13 research outputs found
Probing Allosteric, Partial Inhibition of Thrombin Using Novel Anticoagulants
Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an alternative mechanism called allosteric, partial inhibition, that has shown promise to truly regulate coagulation. Partial inhibition through allosteric mechanisms are well described for membrane-bound and oligomeric proteins. However proteases, specifically monomeric proteases (i.e., thrombin), have not shown this phenomenon until now. A small library of coumarin-based sulfated allosteric modulators (CSAMs) was synthesized to target a surface region called exosite 2; mainly composed of highly positively charged residues surrounded by hydrophobic patches. Studies revealed a non-competitive mechanism of binding with a range of IC50s between 0.2-58 µM combined with inhibitory efficacies (ΔY) between 22-73%; indicative of allosteric, partial inhibition. The KD was determined for the most potent compound (3g; IC50 = 0.2 µM, ΔY = 47%) at 0.15 µM. 3g was observed to bind at exosite 2 through unfractionated heparin competition and thrombin mutant studies. Additional computational studies were in agreement with the mutant and competition studies, showing the sulfate of 3g binding within a pocket containing R126 and R233. Fluorescence quenching and antithrombin inactivation rate studies described a conformational change to thrombin’s active site in the presence of 3g, supporting reduction of thrombin’s catalytic efficiency, without complete inhibition of thrombin’s proteolytic activities. Coupled enzyme assays and gel electrophoresis showed that in the presence of 3g, hydrolysis of fibrinogen (IC50 = 0.51 µM, ΔY = 94%) and protein C activation (IC50 = 1.7 µM, ΔY = 91%) is fully inhibited. Alternatively, FXIII activation was shown to be only partially inhibited by the presence of 3g, and FXI activation did not show any significant activation or inhibition. 3g was also shown to be active in human plasma and whole blood, but requiring much higher concentrations to induce an anticoagulant effect. Mice studies looking at the effects of 3g in vivo showed that even at high concentrations, showed no abnormal bleeding or any other irregularities. This work highlights a novel occurrence regarding thrombin’s allosteric functionality against multiple endogenous substrates. This library of compounds may be useful in the future development of allosteric inhibitors and probes that pose little to no risk of bleeding events by inducing partial inhibition
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Engineering of spectator glycocalyx structures to evaluate molecular interactions at crowded cellular boundaries
In the mucosal epithelium, the cellular glycocalyx can project tens to hundreds of nanometers into the extracellular space, erecting a physical barrier that provides protective functions, mediates the exchange of nutrients and regulates cellular interactions. Little is understood about how the physical properties of the mucosal glycocalyx influence molecular recognition at the cellular boundary. Here, we report the synthesis of PEG-based glycopolymers with tunable glycan composition, which approximate the extended architecture of mucin glycoproteins, and tether them to the plasma membranes of red blood cells (RBC) to construct an artificial mucin brush-like glycocalyx. We evaluated the association of two lectins, ConA and SNA, with their endogenous glycan ligands on the surface of the remodelled cells. The extended glycocalyx provided protection against agglutination of RBCs by both lectins; however, the rate and magnitude of ConA binding were attenuated to a greater degree in the presence of the glycopolymer spectators compared to those measured for SNA. The different sensitivity of ConA and SNA to glycocalyx crowding likely arises from the distinct presentation of their mannoside and sialoside receptors, respectively, within the native RBC glycocalyx
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Efficient Synthesis of Heparinoid Bioconjugates for Tailoring FGF2 Activity at the Stem Cell–Matrix Interface
Heparan sulfate glycosaminoglycans (HS GAGs) attached to proteoglycans harbor high affinity binding sites for various growth factors (GFs) and direct their organization and activity across the cell-matrix interface. Here, we describe a mild and efficient method for generating HS-protein conjugates. The two-step process utilizes a "copper-free click" coupling between differentially sulfated heparinoids primed at their reducing end with an azide handle and a bovine serum albumin protein modified with complementary cyclooctyne functionality. When adsorbed on tissue culture substrates, the glycoconjugates served as extracellular matrix proteoglycan models with the ability to sequester FGF2 and influence mesenchymal stem cell proliferation based on the structure of their HS GAG component
Efficient Synthesis of Heparinoid Bioconjugates for Tailoring FGF2 Activity at the Stem Cell-Matrix Interface
Heparan sulfate glycosaminoglycans (HS GAGs) attached
to proteoglycans harbor high affinity binding sites for various growth factors
(GFs) and direct their organization and activity across the cell-matrix
interface. Here, we describe a mild and efficient method for generating HS-protein
conjugates. The two-step process utilizes a “copper-free” click coupling
between differentially sulfated heparinoids primed at their reducing end with
an azide handle and a bovine serum albumin protein modified with complementary cyclooctyne
functionality. When adsorbed on tissue culture substrates, the glycoconjugates
served as extracellular matrix proteoglycan models with the ability to
sequester FGF2 and influence mesenchymal stem cell proliferation based on the
structure of their HS GAG component
Glycocalyx scaffolding with synthetic nanoscale glycomaterials
We report a method for programming complexity into the glycocalyx of live cells. Via a combination of glycomaterial synthesis and membrane remodeling, we have engineered cells to display native-like, mixed sialoglycan populations, while confining the activity of each glycan into a specific nanoscale presentation
Recent Advances in the Discovery and Development of Factor XI/XIa Inhibitors.
Factor XIa (FXIa) is a serine protease homodimer that belongs to the intrinsic coagulation pathway. FXIa primarily catalyzes factor IX activation to factor IXa, which subsequently activates factor X to factor Xa in the common coagulation pathway. Growing evidence suggests that FXIa plays an important role in thrombosis with a relatively limited contribution to hemostasis. Therefore, inhibitors targeting factor XI (FXI)/FXIa system have emerged as a paradigm-shifting strategy so as to develop a new generation of anticoagulants to effectively prevent and/or treat thromboembolic diseases without the life-threatening risk of internal bleeding. Several inhibitors of FXI/FXIa proteins have been discovered or designed over the last decade including polypeptides, active site peptidomimetic inhibitors, allosteric inhibitors, antibodies, and aptamers. Antisense oligonucleotides (ASOs), which ultimately reduce the hepatic biosynthesis of FXI, have also been introduced. A phase II study, which included patients undergoing elective primary unilateral total knee arthroplasty, revealed that a specific FXI ASO effectively protects patients against venous thrombosis with a relatively limited risk of bleeding. Initial findings have also demonstrated the potential of FXI/FXIa inhibitors in sepsis, listeriosis, and arterial hypertension. This review highlights various chemical, biochemical, and pharmacological aspects of FXI/FXIa inhibitors with the goal of advancing their development toward clinical use