Frank Grams

Structural properties of matrix metalloproteinases

Abstract. Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.

Insights into MMP-TIMP interactions.

ABSTRACT: The proteolytic activity of the matrix metalloproteinases (MMPs) involved in extracellular matrix degradation must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance can result in serious diseases such as arthritis and tumor growth and metastasis. Knowledge of the tertiary structures of the proteins involved in such processes is crucial for understanding their functional properties and to interfere with associated dysfunctions. Within the last few years, several three‐dimensional structures have been determined showing the domain organization, the polypeptide fold, and the main specificity determinants of the MMPs. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure‐based design and improvement of high‐affinity ligands, which might be elaborated into drugs. Very recently, structural information also became available for some TIMP structures and MMP‐TIMP complexes, and these new data elucidated important structural features that govern the enzyme‐inhibitor interaction.

Anti-Invasive, Antitumoral, and Antiangiogenic Efficacy of a Pyrimidine-2,4,6-trione Derivative, an Orally Active and Selective Matrix Metalloproteinases Inhibitor

Purpose: The implication of matrix metalloproteinases (MMPs) in the major stages of cancer progression has fueled interest in the design of synthetic MMP inhibitors (MMPIs) as a novel anticancer therapy. Thus far, drugs used in clinical trials are broad-spectrum MMPIs the therapeutic index of which proved disappointingly low. The development of selective MMPIs for tumor progression-associated MMPs is, thus, likely to offer improved therapeutic possibilities. Experimental Design: The anti-invasive capacity of a series of pyrimidine-trione derivatives was tested in vitro in a chemoinvasion assay, and the most potent compound was further evaluated in vivo in different human tumor xenograft models. The activity of this novel selective MMPI was compared with BB-94, a broad-spectrum inhibitor. Results: Ro-28-2653, an inhibitor with high selectivity for MMP-2, MMP-9, and membrane type 1 (MT1)-MMP, showed the highest anti-invasive activity in vitro. In vivo, Ro-28-2653 reduced the growth of tumors induced by the inoculation of different cell lines producing MMPs and inhibited the tumor-promoting effect of fibroblasts on breast adenocarcinoma cells. Furthermore, Ro-28-2653 reduced tumor vascularization and blocked angiogenesis in a rat aortic ring assay. In contrast, BB-94 up-regulated MMP-9 expression in tumor cells and promoted angiogenesis in the aortic ring assay. Conclusion: Ro-28-2653, a selective and orally bioavailable MMPI with inhibitory activity against MMPs expressed by tumor and/or stromal cells, is a potent antitumor and antiangiogenic agent. In contrast to broad-spectrum inhibitors, the administration of Ro-28-2653 was not associated with the occurrence of adverse side effects that might hamper the therapeutic potential of these drugs.

Structural implications for the role of the N terminus in the ‘superactivation’ of collagenases: A crystallographic study

For the collagenases PMNL‐CL and FIB‐CL, the presence of the N‐terminal Phe79 correlates with an increase in proteolytic activity. We have determined the X‐ray crystal structure of the recombinant Phe79‐Gly242 catalytic domain of human neutrophil collagenase (PMNL‐CL, MMP‐8) using the recently solved model of the Met80‐Gly242 form for phasing and subsequently refined it to a final crystalographic R‐factor of 18.0% at 2.5 Å resolution. The PMNL‐CL catalytic domain is a spherical molecule with a flat active site cleft separating a smaller C‐terminal subdomain from a bigger N‐terminal domain, that harbours two zinc ions, namely a ‘structural’ and a ‘catalytic’ zinc, and two calcium ions. The N‐terminal segment prior to Pro86, which is disordered in the Met80‐Gly242 form, packs against a concave hydrophobic surface made by the C‐terminal helix. The N‐terminal Phe79 ammonium group makes a salt link with the side chain carboxylate group of the strictly conserved Asp232. Stabilization of the catalytic site might be conferred via strong hydrogen bonds made by the adjacent, likewise strictly conserved Asp233 with the characteristic ‘Met‐turn’, which forms the base of the active site residues.

Activation of snake venom metalloproteinases by a cysteine switch-like mechanism

The cDNAs of several snake venom zinc endopeptidases code for a putative propeptide, which includes the conserved cysteine‐containing sequence PKMCGVT. It has been suggested that binding of the cysteine thiol function to the active‐site zinc, resulting in inactivation of the catalytic domain, occurs in a mode similar to the ‘cysteine switch’ mechanism proposed for matrix metalloproteinases. In order to confirm this hypothesis, inhibition kinetics have been performed on the metalloproteinase adamalysin II of the venom of the snake Crotalus adamanteus using several cysteine peptides. Among these the synthetic hexapeptide PKMCGV‐NH2, corresponding to the conserved sequence portion of the known propeptides, was found to be by far the strongest inhibitor of this proteinase with a K i of 3.4 μM. The inhibitory potencies of an equivalent peptide with the l‐Cys replaced by a d‐Cys or by an l‐Ser as well as of reduced glutathione, cysteine and two unrelated cysteine peptides were by one to two orders of magnitudes lower. These findings strongly support a cysteine switch‐like mechanism even for activation of the snake venom metalloproteinases.

Pyrimidine-2,4,6-Triones: a new effective and selective class of matrix metalloproteinase inhibitors.

Abstract Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that have been implicated in various disease processes. Different classes of MMP inhibitors, including hydroxamic acids, phosphinic acids and thiols, have been previously described. Most of these mimic peptides and most likely bind in a similar way to the corresponding peptide substrates. Here we desccribe pyrimidinetriones as a completely new class of metalloprotease inhibitors. While the pyrimidinetrione template is used as the zincchelating moiety, the substituents have been optimized to yield inhibitors comparable in their inhibition efficiency of matrix metalloproteinases to hydroxamic acid derivatives such as batimastat. However, they are much more specific for a small subgroup of MMPs, namely the gelatinases (MMP-2 and MMP-9).

The metzincin-superfamily of zinc-peptidases.

Over the past three years, the three-dimensional structures of a number of zinc proteinases that share the zinc-binding motif HEXXHXXGXXH have been elucidated. These proteinases comprise astacin, a digestive enzyme from crayfish [1,2,3], adamalysin II [4,5] and atrolysin C [6] from snake venom, the Pseudomonas aeruginosa alkaline proteinase [7] and serralysin from Serratia marcescens proteinase [8], the collagenases from human neutrophils [9,10,11]) and fibroblasts [12,13,14,15], human stromelysin 1 [16; K. Appelt, personal communication] and matrilysin [M. Browner, Keystone Symposia, March 5–12, 1994]. These enzymes represent four different families of zinc peptidases: the astacins [3,17], the bacterial serralysins [18], the adamalysins/reprolysins [19,20], and the matrixins (matrix metalloproteinases, MMPs) [21,22].

Research on MMP Inhibitors with Unusual Scaffolds

Synthetic inhibitors of matrix metalloproteases have been developed using hydroxamate, N-carboxyalkyl, phosphonamidate, phosphinate, thiol, and other groups, each as a ligand for the active-site zinc atom of metalloproteases. Several of these inhibitors have been crystallized in complexes with the catalytic domains of various matrix metalloproteinases (MMPs).

Function and Structure of Human Leucocyte Collagenase

Human leucocyte collagenase is one member of the growing protein family of matrix metalloproteinases (MMPs) [Knauper et al., 1990]. It is a calcium-containing Zn-endoproteinase (MMP-8) that cleaves preferentially interstitial native triple-helical type I but also type II and type III collagen into one-quarter and three quarter fragments of the native chain length. If thus differs from the fibroblast interstitial collagenase that preferentially cleaves type III. About one-third of its mass of 65 kDa (for active enzyme) is carbohydrates in contrast to the homologous interstitial collagenase from fibroblasts which carries only a small carbohydrate portion [Tschesche et al., 1992]. The enzyme is stored in the specific granules of granulocytes and is released as a proenzyme, also designated latent enzyme, upon stimulation of the cells by various chemotactic agents, such as formylpeptides, LTB4, C5a, Fla and Zymosan amongst others, [Tschesche et al., 1989 and 1991]. Extracellular activation is then achieved by various different proteinases, such as trypsin, kallikrein, chymotrypsin, cathepsin G [Tschesche et al., 1992] or stromelysin [Knauper et al., 1993]. However, the physiological process of activation is not yet fully understood, since activation was also observed by isolated leucocyte membranes [Tschesche unpublished].