The Specificity of TIMP-2 for Matrix Metalloproteinases Can Be Modified by Single Amino Acid Mutations

Butler, Georgina S.; Hutton, Mike; Wattam, Beth; Williamson, Richard A.; Knäuper, Vera; Willenbrock, Frances; Murphy, Glynis H.

doi:10.1074/jbc.274.29.20391

Cited by 80 publications

(81 citation statements)

References 38 publications

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“…2C). At the similar conditions, n-TIMP-2 and GM6001 inhibited MMP-2/MMP-9/MMP-14 nonselectively (83-100% inhibition), confirming that they are broad-spectrum inhibitors of the MMP family (38,39).…”

Section: Significancementioning

confidence: 63%

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Nam

Rodríguez

Remacle

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

110

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Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 10 9 individual variants) that carried the extended, 23-to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes. family members control various physiological and pathological processes. Multiple diseases are associated with altered MMP expression and aberrant proteolysis, including cancer (1), wound healing (2), inflammatory diseases (3, 4), neurological pain (5, 6), and hypertension (7). There is consensus among researchers that the individual MMPs are promising drug targets in diversified pathologies and that inhibitor specificity is required for selective and successful MMP therapies (8-10).However, achieving target specificity and selectivity in small-molecule MMP inhibitors is remarkably challenging (11,12). Because the catalytic mechanism and catalytic domain fold are conserved among the MMP/ADAM (a disintegrin and metalloproteinase)/ ADAMTS (ADAM with thrombospondin motifs) superfamily members, the available small-molecule inhibitors (most frequently, active-site zinc-chelating hydroxamates) target multiple proteinases, resulting in off-target side effects (8,(12)(13)(14). This aspect is problematic, given that some MMPs (e.g., MMP-14) are always protumorigenic, whereas some other MMPs are antitumorigenic in certain cancer microenvironments (15, 16). As a result, broad-spectrum hydroxamates failed in cancer clinical trials due to their low overall efficacy and side effects (13). Alternatively, antibody-based MMP inhibitors are emerging as both research tools and potential therapeutic agents (10, 17-21) because of (i) high affinity and specificity due to the large antigen-antibody interaction area and multiple complementarity-determining regions (CDRs), (ii) long half-life and well-defined action mechanisms, (iii) low immunogenicity and toxicity, and (iv) multiple MMPs potentially targe...

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Section: Significancementioning

confidence: 63%

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Nam

Rodríguez

Remacle

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

110

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“…In our modeled TIMP-1-MT3-MMP complex, only the hydrophobic part of the Glu156 T1 side-chain would get into contact with Phe267 MT3 (Phe is conserved in all human MT-MMPs except for MT4-MMP, where it is a Tyr). Furthermore, an MT1/3-MMP complex with TIMP-1 would lack the favorable interactions made by the extended sA-sB loop of TIMP-2, which in the MT1-MMP-TIMP-2 complex slots into the surface groove left by the MT-loop, the sidechains of Asp212 MT1 and Phe250 MT1 , and the S-loop of MT1-MMP 25 The importance of these interactions for the MT-MMP-TIMP-2 complex stability is shown by the 40-fold reduction in the apparent Ki value of the Tyr36 T2 Gly sA-sB loop tip mutants (of N-TIMP-2) for association with MT1-MMP 18 These interactions mediated through the sA-sB loop might also pull the "left" side of TIMP-2 (Figure 3(a)) towards the MT1-MMP molecule, thus preventing the approach of the "right" side, which in the modeled TIMP-1 complex shows clashes (see above). In summary, the crystal structures of MT1-and MT3-MMP are not in agreement with the favorable binding of TIMP-1, somewhat explaining the extremely low association reaction/affinity.…”

Section: Timp-1 Docking To Mt-mmps Shows Obstacles For Interactionmentioning

confidence: 99%

“…11 MT1-MMP seems to be the major activator of proMMP-2, mainly via presentation of proMMP-2 by a MT1-MMP-TIMP-2 "receptor". [15][16][17][18] However, the catalytic domains or the fulllength molecules of the other MT-MMPs [19][20][21][22][23] have also been shown in vitro or expressed in mammalian cells, respectively, to activate proMMP-2. Apparently, MT2-MMP is able to perform this activation in a TIMP-2-independent manner.…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of the Catalytic Domain of MMP-16/MT3-MMP: Characterization of MT-MMP Specific Features

Lang

Braun

Sounni

et al. 2004

Journal of Molecular Biology

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Membrane-type matrix metalloproteinases (MT-MMPs) have attracted strong attention, because four of them can activate a key player in the tumor scenario, proMMP-2/progelatinase A. In addition to this indirect effect on the cellular environment, these MT-MMPs degrade extracellular matrix proteins, and their overproduction is associated with tumor growth. We have solved the structure of the catalytic domain (cd) of MT3-MMP/MMP-16 in complex with the hydroxamic acid inhibitor batimastat. CdMT3-MMP exhibits a classical MMP-fold with similarity to MT1-MMP Nevertheless, it also shows unique properties such as a modified MT-specific loop and a closed S1' specificity pocket, which might help to design specific inhibitors. Some MT-MMP-specific features, derived from the crystal structures of MT-1-MMP determined previously and MT3-MMP, and revealed in recent mutagenesis experiments, explain the impaired interaction of the MT-MMPs with TIMP-1. Docking experiments with proMMP-2 show some exposed loops including the MT-loop of cdMT3-MMP involved in the interaction with the proMMP-2 pro-domain in the activation encounter complex. This model might help to understand the experimentally proven importance of the MT-loop for the activation of proMMP-2.

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“…The truncated N-terminal domains of TIMPs (N-TIMPs) and isolated MMP catalytic domains (MMPcd) have been extensively used in studies of the interactions between TIMPs and MMPs (12)(13)(14)(15)(16)(17)(18)(19) and the majority of the intermolecular interactions in structurally characterized inhibitory TIMP⅐MMP complexes involve residues in these domains.…”

mentioning

confidence: 99%

“…Although it is possible that interactions involving the TIMP C-domain might modulate the affinity for some MMPs, they are not necessary for binding because N-TIMPs are fully active as MMP inhibitors (12)(13)(14)(15)(16)(17)(18)(19). The one clear example of selectivity in TIMP/MMP interactions is the weak affinity of TIMP-1 for the membrane-type MMPs, including MT1-MMP and MMP-19, whereas TIMP-2 is a potent inhibitor of these enzymes (3).…”

mentioning

confidence: 99%

Entropy Increases from Different Sources Support the High-affinity Binding of the N-terminal Inhibitory Domains of Tissue Inhibitors of Metalloproteinases to the Catalytic Domains of Matrix Metalloproteinases-1 and -3

Wei

Doren

et al. 2011

Journal of Biological Chemistry

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The matrix metalloproteinases (MMPs) 3 catalyze the turnover of components of the extracellular matrix and have important roles in tissue remodeling, wound healing, embryo implantation, cell migration, and shedding of cell surface proteins (1, 2). MMP-1 is a well characterized collagenase that catalyzes the turnover of collagen fibrils in the matrix, whereas MMP-3 (stromelysin 1) cleaves multiple extracellular matrix components and functions in tissue remodeling and other processes (2). Collagenolysis is a key feature of biological processes including development, morphogenesis, and wound repair yet unregulated collagen breakdown contributes to important diseases including cancer, arthritis, emphysema, and fibrosis (1). An understanding of the molecular basis of the regulation of MMP activities is crucial for understanding MMP-associated diseases and developing therapies for them. The tissue inhibitors of metalloproteinases (TIMP-1 to Ϫ4) are a family of four endogenous MMP inhibitors that can form high affinity 1:1 complexes with most MMPs. TIMP-3 also inhibits some members of the distantly related disintegrin metalloproteinase (ADAM) and disintegrin metalloproteinase with thrombospondin type 1 motif (ADAMTS) families (2). A loss of balance between the TIMPs and their target proteases is linked to diseases such as cancer and arthritis (2, 3).TIMPs are slow, tight-binding inhibitors of the MMPs with K i values typically in the sub-to low nanomolar range (3). Mammalian TIMPs have two domains, a larger (ϳ125 residue) N-terminal domain that can be expressed separately and carries the MMP inhibitory activity (2, 3) and a smaller C-terminal domain that is absent from the TIMPs of some invertebrates (3). In the crystal structures of the MMP-3⅐TIMP-1, MMP-1⅐N-TIMP-1, MT1-MMP⅐TIMP-2, and MMP-13⅐TIMP-2 complexes (4 -7), most of the MMP interaction surface is located within the N-domains of the TIMPs; as shown in Fig. 1, the N-terminal region (residues 1-5) inserts into the active site of the MMP, whereas the ␣-amino group together with the carbonyl oxygen of Cys 1 coordinate the catalytic zinc (4 -7). Modification of the ␣-amino group by addition of an alanine (8, 9), carbamylation (10), or acetylation (11) radically reduces the inhibitory activities of TIMPs for MMPs. In the all inhibitory TIMP⅐MMP complexes, the side chain of residue 2 of the TIMP (Ser or Thr in vertebrate TIMPs) sits over the mouth of the key S 1 Ј subsite of the MMP active site (4 -7), and substitution by glycine results in large loss of affinity for most MMPs (9, 12). However, both the Ala extension and Thr 2 to Gly mutation in N-TIMP-3 have little effect on the inhibition of ADAM-17, 13). The inhibitory domain of TIMP has an OB-fold structure, a 5-stranded ␤-barrel structure with two small helices. Other regions in TIMPs that contact MMPs include the connector between the C and D ␤ strands and the loops connecting the A to B, and E to F strands (4 -7). In * This work was supported, in whole or in part, by National Institutes of Health Grants R01 AR...

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The Specificity of TIMP-2 for Matrix Metalloproteinases Can Be Modified by Single Amino Acid Mutations

Cited by 80 publications

References 38 publications

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Crystal Structure of the Catalytic Domain of MMP-16/MT3-MMP: Characterization of MT-MMP Specific Features

Entropy Increases from Different Sources Support the High-affinity Binding of the N-terminal Inhibitory Domains of Tissue Inhibitors of Metalloproteinases to the Catalytic Domains of Matrix Metalloproteinases-1 and -3

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