety at N-4 position is described. A model oligodeoxyribonucleotide containing the specified nucleoside unit has been synthesised. A synthesis of a polyaminooligonucleotide combinatorial library was carried out. The analysis of the above library clearly shows that the presence of spermine moieties in oligodeoxyribonucleotides increases stability of their duplexes.
Controlling and monitoring impurities in APIs and finished drug products is a crucial issue in drug development and manufacturing. Part I of this article, published in the February 2012 issue of Pharmaceutical Technology, discussed the various types of and sources of impurities with specific case studies (1). Part II, published in the March 2012 issue, examined chiral, polymorphic, and genotoxic impurities (2). In Part III, the authors examine various degradation routes of APIs, impurities arising from API–excipient interaction during formulation, metabolite impurities, various analytical methodologies to measure impurity levels, and ways to control impurities in pharmaceuticals.
With the use of an NMR-based method, potent (IC50 < 25 nM) nonpeptide inhibitors of the matrix metalloproteinase stromelysin (MMP-3) were discovered. The method, called SAR by NMR (for structure−activity relationships by nuclear magnetic resonance), involves the identification, optimization, and linking of compounds that bind to proximal sites on a protein. Using this technique, two ligands that bind weakly to stromelysin (acetohydroxamic acid, KD = 17 mM; 3-(cyanomethyl)-4‘-hydroxybiphenyl, KD = 0.02 mM) were identified. On the basis of NMR-derived structural information, the two fragments were connected to produce a 15 nM inhibitor of this enzyme. This compound was rapidly discovered (less than 6 months) and required only a minimal amount of chemical synthesis. These studies indicate that the SAR by NMR method can be effectively applied to enzymes to yield potent lead inhibitorsan important part of the drug discovery process.
Modification of the N-carboxyalkylamine 3 by independent replacement of the P1′ NH group for CH2 and introduction of P1′ gem-cyclohexyl substitution affords compounds 5 and 6a which retain appreciable activity against MMP-1 (IC50s = 0.023 μM and 0.09 μM, respectively). The glutaramide 7a which incorporates both these structural changes also retains potent activity (IC50 = 0.038 μM).
Several carboxylate derivatives with variation at the P1′ residue were synthesized and evaluated as stromelysin (MMP-3) inhibitors. Compounds containing a biphenyl moiety at P1′ were found to be potent inhibitors of MMP-3. An X-ray crystal structure of the most potent compound, carboxylate 19, revealed an important interaction between the inhibitor's biphenyl and histidine 224 in the S1′ pocket of MMP-3.
Matrix metalloproteinases (MMPs) play an important role in many physiological and pathological processes. Development of MMP inhibitors, in particular collagenase inhibitors, for the treatment of arthritis has been more challenging, undoubtedly. Small-molecular-weight collagenase inhibitors may be classified into several different arbitrary structural classes, depending on the catalytic zinc-binding function as well as other structural elements of the inhibitors. This chapter tries to make an attempt in providing the reader with an overall flavor of the type of scaffolds reported in the past few years along with the molecular modeling studies.
A ligand-based 3D-QSAR study for the identification of MMP3 inhibitors was developed by applying an innovative alignment method capable of taking into account information obtained from available X-ray MMP3 structures. Comparison of the obtained model with data recently published using a docking-based alignment method indicated that the ligand-based 3D-QSAR model provided better predictive ability. A second external test set of 106 MMP3 inhibitors further confirmed the predictive ability of the 3D-QSAR model. Finally, certain iminodiacetyl-based hydroxamate-benzenesulfonamide conjugates, which were predicted to be active by the 3D-QSAR model, were tested in vitro for MMP3 inhibition; some provided low nanomolar activity. As such, our results suggest that the multitemplate alignment method is capable of improving the quality of 3D-QSAR models and therefore could be applied to the study of other systems. Furthermore, since MMP3 is an important target toward the treatment of arthritis, this model could be applied to the design of new active MMP3 inhibitors.
Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).
Structure-activity relationships of a lead hydroxamic acid inhibitor of recombinant human stromelysin were systematically defined by taking advantage of a concise synthesis that allowed diverse functionality to be explored at each position in a template. An ex vivo rat model and an in vivo rabbit model of stromelysin-induced cartilage degradation were used to further optimize these analogs for oral activity and duration of action. The culmination of these modifications resulted in CGS 27023A, a potent, orally active stromelysin inhibitor that blocks the erosion of cartilage matrix.
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.
A set of 90 novel 2-(arylsulfonyl)-1,2,3, 4-tetrahydroisoquinoline-3-carboxylates and -hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) was designed, synthesized, and investigated by 3D-QSAR techniques (CoMFA, CoMSIA) and X-ray structure analysis. Docking studies of a reference compound are based on crystal structures of MMP-8 complexed with peptidic inhibitors to propose a model of its bioactive conformation. This model was validated by a 1. 7 A X-ray structure of the catalytic domain of MMP-8. The 3D-QSAR models based on a superposition rule derived from these docking studies were validated using conventional and cross-validated r2 values using the leave-one-out method, repeated analyses using two randomly chosen cross-validation groups plus randomization of biological activities. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which were found to correspond to experimentally determined MMP-8 catalytic site topology in terms of steric, electrostatic, and hydrophobic complementarity. Subsets selected as smaller training sets using 2D fingerprints and maximum dissimilarity methods resulted in 3D-QSAR models with remarkable correlation coefficients and a high predictive power. This allowed to compensate the weaker zinc binding properties of carboxylates by introducing optimal fitting P1' residues. The final QSAR information agrees with all experimental data for the binding topology and thus provides clear guidelines and accurate activity predictions for novel MMP-8 inhibitors.
Two series of compounds synthesized as specific matrix metalloproteinase (MMP) inhibitors have been evaluated for their inhibition of non-MMPs. In a series of substituted succinyl hydroxamic acids, some were found to be significant (IC50 < 1 microM) inhibitors of leucine (microsomal) aminopeptidase, neprilysin (3.4.24.11), and thermolysin. Macrocyclic compounds in which the alpha carbon of the succinyl hydroxamate is linked to the side chain of the P2' amino acid were found to be good inhibitors of aminopeptidase, but not of neprilysin or thermolysin. Compounds of neither series were found to be significant inhibitors of angiotensin converting enzyme or carboxypeptidase A.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
The design, synthesis and structure-activity relationship (SAR) of a series of nonpeptidic 2-arylsulfonyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates and-hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) is described here. Based on available X-ray structures of MMP-8/inhibitor complexes, our structure-based design strategy was directed to complement major protein-ligand interaction regions mainly in the S1' hydrophobic specificity pocket close to the catalytic zinc ion. Here, the rigid 1,2,3,4-tetrahydroisoquinoline scaffold (Tic) provides ideal geometry to combine hydroxamates and carboxylates as typical zinc complexing functionalities, with a broad variety of S1' directed mono- and biaryl substituents consisting of aromatic rings perfectly accommodated within this more hydrophobic region of the MMP-8 inhibitor binding site. The effect of different S1' directed substituents, zinc-complexing groups, chirality and variations of the tetrahydroisoquinoline ring-system is investigated by systematic studies. X-ray structure analyses in combination with 3D-QSAR studies provided an additional understanding of key determinants for MMP-8 affinity in this series. The hypothetical binding mode for a typical molecule as basis for our inhibitor design was found in good agreement with a 1.7 A X-ray structure of this candidate in complex with the catalytic domain of human MMP-8. After analysis of all systematic variations, 3D-QSAR and X-ray structure analysis, novel S1' directed substituents were designed and synthesized and biologically evaluated. This finally results in inhibitors, which do not only show high biological affinity for MMP-8, but also exhibit good oral bioavailability in several animal species.
The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix (required for cell migration) and growth factors and their receptors. The overexpression of MMP-3 (stromelysin-1) and MMP-13 (collagenase-3) is associated with nonhealing wounds, whereas active MMPs-1, -2, -9, and -14 are required for normal wound healing to occur. We describe the synthesis and enzyme inhibition profile of (3R)-3-[([(1S)-2,2-dimethyl-1-(([(1S)-2-methoxy-1-phenylethyl]amino)carbonyl)propyl]amino)carbonyl]-6-(3-methyl-4-phenylphenyl)hexanoic acid (UK-370,106, 7), which is a potent inhibitor of MMP-3 (IC(50) = 23 nM) with >1200-fold weaker potency vs MMP-1, -2, -9, and -14. MMP-13, which may also contribute to the pathology of chronic wounds, was inhibited about 100-fold less potently by compound 7. Compound 7 potently inhibited cleavage of [(3)H]-fibronectin by MMP-3 (IC(50) = 320 nM) but not cleavage of [(3)H]-gelatin by either MMP-2 or -9 (up to 100 microM). Compound 7 had little effect, at MMP-3 selective concentrations, on keratinocyte migration over a collagen matrix in vitro, which is a model of the re-epithelialization process. Following iv (rat) or topical administration to dermal wounds (rabbit), compound 7 was cleared rapidly (t(1/2) = 23 min) from plasma, but slowly (t(1/2) approximately 3 days) from dermal tissue. In a model of chronic dermal ulcers, topical administration of compound 7 for 6 days substantially inhibited MMP-3 ex vivo. These data suggest compound 7 is sufficiently potent to inhibit MMP-3-mediated matrix degradation while leaving unaffected cellular migration mediated by MMPs 1, 2, and 9. These properties make compound 7 a suitable candidate for progression to clinical trials in human chronic dermal wounds, such as venous ulcers.
Matrix metalloproteinases form a proteinase family with at least 20 members, which are involved in several pathological conditions and which fulfill a large number of physiological functions. Gelatinase A/MMP-2 is a constitutively produced homeostatic enzyme, whereas gelatinase B/MMP-9 is upregulated in acute and chronic inflammations and forms a target for the development of therapeutic inhibitors. We have used a recently developed assay with fluorescent gelatin to analyze gelatinase inhibitors. A peptidomimetic, based on the consensus sequence of the cleavage sites in type II collagen, and various derivatives of a neutralizing antibody were compared as gelatinase inhibitors. A single-chain variable fragment (scFv) derived from the gelatinase B-selective monoclonal antibody REGA-3G12 was tagged with oligohistidine and was also compared with the untagged scFv. Both scFv derivatives inhibited gelatinase B but the peptidomimetic was inefficient. As an extra control and serendipitously it was found that polyhistidine is an inhibitor of gelatinases, presumably by altering the active site by chelation of the catalytic Zn2+.
The matrix metalloproteinases (MMPs) are a family of more than 20 enzymes that are intimately involved in tissue remodelling. These zinc-containing endopeptidases consist of several subsets of enzymes, including collagenase, stromelysins and gelatinases and are involved in the degradation of the extracellullar matrix (ECM) that forms the connective material between cells and around tissues. Disease processes associated with the MMPs are generally related to imbalance between the inhibition and activation of MMPs resulting in excessive degradation of the ECM. These indications include osteoarthritis rheumatoid arthritis, tumour metastasis and congestive heart failure. Inhibitors for these enzymes have been developed for the treatment of a starthingly wide array of disease process where matrix remodelling plays a key role. There are three major components to most MMP inhibitors- the zinc binding group ZBG, the peptidic backbone and the pocket occupying side chain. Most MMPs inhibitors are classified according to their ZBG. Inhibitors interactions at active-site zinc plays a critical role in defining the binding mode and relative inhibitor potency. The majority of MMP inhibitors reported in the literature contain an effective zinc binding group (e.g. hydroxamic acid, carboxylic acid, sulfhydryl group) that is either generally substituted with a peptide-like structure that mimics the substrates that they cleave or appended to smaller side chains that may interact with specific subsites (e.g., P1', P2', P3') within the active site. Although carboxylates exhibit weaker zinc binding properties than hydroxamates, they are known to show better oral bioavailability and are less prone to metabolic degradation. The expected loss of binding affinity after replacement of hydroxamates against carboxylates is faced by adequate choice of elongated S1' directed substituents. The need for novel selective MMP inhibitors makes them an attractive target for the QSAR and molecular modelling. 3-D QSAR models were derived using CoMFA, CoMSIA and GRID approaches leading to the identification of binding regions where steric, electronic or hydrophobic effects are important for affinity. Some structural requirements essential for achieving high binding affinity and selectivity are: an acidic unit tightly anchored through four contact points, bidentate chelation of Zn2+, carbonyl groups for hydrogen bonding, more than two extra units for hydrogen bonds, a hydrophobic moiety.
Matrix metalloproteinases are an important group of zinc enzymes responsible for degradation of the extracellular matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling and in many disease processes such as arthritis, cancer, periodontitis, and osteoporosis. A matrixin family is defined, comprising at least seven members that range in size from Mr 28,000 to 92,000 and are related in gene sequence to collagenase. All family members are secreted as zymogens that lose peptides of about 10,000 daltons upon activation. Latency is due to a conserved cysteine that binds to zinc at the active center. Latency is overcome by physical (chaotropic agents), chemical (HOCl, mercurials), and enzymatic (trypsin, plasmin) treatments that separate the cysteine residue from the zinc. Expression of the metalloproteinases is switched on by a variety of agents acting through regulatory elements of the gene, particularly the AP-1 binding site. A family of protein inhibitors of Mr 28,500 or less binds strongly and stoichiometrically in noncovalent fashion to inhibit members of the family. The serum protein alpha 2-macroglobulin and relatives are also strongly inhibitory.
Human rheumatoid synovial cells in culture stimulated with the conditioned culture medium of rabbit macrophages secrete three distinct latent metalloproteinases. One of them, a proteinase that digests proteoglycan and other connective tissue matrix components, was purified as two active forms after activation with 4-aminophenylmercuric acetate. The two forms were homogeneous on sodium dodecyl sulfate-gel electrophoresis with Mr = 45,000 and Mr = 28,000, whereas the latent precursor was estimated to have Mr = 51,000 by gel permeation chromatography. Both active enzymes had optimal activity at pH 7.5-7.8 and were inhibited by EDTA and 1,10-phenanthroline but not by inhibitors for cysteine, serine, or aspartic proteinases. Removal of Ca2+ from the enzyme solution resulted in a complete loss of activity that could be fully restored by the addition of 1 mM Ca2+. The activity of the apoenzyme was restored by the addition of 0.5 mM Zn2+, 5 mM Co2+, or 5 mM Mn2+ in the presence of Ca2+ but not by each metal ion alone. The identical digestion patterns of reduced, carboxymethylated protein substrates indicated that both active forms of the enzyme have the same substrate specificity. The enzyme degraded cartilage proteoglycans, type I gelatin, type IV collagen, laminin, and fibronectin, and removed the NH2-terminal propeptides from chick type I procollagen. This enzyme may play a role in the normal turnover of the connective tissue matrix as well as in the joint destruction of chronic synovitis.
A series of N-carboxyalkyl dipeptides were synthesized to evaluate their inhibitory activities against human stromelysin-1(MMP-3), collagenase(MMP-1), and gelatinase-A(MMP-2). Structures with a homophenylalanine residue at P1′ substituted at the para position with small alkyl groups are potent inhibitors of (MMP-3) and (MMP-2) (Ki′ s 2–40 nM), but weak inhibitors of (MMP-1).
Aroyl and arylacyl aminoalkyl substitutents at the P1 position of N-carboxyalkyl dipeptides were found to enhance potency and selectivity for stromelysin-1 (MMP-3). In particular, the phthalimidobutyl and phenylpropanoylaminopropyl groups offered inhibitors of MMP-3 with Ki's of ∼ 10nM.
The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a proenzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three α-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.
Recombinant human tissue inhibitor of metalloproteinase (rhTIMP) suppressed the ability of native human stromelysin to degrade [3H]transferrin in vitro. Maximum inhibition occurred at molar ratios (TIMP: stromelysin) of 2:1 and 1:1. Reduced and alkylated tissue inhibitor of metalloproteinases (TIMP) lost its ability to suppress stromelysin activity. rhTIMP also inhibited stromelysin from degrading proteoglycan monomer in vitro. When injected into the rat pleural cavity prior to stromelysin, rhTIMP inhibited the ability of the enzyme to degrade aggregating cartilage proteoglycan monomer. Marked inhibition of stromelysin-mediated proteoglycan degradation in vivo occurred at molar ratios (TIMP: enzyme) of 2:1 and 1:1, with less inhibition at molar ratios of 0.5:1 and 0.25:1. Reduction and alkylation prevented rhTIMP from suppressing stromelysin-mediated degradation of proteoglycan monomer in vivo. By comparison, an equimolar concentration of the serine proteinase inhibitor, alpha 1-proteinase inhibitor (alpha 1-PI), did not inhibit stromelysin activity in the rat pleural cavity. This study demonstrates that rhTIMP is effective in inhibiting native human stromelysin both in vitro and in vivo.
The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 A resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded beta-sheet and three alpha-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase.
An extensive study of the requirements for effective binding of N-carboxyalkyl peptides to human stromelysin, collagenase, and to a lesser extent, gelatinase A has been investigated. These efforts afforded inhibitors generally in the 100-400 nM range for these matrix metalloproteinases. The most significant increase in potency was obtained with the introduction of a beta-phenylethyl group at the P1' position, suggesting a small hydrophobic channel into the S1' subsite of stromelysin. One particular compound, N-[1(R)-carboxyethyl]-alpha(S)-(2-phenylethyl)glycyl-L-leucine,N- phenylamide (79a), is relatively selective for rabbit stromelysin with a K(i) = 6.5 nM and may prove useful for elucidating the role of endogenously-produced stromelysin in lapine models of tissue degradation.
The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.