Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates

Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods

“…Forschungsartikel than the parent bivalent inhibitors (TTI: K i = 0.59 AE 0.05 pM, [12] Va r: K i = 3.8 AE 0.6 pM, Ano: K i = 7.7 AE 1.1 pM). [8] This enhanced inhibition was particularly pronounced in the case of TTI-Ano inhibitor 5 which displayed 30-fold improved thrombin inhibitory activity compared to sulfated TTI and activity 385-fold greater than singly sulfated Ano from A. albimanus (despite bearing only 23 of the 61 residues found in the native inhibitor).…”

“…Thet arget fragments included TTI(1-13) sulfopeptide selenoester 6,V ar(15-32) sulfopeptide 7 and bifunctional diselenide dimer fragments 8-10 bearing aC-terminal thioester and an N-terminal b-selenoaspartate [(b-Se)Asp] [18] moiety. These latter bifunctional fragments were comprised of TTI-(14-31) and Va r (12)(13)(14) fragments,connected through l-Ala, sarcosine or Aib amino acid linkers (Scheme 1). It should be noted that the sulfotyrosine residues within sulfopeptides fragments 6 and 7 were incorporated with neopentyl (nP) sulfate ester protection, enabling the acid labile sulfate esters to remain intact during Fmoc-SPPS and reversed-phase HPLC purification (see Supporting Information for synthetic details).…”

“…Based on their mechanistically and spatially distinct thrombin binding modes and potent anticoagulant activities, we selected three inhibitors to serve as scaffolds from which the proposed trivalent hybrid inhibitors could be constructed. Specifically,w es elected the exosite I-binding inhibitors, variegin (Var) [5,6] and af ragment of anophelin (Ano), [7,8] bearing asingle sulfotyrosine residue,and the bivalent exosite II binder,T TI, [12,13] possessing two sulfotyrosine residues. Using crystal structures of a-thrombin complexed with either Va r, Ano from A. albimanus or TTI, we proposed aseries of hybrid inhibitors 1-5 designed to bind to the active site,while straddling both exosite Iand exosite II of the protease.Itwas envisaged that the trivalent binding of these molecules would lead to enhanced inhibitory potencya gainst thrombin compared to the individual bivalent inhibitors.…”

“…Very recently, we uncovered the crucial role of this Tyr sulfation within a novel class of bivalent thrombin inhibitors that inhibit thrombin activity through a unique binding mode, by occupying both the catalytic site and exosite II. Examples include the tick‐derived madanins (from Haemaphysalis longicornis ), [10] hyalomins (from Hyalomma marginatum rufipes ) and andersonins (from Dermacentor andersoni ), [11] and the tsetse fly‐derived thrombin inhibitor (TTI) (from Glossina morsitans morsitans ) [12] . For each of these peptides, Tyr sulfation at two residues within their respective sequences was shown to significantly enhance thrombin inhibitory and anticoagulant activity, partially attributed to increased electrostatic affinity to the positively charged exosite II.…”

Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods

“…Research Articles than the parent bivalent inhibitors (TTI: K i = 0.59 AE 0.05 pM, [12] Va r: K i = 3.8 AE 0.6 pM, Ano: K i = 7.7 AE 1.1 pM). [8] This enhanced inhibition was particularly pronounced in the case of TTI-Ano inhibitor 5 which displayed 30-fold improved thrombin inhibitory activity compared to sulfated TTI and activity 385-fold greater than singly sulfated Ano from A. albimanus (despite bearing only 23 of the 61 residues found in the native inhibitor).…”