Engineered Antagonists of uPA and PAI-1

Stoppelli, Maria Patrizia; Andersen, Lone B.; Votta, Giuseppina; Andreasen, Peter A.

doi:10.1007/978-0-387-69057-5_35

Cited by 3 publications

(4 citation statements)

References 229 publications

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“…In contrast, there was no effect of mAb-118, which inhibits neither pro-uPA activation nor plasminogen activation catalyzed by two-chain uPA. 5 Likewise, there was no effect of IgG purified from mouse plasma (data not shown). Importantly, none of these treatments affected primary tumor growth.…”

Section: The Epitope Of Mab-112 Is Localized To the Autolysis Loop-mentioning

confidence: 89%

“…Anti-uPA mAb-118 was from the same fusion as mAb-112 and found to bind the serine protease domain but did not affect uPA enzyme activity. 5 Surface Plasmon Resonance Analysis-Surface plasmon resonance (SPR) analyses were performed on a BIACORE T100 instrument using CM5 sensor chips, flow rates of 30 l/min, and HBS-B with 0.05% Tween 20. Concentrations of pro-uPA in conditioned media from HEK 293T cells were determined by measuring the initial rate of binding to a chip with 200 response units of anti-uPA mAb-6 (12) using a standard curve of purified pro-uPA.…”

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confidence: 99%

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A Novel Mode of Intervention with Serine Protease Activity

Blouse

Bøtkjær

Deryugina

et al. 2009

Journal of Biological Chemistry

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Serine proteases are secreted from cells as single-chain zymogens, typically having activities orders of magnitude lower than those of the mature two-chain enzymes. Activation occurs by a conformational change initiated by cleavage of a specific peptide bond. We have derived a monoclonal antibody (mAb-112) which binds with subnanomolar affinity to pro-uPA, the zymogen form of urokinase-type plasminogen activator (uPA). We mapped the epitope of the antibody to the autolysis loop, one of the structural elements known to change conformation during zymogen activation. A mechanistic evaluation with biophysical methods elucidated a novel bifunctional inhibitory mechanism whereby mAb-112 not only delays the proteolytic conversion of single-chain pro-uPA into the two-chain form but also subsequently averts the conformational transition to a mature protease by sequestering the two-chain form in a zymogen-like, noncatalytic state. Functional studies employing two variants of human HT-1080 cells, exhibiting high and low levels of dissemination in a chorioallantoic membrane assay, demonstrate that mAb-112 is an effective inhibitor of tumor cell intravasation. These findings show that pharmacological interference with zymogen activation is a plausible and robust means to regulate uPA activity and the downstream effects of plasminogen activation in the spread of cancer and other processes of pathological tissue remodeling. A strategy that targets regions related to pro-enzyme activation likely provide a unique inhibitorprotease interaction surface and is, thus, expected to enhance the chances of engineering high inhibitor specificity. Our results provide new information about the structural flexibility underlying the equilibrium between active and inactive forms of serine proteases.In nature a key mechanism for regulation of serine proteases with a trypsin-like fold is the activation of secreted zymogens or proenzymes, which typically have activities orders of magnitude lower than the mature enzymes. Zymogen activation is the central step in natural protease cascade regulation, allowing for rapid amplification of the activation signal. The catalytic activity of a zymogen relative to the mature protease can generally be thought of as a problem of equilibrium between active and inactive conformational states of the protease domain. Zymogen activation generally occurs by cleavage of the bond between amino acid residues 15 and 16.2 The liberated amino terminus inserts into a hydrophobic binding cleft of the catalytic domain forming, in addition to hydrophobic interactions, a salt bridge to the side chain of Asp 194 which stabilizes the substrate binding pocket and oxyanion hole in a catalytically productive conformation. Conformational changes after cleavage involves four disordered regions of the activation domain, including the activation loop (residues 16 -21), the autolysis loop (residues 142-152), the oxyanion stabilizing loop (residues 184 -194), and the S1 entrance frame (residues 216 -223) (Fig. 1A) (for reviews, see Refs...

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Section: The Epitope Of Mab-112 Is Localized To the Autolysis Loop-mentioning

confidence: 89%

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confidence: 99%

A Novel Mode of Intervention with Serine Protease Activity

Blouse

Bøtkjær

Deryugina

et al. 2009

Journal of Biological Chemistry

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“…A number of inhibitors of the proteolytic activity of uPA have been developed, including small organochemical molecules, peptides, and monoclonal antibodies, both with a view to their use for elucidating the pathophysiological functions of uPAs various molecular interactions and to generate leads for drug development (for a review see [40]). The most specific inhibitors to date appear to be those derived from antibodies and peptidylic inhibitors, which utilize binding sites involving surface loops of uPA and extended exosite interactions that drive selectivity and specificity.…”

Section: Discussionmentioning

confidence: 99%

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

Bøtkjær

Fogh

Bekes

et al. 2011

Biochemical Journal

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SUMMARY Tight regulation of serine proteases is essential for their physiological functions and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of urokinase-type plasminogen activator (uPA) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA’s activity. We have now characterised the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, by immunofluorescence, the conformation-specific antibodies, mAb-112 and mAb-12E6B10, enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface-loops that undergo conformational changes during zymogen activation.

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“…Other PAI-1 inactivators, including various peptides corresponding to the reactive center loop in PAI-1, have been found to be able to insert between ␤-strands 3A and 5A, inhibit RCL insertion during reaction with target proteases, and thus induce substrate behavior (Eitzman et al, 1995;Xue et al, 1998). A number of organochemical compounds were reported to inactivate the antiproteolytic activity of PAI-1 by a variety of mechanisms (for reviews, see Durand et al, 2004;Andreasen, 2007;Stoppelli et al, 2008). High concentrations of nonionic detergents were found to induce substrate behavior and then latency transition (Ehnebom et al, 1997;Gils and Declerck, 1998;Andreasen et al, 1999;Gils et al, 2000Gils et al, , 2003.…”

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confidence: 99%

A Peptide Accelerating the Conversion of Plasminogen Activator Inhibitor-1 to an Inactive Latent State

Mathiasen

Dupont²,

Christensen³

et al. 2008

Mol Pharmacol

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The serpin plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and a potential therapeutic target in cancer and cardiovascular diseases. Accordingly, formation of a basis for development of specific PAI-1-inactivating agents is of great interest. One possible inactivation mode for PAI-1 is conversion to the inactive, so-called latent state. We have now screened a phage-displayed peptide library with PAI-1 as bait and isolated a 31-residue cysteine-rich peptide that will be referred to as paionin-4. A recombinant protein consisting of paionin-4 fused to domains 1 and 2 of the phage coat protein g3p caused a 2-to 3-fold increase in the rate of spontaneous inactivation of PAI-1. Paionin-4-D1D2 bound PAI-1 with a K D in the high nanomolar range. Using several biochemical and biophysical methods, we demonstrate that paionin-4-D1D2-stimulated inactivation consists of an acceleration of conversion to the latent state. As demonstrated by site-directed mutagenesis and competition with other PAI-1 ligands, the binding site for paionin-4 was localized in the loop between ␣-helix D and ␤-strand 2A. We also demonstrate that a latency-inducing monoclonal antibody has an overlapping, but not identical binding site, and accelerates latency transition by another mechanism. Our results show that paionin-4 inactivates PAI-1 by a mechanism clearly different from other peptides, small organochemical compounds, or antibodies, whether they cause inactivation by stimulating latency transition or by other mechanisms, and that the loop between ␣-helix D and ␤-strand 2A can be a target for PAI-1 inactivation by different types of compounds.Plasminogen activator inhibitor type-1 (PAI-1) is a fast and specific inhibitor of the urokinase-type plasminogen activator (uPA) and the tissue-type plasminogen activator. PAI-1 is therefore an important regulator of the physiological and pathophysiological functions of plasminogen activation. A high level of PAI-1 in blood is associated with an increased risk of thrombotic events (for review, see Vaughan, 1998). PAI-1 is therefore a potential target for antithrombotic therapy. A high level of PAI-1 in tumors is associated with a poor prognosis, and several studies have suggested that PAI-1 is contributory to tumor invasion and metastasis (for review, see Andreasen et al., 1997Andreasen et al., , 2000Durand et al., 2004;Andreasen, 2007). PAI-1 is therefore a potential target for anticancer therapy.PAI-1 belongs to the serpins, a protein family of which the best characterized members are known to be inhibitors of serine proteases. Serpins are globular proteins consisting of three ␤-sheets (A, B, and C) and nine ␣-helices (A-I). Of crucial importance for the inhibitory mechanism of serpins is the surface-exposed reactive center loop (RCL), tethered between ␤-strands 1C and 5A. The active site of the protease attacks the P1-P1Ј bond in the RCL as a substrate, but at the enzyme-acyl intermediate stage, where the active site serine of the protease and the P1 r...

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Engineered Antagonists of uPA and PAI-1

Cited by 3 publications

References 229 publications

A Novel Mode of Intervention with Serine Protease Activity

A Novel Mode of Intervention with Serine Protease Activity

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

A Peptide Accelerating the Conversion of Plasminogen Activator Inhibitor-1 to an Inactive Latent State

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