title: Structural and Functional Characterization of Hirudin P6 Derived Novel Bivalent Thrombin Inhibitors - Studying the Effect of Linker Length and Glycosylation on Their Function.
abstract: HirudinP6 is a glycosylated and sulfated high affinity thrombin inhibitory protein isolated from Hirudineria manillensis. In this study, designing of novel bivalent thrombin inhibitory peptides based on this hirudin isoform is described. The structural and functional impact of varying linker length and glycosylation on their inhibitory potencies and binding kinetics were assessed. The bivalent peptides were obtained by tethering an active site blocking fPRP motif with the carboxy terminal 22 residue segment of hirudin P6 (HP642-63 ) by varying number of glycine residues in the linker region. Among them, analog BiG1 -HP6 inhibited thrombin with a Ki of 5.12 nm which was comparable to that of glycosylated (disaccharide bearing) and non-sulfated full length hirudin P6 protein (Ki = 6.38 nm). Binding kinetics studies revealed increasing linker length can decrease the association rates of peptide─thrombin interactions. Similarly, glycosylation was found to negatively modulate the inhibitory potencies of these peptides by decreasing their rates of association with thrombin. Molecular docking studies revealed that increasing linker length can compromise the electrostatic interactions with the prime subsite residues of thrombin and provided structural explanation for the observed effect of linker length on association rates. These findings thus enhance our understanding of thrombin─(glyco)peptide interactions and provide key insights into the designing of efficient thrombin inhibitors and allosteric modulators of therapeutic potential.
authors: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
authors: Prv
authors: P
authors: Shabareesh
authors: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
authors: Kanwal J
authors: KJ
authors: Kaur
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D000845:Annelida / Q000737:chemistry; D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D020134:Catalytic Domain; D015195:Drug Design; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006031:Glycosylation; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000991:Antithrombins; D006020:Glycopeptides; D006629:Hirudins; D008024:Ligands; D010446:Peptide Fragments; C000615610:hirudin (42-63); D013912:Threonine; D013917:Thrombin
keywords: binding kinetics; bivalent peptides; glycopeptide; protein - peptide interactions; thrombin inhibition