Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1.
XR5118 [(3 Z,6 Z )-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 P(1) 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 beta-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 alpha-helix F, situated above beta-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to beta-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.
Department of Molecular Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 C Aarhus, Denmark.
Anja P
AP
Einholm
Katrine E
KE
Pedersen
Troels
T
Wind
Paulina
P
Kulig
Michael T
MT
Overgaard
Jan K
JK
Jensen
Julie S
JS
Bødker
Anni
A
Christensen
Peter
P
Charlton
Peter A
PA
Andreasen
D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010879:Piperazines / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics; D019096:Vitronectin / Q000378:metabolism*
D010879:Piperazines; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; C107761:XR 5118