Structural analysis of antibody-based profibrinolytics

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily, is the main physiologic inhibitor of plasminogen activators (PAs). Therefore, PAI-1 carries a flexible reactive center loop (RCL) that presents a substrate-mimicking peptide sequence that inserts into the active site of the PA. After initial complex formation, the reactive center in the RCL is cleaved by the PA which induces a conformational change to translocate the PA to the opposite side of the PAI-1 molecule. There, the active site of the PA is distorted when the PA is tightly pressed to the surface of the PAI-1 molecule. By forming this irreversible PAI-1/PA complex, PAI-1 inhibits the PA-mediated plasmin formation and the subsequent dissolution of the fibrin network that constitutes blood clots. As one of the key inhibitory proteins of the fibrinolytic PA system, PAI-1 has a regulatory role in hemostasis by preventing hyperfibrinolysis and excessive bleeding. Furthermore, by having pleiotropic functions in and beyond the PA system, PAI-1 is critically involved in the normal physiological processes associated with wound healing, angiogenesis, cell migration, and inflammation. However, emerging evidence points to a link between elevated PAI-1 levels and a variety of pathologies, including cardiovascular disease, thrombosis, cancer, fibrosis, neurodegenerative diseases, and age-related pathologies. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1-related pathologies. A very diverse collection of PAI-1 inhibitors has been developed, including peptides, RNA aptamers, small organochemical molecules, antibodies, antibody fragments, and nanobodies. Despite ongoing efforts, no PAI-1 inhibitors were approved to date for therapeutic use in humans. Even though several antagonists have been extensively characterized in vitro and in vivo, little is known on the exact molecular mechanism by which these inhibitors bind PAI-1 and modulate its activity. A better understanding of these mechanisms is therefore necessary to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of peptide-based or small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions. In this respect, we aimed to elucidate the molecular mechanism of antibody-based or small molecule PAI-1 inhibitors.

In the first two chapters of this work, we elucidated the X-ray crystallographic structures of PAI-1 in complex with three inhibitory nanobodies, Nb42 and Nb64 (chapter I), and Nb93 (chapter II). Together with biochemical and biophysical characterization, we were able to substantiate three distinct mechanisms by which they modulate PAI-1 activity. Both Nb42 and Nb93 were shown to directly interfere with the initial interactions between PAI-1 and PAs, albeit in two different ways. The structures revealed that Nb42 binds a surface on PAI-1 that is normally engaged in exosite interactions with PAs, i.e., interactions not involving the reactive center of PAI-1 or the active site of the PA. By interfering with this exosite interaction, Nb42 dramatically increased the rate of RCL insertion, reflecting its destabilizing effect on the initial PAI-1/PA complex. In contrast, Nb93 directly blocks the access of PAs to both the reactive center and the exosite binding regions in PAI-1. The third nanobody, Nb64, binds near or at the position of the PAs in the final inhibitory complex, and thus on the opposite side of PAI-1 with respect to the RCL and the binding sites for Nb42 and Nb93. Neither the formation of the initial PAI-1/PA complex nor the kinetics for the PAI-1/PA reaction was affected by Nb64. Therefore, Nb64 most likely prevents the distortion of the PA active site leading to the release of PA from cleaved PAI-1, thereby converting PAI-1 from an inhibitor to a substrate for the PA.

In chapter III we aimed to determine the crystal structure of PAI-1 in complex with the single-chain variable fragment (scFv) of substrate-inducing monoclonal antibody MA-33H1F7. As we were able to determine the structure of scFv-33H1F7, we used antibody-protein docking to model its complex with PAI-1. Biochemical information was used for knowledge-based filtering of the generated models. The ultimately selected model appeared to be in good agreement with several lines of experimental evidence.

In chapter IV, the X-ray crystallographic structure of PAI-1 in complex with small molecule inhibitor TM5484 revealed a binding site at the surface of PAI-1 overlapping with the binding site for vitronectin, a biological ligand of PAI-1. Of note, this observed binding site is substantially different from the presumed binding site described in the literature. However, the binding site proposed by the structure may form a rational explanation for the observed inhibitory mechanism in biochemical assays, i.e., inducing substrate behavior of PAI-1 and impairing its interaction with PAs. By binding at the vitronectin binding site and rigidifying the flexible joint region, TM5484 may induce substrate behavior and convert PAI-1 into a non-reactive form through allosteric modulation.

In conclusion, structural studies complemented with biochemical assays provided insight into the inhibitory mechanisms of PAI-1 inhibitory nanobodies and one class of small molecule PAI-1 antagonists. Furthermore, the obtained structures of the PAI-1/inhibitor complexes may provide an excellent starting point for the design of novel profibrinolytic therapeutics.