XR5118 [(3Z,6Z)-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P1 residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpin's large central β-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of α-helix F, situated above β-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to β-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target.
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August 2003
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Research Article|
August 01 2003
Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1
Anja P. EINHOLM;
Anja P. EINHOLM
1
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
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Katrine E. PEDERSEN;
Katrine E. PEDERSEN
1
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
2To whom correspondence should be addressed (e-mail pa@mb.au.dk).
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Troels WIND;
Troels WIND
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
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Paulina KULIG;
Paulina KULIG
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
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Michael T. OVERGAARD;
Michael T. OVERGAARD
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
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Jan K. JENSEN;
Jan K. JENSEN
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
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Julie S. BØDKER;
Julie S. BØDKER
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
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Anni CHRISTENSEN;
Anni CHRISTENSEN
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
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Peter CHARLTON;
Peter CHARLTON
†Xenova, 957 Buckingham Avenue, Slough, Berkshire, SL1 4NL, U.K.
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Peter A. ANDREASEN
Peter A. ANDREASEN
2
∗Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark
2To whom correspondence should be addressed (e-mail pa@mb.au.dk).
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Publisher: Portland Press Ltd
Received:
December 04 2002
Revision Received:
March 27 2003
Accepted:
April 30 2003
Accepted Manuscript online:
April 30 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 373 (3): 723–732.
Article history
Received:
December 04 2002
Revision Received:
March 27 2003
Accepted:
April 30 2003
Accepted Manuscript online:
April 30 2003
Citation
Anja P. EINHOLM, Katrine E. PEDERSEN, Troels WIND, Paulina KULIG, Michael T. OVERGAARD, Jan K. JENSEN, Julie S. BØDKER, Anni CHRISTENSEN, Peter CHARLTON, Peter A. ANDREASEN; Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1. Biochem J 1 August 2003; 373 (3): 723–732. doi: https://doi.org/10.1042/bj20021880
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