Machteld Sillen1, Stephen D Weeks2, Xiaohua Zhou1, Andrey A Komissarov3, Galina Florova3, Steven Idell3, Sergei V Strelkov2, Paul J Declerck1. 1. Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium. 2. Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium. 3. Department of Cellular and Molecular Biology, The University of Texas Health Science Center, Tyler, TX, USA.
Abstract
BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasminogen activators (PAs) tissue-type PA (tPA) and urokinase-type PA (uPA) plays a crucial role in many (patho)physiological processes (e.g., cardiovascular disease, tissue fibrosis) as well as in many age-related pathologies. Therefore, much effort has been put into the development of small molecule or antibody-based PAI-1 inhibitors. OBJECTIVE: To elucidate the molecular mechanism of nanobody-induced PAI-1 inhibition. METHODS AND RESULTS: Here we present the first crystal structures of PAI-1 in complex with two neutralizing nanobodies (Nbs). These structures, together with biochemical and biophysical characterization, reveal that Nb VHH-2g-42 (Nb42) interferes with the initial PAI-1/PA complex formation, whereas VHH-2w-64 (Nb64) redirects the PAI-1/PA interaction to PAI-1 deactivation and regeneration of active PA. Furthermore, whereas vitronectin does not have an impact on the inhibitory effect of Nb42, it strongly potentiates the inhibitory effect of Nb64, which may contribute to a strong inhibitory potential of Nb64 in vivo. CONCLUSIONS: These findings illuminate the molecular mechanisms of PAI-1 inhibition. Nb42 and Nb64 can be used as starting points to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions.
BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasminogen activators (PAs) tissue-type PA (tPA) and urokinase-type PA (uPA) plays a crucial role in many (patho)physiological processes (e.g., cardiovascular disease, tissue fibrosis) as well as in many age-related pathologies. Therefore, much effort has been put into the development of small molecule or antibody-based PAI-1 inhibitors. OBJECTIVE: To elucidate the molecular mechanism of nanobody-induced PAI-1 inhibition. METHODS AND RESULTS: Here we present the first crystal structures of PAI-1 in complex with two neutralizing nanobodies (Nbs). These structures, together with biochemical and biophysical characterization, reveal that Nb VHH-2g-42 (Nb42) interferes with the initial PAI-1/PA complex formation, whereas VHH-2w-64 (Nb64) redirects the PAI-1/PA interaction to PAI-1 deactivation and regeneration of active PA. Furthermore, whereas vitronectin does not have an impact on the inhibitory effect of Nb42, it strongly potentiates the inhibitory effect of Nb64, which may contribute to a strong inhibitory potential of Nb64 in vivo. CONCLUSIONS: These findings illuminate the molecular mechanisms of PAI-1 inhibition. Nb42 and Nb64 can be used as starting points to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions.
Authors: San Hadži; Abel Garcia-Pino; Sarah Haesaerts; Dukas Jurenas; Kenn Gerdes; Jurij Lah; Remy Loris Journal: Nucleic Acids Res Date: 2017-05-05 Impact factor: 16.971
Authors: Mirjam E Meltzer; Ton Lisman; Philip G de Groot; Joost C M Meijers; Saskia le Cessie; Carine J M Doggen; Frits R Rosendaal Journal: Blood Date: 2010-04-12 Impact factor: 22.113
Authors: D A Lawrence; S Palaniappan; S Stefansson; S T Olson; A M Francis-Chmura; J D Shore; D Ginsburg Journal: J Biol Chem Date: 1997-03-21 Impact factor: 5.157
Authors: Britt Van De Craen; Ilse Scroyen; Rana Abdelnabi; Els Brouwers; H Roger Lijnen; Paul J Declerck; Ann Gils Journal: Thromb Res Date: 2011-03-09 Impact factor: 3.944
Authors: Ross W Cheloha; Thibault J Harmand; Charlotte Wijne; Thomas U Schwartz; Hidde L Ploegh Journal: J Biol Chem Date: 2020-08-31 Impact factor: 5.157
Authors: Machteld Sillen; Toshio Miyata; Douglas E Vaughan; Sergei V Strelkov; Paul J Declerck Journal: Int J Mol Sci Date: 2021-02-02 Impact factor: 5.923