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

Abstract

Blood feeding arthropods,such as leeches,ticks, flies and mosquitoes,p rovide ap rivileged source of peptidic anticoagulant molecules.T hese primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite Io re xosite II. Herein, we describe the rational design of an ovel class of trivalent thrombin inhibitors that simultaneously blockb oth exosites as well as the active site.These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in onepot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against a-thrombin and were selective over related coagulation proteases.Alead hybrid inhibitor possessed potent anticoagulant activity,blockade of both thrombin generation and platelet aggregation in vitro and efficacy in am urine thrombosis model at 1mgkg À1 .T he rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for ar ange of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions,i na ddition to an active site.

O-Glycosylation Induces Amyloid-β To Form New Fibril Polymorphs Vulnerable for Degradation

“…To obtain homogeneously glycosylated Aβ (1–42) for further investigation, we envisioned that solid phase peptide synthesis (SPPS) could be applied, which requires the carbohydrate-containing tyrosine as a building block. Such a “cassette” strategy has been previously applied in preparing a number of proteins with PTMs, among which studies of tyrosine O-sulfations and O-phosphorylation are particularly inspiring. While O-glycosylations in eukaryotic proteins are mostly located on Ser/Thr residues, previous studies have indicated that the mucin-type O -glycans on Aβ-derived degradation fragments are sialylated Hex-HexNAc and attached to tyrosine 10 through an α-glycosidic bond .…”

Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis

“…Furthermore, unlike hyalomin (from the tick H. marginatum rufipes), madanin (from the tick H. longicornis), avathrin, and variegin, XC-43 is not cleaved by thrombin, suggesting that it retains full inhibitory activity and affinity for thrombin after interaction with the enzyme. Recently Agten et al (39) developed synthetic inhibitors by fusing the exosite II-binding region of the tsetse thrombin inhibitor (TTI) (40) with variegin or anophelin generating a molecule with high affinity for thrombin that binds both exosites in addition to the catalytic site. A similar strategy using XC-43 instead of variegin would be interesting to pursue, since the flea-derived peptide is not cleaved by thrombin and therefore could result in a more stable complex.…”

“…Recently Agten et al. ( 39 ) developed synthetic inhibitors by fusing the exosite II-binding region of the tsetse thrombin inhibitor (TTI) ( 40 ) with variegin or anophelin generating a molecule with high affinity for thrombin that binds both exosites in addition to the catalytic site. A similar strategy using XC-43 instead of variegin would be interesting to pursue, since the flea-derived peptide is not cleaved by thrombin and therefore could result in a more stable complex.…”

Synthetic Thiol and Selenol Derived Amino Acids for Expanding the Scope of Chemical Protein Synthesis

“…Remarkably, this method involves the simple mixing of peptide selenoesters with the peptide diselenide dimer in ligation buffer without the need of reducing agents ( Scheme 1F ). The additive-free reaction dubbed diselenide–selenoester ligation (DSL) represents an unprecedented reactivity of Sec that gateways to rapid generation of polypeptide and protein therapeutic leads bearing unique and defined modifications ( Watson et al, 2018 ; Watson et al, 2019 ; Agten et al, 2021 ). These pioneer works have also fuelled the development of synthetic routes to assess suitably protected β/γ/δ-selenyl derived amino acids that can be employed in traceless selenium-mediated peptide ligation for total synthesis of protein targets bearing distinct modifications.…”