The Mechanism of Inhibition of Antibody-based Inhibitors of Membrane-type Serine Protease 1 (MT-SP1)

Farady, Christopher J.; Sun, Jingbo; Darragh, Molly R.; Miller, Susan M.; Craik, Charles S.

doi:10.1016/j.jmb.2007.03.078

Cited by 50 publications

(61 citation statements)

References 45 publications

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“…In the latter, the protruding domains encoded by the ultralong, up to 60-residue, CDR-H3s are frequent (45,46). In agreement, structural studies of the inhibitory antibodies isolated from human naive libraries also suggested that insertion of the long CDR-H3 variable loops (up to 19 residues) into the substrate-binding pocket is required to achieve efficient inhibition of membrane type serine protease 1 (matriptase) (37,47). Although the alternative inhibitory mechanisms are also well known, including those mechanisms that inactivate enzymes by inducing conformational changes or by blocking substrate access (48,49), multiple inhibitory antibodies exhibit unusually long CDR-H3s, implying that the extended CDR-H3s are vital for enzyme inhibition.…”

Section: Discussionmentioning

confidence: 69%

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: Discussionmentioning

confidence: 69%

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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“…The cytochrome c can activate caspase-9 with subsequent caspase-3 that results in DNA fragmentation in mammalian cells [38]. It has been reported that the antibody of specific protein suppresses the function of corresponding antigen by blocking access to the catalytic site [85,86]. In this study, Anti-Cyt C inhibited the • OH-induced activation of caspase-9 in the erythrocytes.…”

Section: • Oh-induced Apoptosis In Fish Erythrocytesmentioning

confidence: 59%

The metabolites of glutamine prevent hydroxyl radical-induced apoptosis through inhibiting mitochondria and calcium ion involved pathways in fish erythrocytes

Jiang

Liu

et al. 2016

Free Radical Biology and Medicine

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a b s t r a c tThe present study explored the apoptosis pathways in hydroxyl radicals ( • OH)-induced carp erythrocytes. Carp erythrocytes were treated with the caspase inhibitors in physiological carp saline (PCS) or Ca 2 þ -free PCS in the presence of 40 μM FeSO 4 /20 μM H 2 O 2 . The results showed that the generation of reactive oxygen species (ROS), the release of cytochrome c and DNA fragmentation were caspase-dependent, and Ca 2 þ was involved in calpain activation and phosphatidylserine (PS) exposure in • OH-induced carp erythrocytes. Moreover, the results suggested that caspases were involved in PS exposure, and Ca 2 þ was involved in DNA fragmentation in • OH-induced fish erythrocytes. These results demonstrated that there might be two apoptosis pathways in fish erythrocytes, one is the caspase and cytochrome c-dependent apoptosis that is similar to that in mammal nucleated cells, the other is the Ca 2 þ -involved apoptosis that was similar to that in mammal non-nucleated erythrocytes. So, fish erythrocytes may be used as a model for studying oxidative stress and apoptosis in mammal cells. Furthermore, the present study investigated the effects of glutamine (Gln)'s metabolites [alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala 10 Pro 4 Cit 1 )] on the pathways of apoptosis in fish erythrocytes. The results displayed that Ala, Cit, Pro and Ala 10 Pro 4 Cit 1 effectively suppressed ROS generation, cytochrome c release, activation of caspase-3, caspase-8 and caspase-9 at the physiological concentrations, prevented Ca 2 þ influx, calpain activation, PS exposure, DNA fragmentation and the degradation of the cytoskeleton and oxidation of membrane and hemoglobin (Hb) and increased activity of anti-hydroxyl radical (AHR) in • OH-induced carp erythrocytes. Ala 10 Pro 4 Cit 1 produced a synergistic effect of inhibited oxidative stress and apoptosis in fish erythrocytes. These results demonstrated that Ala, Cit, Pro and their combination can protect mammal erythrocytes and nucleated cells against oxidative stress and apoptosis. The studies supported the use of Gln, Ala, Cit and Pro as oxidative stress and apoptosis inhibitors in mammal cells and the hypothesis that the inhibited effects of Gln on oxidative stress and apoptosis are at least partly dependent on that of its metabolites in mammalian.

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“…Although a relatively small molecule (∼18-25kD), a scFv retains the specificity of the parent antibodies from which it was derived. Currently, specific scFvs are in use in vitro that inhibit proteases such as membrane type serine protease-1, which has been implicated in ovarian and prostate tumor progression (Farady et al, 2007), as well as to study the pleiotropic functions of substances such as IL-13, to better understand their role in immune function (Knackmuss et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Highly specific inhibition of C1q globular-head binding to human IgG: A novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment

Duvall

Tomlinson

Boackle

2008

Molecular Immunology

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We sought to specifically regulate the binding of human C1q, and thus the activation of the first complement component, via the construction of a single chain antibody variable binding region fragment (scFv) targeting the C1q globular heads. Here we describe details of the construction, expression and evaluation of this scFv, which was derived from a high affinity hybridoma (Qu) specific for the C1q globular heads. The scFv was comprised of the Qu variable-heavy chain domain (VH) linked to the Qu variable-light chain domain (VL) and was termed scFv-QuVHVL. When mixed with either purified C1q or with human serum as a source of C1, scFv-QuVHVL bound to C1q and competitively restricted the interaction of C1q or C1 with immobilized IgG or with IgG1 antibody-coated cells, and prevented the activation of native C1 in human serum as determined by analyses of C1-mediated C4 deposition and fluid phase C4 conversion. However scFv-QuVHVL could be manipulated to become a C1 activator when it was irreversibly immobilized onto microtiter ELISA plates, prior to contact with human serum complement. This functional dichotomy can be a useful tool in selectively elucidating, differentiating, inducing or inhibiting specific roles of human C1q and the classical complement pathway in complement-mediated physiological processes. We project that once fully humanized, fluid phase scFv-QuVHVL could become a useful therapeutic in limiting inadvertent host tissue damage elicited by the classical complement pathway.

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The Mechanism of Inhibition of Antibody-based Inhibitors of Membrane-type Serine Protease 1 (MT-SP1)

Cited by 50 publications

References 45 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

The metabolites of glutamine prevent hydroxyl radical-induced apoptosis through inhibiting mitochondria and calcium ion involved pathways in fish erythrocytes

Highly specific inhibition of C1q globular-head binding to human IgG: A novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment

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