Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.17 (MMP-3)
 3,419 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To probe the specificity of the metalloendoproteinase stromelysin toward peptide substrates, we determined kc/Km values for the stromelysin-catalyzed hydrolyses of peptides whose design was based loosely on the structure of a known SLN substrate, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2, hydrolysis at Gln-Phe, kc/Km = 1700 M-1 s-1). Several noteworthy points emerge from this study: (i) Catalytic efficiency is dependent on peptide chain length with N-terminal truncation of substance P resulting in more pronounced rate-constant reductions than C-terminal truncation. These results suggest the existence of an extended active site for stromelysin. (ii) Preferences at positions P3, P2, P1, P1', and P2' are for the hydrophobic amino acids Pro, Leu, Ala, Nva, and Trp, respectively. (iii) Investigation of specificity at P3' supports our earlier hypothesis that SLN has a requirement for a hydrogen-bond donor at this position in its substrates. Based on these observations, we designed and had synthesized the fluorogenic substrate N-(2,4-dinitrophenyl)Arg-Pro-Lys-Pro-Leu-Ala-Nva-TrpNH2, whose stromelysin-catalyzed hydrolysis can be monitored continuously (kc/Km = 45,000 M-1 s-1).
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PMID:Substrate specificity of the human matrix metalloproteinase stromelysin and the development of continuous fluorometric assays. 147 98

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).
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PMID:Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides. 908 93

The active site of the catalytic domain of stromelysin-1 (matrix metalloproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the environmentally sensitive fluorescent reporter molecule bisANS. Wavelength-dependent acrylamide quenching identified three distinct emitting tryptophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Significant changes in the tryptophan fluorescence polarization occur upon binding by any of the three hydroxamate inhibitors Batimastat, CAS108383-58-0, and Celltech CT1418, all of which bind in the P2'-P3' region of the active site. In contrast, the inhibitor CGS27023A, which is thought to bind in the P1-P1' region, does not induce any change in tryptophan fluorescence polarization. The use of the fluorescent probe bisANS revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysin with a dissociation constant of Ki=22 microM. In addition to this binding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40+/-0.17 microM. The auxiliary site is open, hydrophobic, and near the fluorescing tryptophans. The binding of bisANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.
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PMID:The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3). 985 86

Examination of the S1 area of the active site of pro-stromelysin has led us to the design of novel and potent inhibitors of matrix metalloproteinases containing constrained quaternary-hydroxy group at P1. The synthesis and biological activity of these compounds with variations at P1', P2', and P3' will be described.
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PMID:Structure-based design and synthesis of a series of hydroxamic acids with a quaternary-hydroxy group in P1 as inhibitors of matrix metalloproteinases. 987 51

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthritis and cancer. Among these enzymes, stromelysin-1 can also activate the proenzymes of other MMPs, making it an attractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze the binding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses a hydroxamic acid group (pK(a) congruent with 8.7) to bind the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent with 4.7). Binding affinity increased dramatically as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino acid residue of the enzyme also becomes protonated upon binding. Similar experiments carried out with the H224N mutant gave strong evidence that this residue is histidine 224. DeltaG, DeltaH, DeltaS, and DeltaC(p) were determined for the three inhibitors at pH 6.7, and DeltaC(p) was used to obtain estimates of the solvational, translational, and conformational components of the entropy term. The results suggest that: (1) a polar group at the P1 position can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' substituents of certain sizes may trigger an entropically unfavorable conformational change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities alone.
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PMID:Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry. 1052 Dec 66

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 10(8) independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2, 4-dinitrophenyl-GPLGMRGL-NH(2) (CP), has a k(cat)/K(m) value of 4.22 x 10(6) m(-)(1) s(-)(1) for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.
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PMID:Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. 1090 30

Structure-activity relationships are described for a series of succinyl hydroxamic acids 1a-o and their carboxylic acid analogues 2a-o as inhibitors of matrix metalloproteases MMP-3 and MMP-2. For this series (P1' = (CH2)3Ph, P2' = t-Bu) selectivity for the inhibition of MMP-2 was found to be strongly dependent on P3'.
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PMID:Selectivity of inhibition of matrix metalloproteases MMP-3 and MMP-2 by succinyl hydroxamates and their carboxylic acid analogues is dependent on P3' group chirality. 1122 73

Potent and selective inhibition of matrix metalloproteinases was demonstrated for a series of sulfonamide-based hydroxamic acids. The design of the heterocyclic sulfonamides incorporates a six- or seven-member central ring with a P2' substituent that can be modified. Binding interactions of this substituent at the S2' site are believed to contribute to high inhibitory potency against stromelysin, collagenase-3 and gelatinases A and B, and to provide selectivity against collagenase-1 and matrilysin. An X-ray structure of a stromelysin inhibitor complex was obtained to provide insights into the SAR and selectivity trends observed for the series.
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PMID:Heterocycle-based MMP inhibitors with P2' substituents. 1132 77

The substrate specificity of rat brain neurolysin was rapidly modified by semirational mutagenesis coupled with a yeast molecular display system. Neurolysin mainly recognizes substrates with sequential six residues close to the scissile bond in polypeptides, cleaving a peptide bond in the center position of the six residues. To alter the recognition of the P2' amino acid of substrates by neurolysin, six residues of neurolysin, Asp467, Arg470, Glu510, Tyr606, Tyr610 and Tyr611, which might be involved in the formation of the neurolysin S2' subsite, were individually and comprehensively substituted. The protein libraries of mutant neurolysins comprising 120 species were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) and MMP-3-specific substrates, which consisted of similar amino acids, except for alanine (for MMPs-2/9) or glutamic acid (for MMP-3) at the P2' amino acid position. Among mutant neurolysins, the Y610L mutant neurolysin exhibited a marked change in substrate specificity. Steady-state kinetic analysis of the purified Y610L mutant neurolysin revealed that the binding efficiency toward the MMP-3-specific substrate was about 3-fold higher than that toward the MMP-2/9-specific substrate. These results indicate that Tyr610 of neurolysin is the important residue to recognize the P2' amino acid of substrates.
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PMID:Alteration of substrate specificity of rat neurolysin from matrix metalloproteinase-2/9-type to -3-type specificity by comprehensive mutation. 1849 80

To demonstrate the practical use of a novel high-throughput screening system by single cells constructed by the molecular display method, a yeast cell chip microchamber array was developed. As applications, peptides, peptidases, and antibodies were examined. Neurolysin originally recognizes substrates with six-amino-acid-long residues, cleaving a peptide bond in the center position of the substrate amino acid sequence. To alter the recognition of the P2' amino acid of substrates by neurolysin, six residues of neurolysin which might be involved in the formation of the neurolysin S2' subsite were individually and comprehensively substituted by semirational mutagenesis coupled with the yeast molecular display system. The protein libraries of mutant neurolysins were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- and MMP-3-specific substrates. Among mutant neurolysins, one mutant neurolysin with a marked change in substrate specificity was successfully obtained. Furthermore, skillful display of antibodies (H and L chains) on the cell surface of yeast cells suggested the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach. Accordingly, the combination of the molecular display and combinatorial bioengineering would lead to produce novel medicines.
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PMID:[Novel high-throughput system for production of new medicines-integration and combination with molecular display and combinatorial bioengineering]. 1988 Nov 98


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