Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1

Abstract

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.

ARNTL2 and SERPINE1: potential biomarkers for tumor aggressiveness in colorectal cancer

“…Plasmin proteolysis must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation, substantiating the clinical paradox where by PAI-1 promotes tumor progression, and contraindicating the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors (Bajou et al 2001). Inactivation of PAI-1 might be a potential therapeutic tool in colorectal cancer, and in the last 10 years, PAI-1 has been the target of experimental therapeutic strategies using inhibitors such as XR5118, a diketopiperazine-based low molecular weight inhibitor (Einholm et al 2003), the orally active tiplaxtinin (Leik et al 2006), or polyphenolic inhibitors (Cale et al 2010), which could modulate pharmacologically PAI-1 effects in tumor angiogenesis.…”

Small molecules inhibitors of plasminogen activator inhibitor-1 – An overview

“…In rats, a 41% increase in clot lysis was observed at 5 mg/kg and an infusion doubled the time of vessel occlusion. It was later shown [55] that interactions of XR5118 (12) with PAI-1 inactivate the serpin activity by inducing a different conformation from the one of the latent form. It was suggested that the binding site was in an area between b-sheet A and a-helix F, nearby the one proposed for vitronectin.…”

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

“…These associations have made PAI-1 an attractive pharmaceutical target. However, despite extensive studies, only a few small molecule inhibitors have been identified thus far (7)(8)(9)(10)(11)(12)(13)(14)(15)(16), and the majority of these are poor pharmaceutical candidates as they have relatively low affinity for PAI-1 and are unable to inactivate PAI-1 bound to its plasma cofactor vitronectin.…”