title: Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase.
abstract: Heparanase is a β-d-glucuronidase which cleaves heparan sulfate chains in the extracellular matrix and on cellular membranes. A dysregulated heparanase activity is intimately associated with cell invasion, tumor metastasis and angiogenesis, making heparanase an attractive target for the development of anticancer therapies. SST0001 (roneparstat; Sigma-Tau Research Switzerland S.A.) is a non-anticoagulant 100% N-acetylated and glycol-split heparin acting as a potent heparanase inhibitor, currently in phase I in advanced multiple myeloma. Herein, the kinetics of heparanase inhibition by roneparstat is reported. The analysis of dose-inhibition curves confirmed the high potency of roneparstat (IC50 ≈ 3 nM) and showed, at higher concentrations, a Hill coefficient consistent with the engagement of two molecules of inhibitor. A homology model of human heparanase GS3 construct was built and used for docking experiments with inhibitor fragments. The model has high structural similarity with the recently reported crystal structure of human heparanase. Different interaction schemes are proposed, which support the hypothesis of a complex binding mechanism involving the recruitment of one or multiple roneparstat chains, depending on its concentration. In particular, docking solutions were obtained in which (i) a single roneparstat molecule interacts with both heparin-binding domains (HBDs) of heparanase or (ii) two fragments of roneparstat interact with either HBD-1 or HBD-2, consistent with the possibility of different inhibitor:enzyme binding stoichiometries. This study provides unique insights into the mode of action of roneparstat as well as clues of its interaction with heparanase at a molecular level, which could be exploited to design novel potential inhibitor molecules.
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze 27/A, Parma 43124, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Pala
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze 27/A, Parma 43124, Italy silvia.rivara@unipr.it ferdinando.milazzo@sigma-tau.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Rivara
authors.affiliation: Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze 27/A, Parma 43124, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Mor
authors.affiliation: R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, Pomezia, Roma 00071, Italy silvia.rivara@unipr.it ferdinando.milazzo@sigma-tau.it.
authors.forename: Ferdinando Maria
authors.initials: FM
authors.lastname: Milazzo
authors.affiliation: Takis s.r.l., Via Castel Romano 100, Roma 00128, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Roscilli
authors.affiliation: Takis s.r.l., Via Castel Romano 100, Roma 00128, Italy.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Pavoni
authors.affiliation: R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, Pomezia, Roma 00071, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Giannini
mesh_terms: D061271:Acidobacteria / Q000737:chemistry / Q000201:enzymology; D020816:Amino Acid Motifs; D001665:Binding Sites; D002240:Carbohydrate Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry*; D000077425:Fondaparinux; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013816:Thermodynamics; D000099175:Heparanase
chemical_list: D004791:Enzyme Inhibitors; D000077425:Fondaparinux; D005966:Glucuronidase; D006493:Heparin; D011134:Polysaccharides; D011994:Recombinant Proteins; D000099175:Heparanase; C559639:SST 0001
keywords: SST0001; heparanase; homology modeling; kinetic inhibition analysis; roneparstat