TFPI is an important inhibitor of the extrinsic coagulation pathway. It efficiently inhibits TF-FVIIa and FXa by quaternary complex formation. Plasma contains various truncated forms of TFPI which ...are poor inhibitors, and full length (fl)TFPI (0.3 – 0.5 nM) which is the most active TFPI in plasma. flTFPI is released from platelets upon activation, and increases flTFPI concentrations locally up to 30-fold. Most intravascular TFPI (∼80%) is associated with endothelial cells. Both endothelial forms, TFPIa and TFPIb, are similarily effective inhibitors of FX activation on the endothelial cell surface. Inhibition of TFPI in hemophilia models with blocking antibodies, aptamers or peptide inhibitors improves hemostasis and may become an option to treat hemophilia. Recently, we presented peptide inhibitors of TFPI that enhance coagulation in hemophilia models. Two optimized peptides, JBT-A7 and JBT-B5, efficiently blocked inhibitory activity of TFPI and bound to distinct binding sites.
We demonstrated the crystal structure of JBT-A7, a linear TFPI inhibitory peptide composed of 20 amino acids, bound to NtermK1 (TFPI 1-83). JBT-B5, a cyclic TFPI inhibitory peptide of 23 amino acids, co-crystallized with TFPI KD1-KD2 (TFPI 22-150). Overlaying the KD1 structure in the KD1-KD2/JBT-B5 and the NTermK1/JBT-A7 complex provided atomic details for linking the two peptide entities. Binding of peptides to TFPI and TFPI fragments was studied by BioCore. The TFPI inhibitory potential of the resulting fusion peptide was tested in model systems (FXa inhibition and TF-FVIIa catalyzed FX activation) and global hemostatic assays (TF-triggered thrombin generation) using hemophilia plasma. To model situations of increased TFPI concentration, both model and plasma assays were carried out at TFPI concentrations up to 10 nM, which is 40-50-fold higher than the physiological flTFPI plasma concentration. To characterize the inhibition of platelet TFPI, we used platelets isolated from blood samples and platelet rich plasma from different donors. Binding of a biotinylated fusion peptide on living HUVE cells was assessed by fluorescence activated cell sorting (FACS) and fluorescence microscopy. Inhibition of cell surface TFPI was analyzed on cultivated HUVECs stimulated with TNFa for TF expression. We monitored FXa generation by the TFPI-dependent cell surface FX activation complex by conversion of an FXa-specific fluorogenic substrate.
The overlay of the crystal structures of KD1-KD2/JBT-B5 and the NTermKD1/JBT-A7 complexes revealed non-overlapping epitopes and close proximity of the termini of both peptides. The distance could be bridged by an approximately ten amino acid linker. A fusion peptide with a 10-serine-linker was synthesized and showed highly improved dissociation in Biacore experiments and most efficiently inhibited TFPI activity in the model assays. In contrast, single peptides only partially inhibit TFPI especially at high TFPI concentrations. In thrombin generation assays using hemophilia plasma, the fusion peptide showed a substantially higher ability than the single peptides to increase the thrombin peak even at elevated TFPI. The fusion peptide efficiently inhibited TFPI released from platelets and improved thrombin generation in TFPI deficient plasma reconstituted with platelets as the only source of TFPI released upon platelet activation. The fusion peptide was also shown to bind TFPI on the surface of living HUVECs. This is consistent with its binding epitopes on KD1 and KD2 which result in inhibition of cell surface TFPI in a cell based FX activation assay. Thus, we demonstrate that a molecular fusion peptide most efficiently inhibits all physiologic forms of TFPI.
X-ray structures of binary and ternary peptide TFPI complexes provided atomic details for linking two single peptides to generate a fusion peptide that most efficiently blocks TFPI in plasma, released from platelets and associated with endothelial cells. It most efficiently neutralizes TFPI even at substantially elevated concentrations occurring at sites of platelet activation. Our observations support the notion that targeting TFPI with TFPI inhibitors is a promising novel strategy to mitigate the bleeding risk in hemophilia patients.
Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter:Baxter Innovations GmbH, Vienna, Austria: Research Funding. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.
Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via ...formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.
Background: Tissue factor pathway inhibitor (TFPI) inhibits coagulation factors Xa and VIIa.
Results: A de novo synthesized 20-mer peptide that binds to TFPI was structurally and functionally characterized.
Conclusion: The peptide binds to the Kunitz domain 1 of TFPI and blocks inhibition of factor Xa and factor VIIa by TFPI.
Significance: The peptide can potentially prevent bleeding in hemophilia patients.
Combining structural features of integrin α5β1 antagonists leads to the generic structure
3. The compounds expressing high integrin α5β1 binding affinities, low systemic clearances and long terminal ...half-lifes in male Wistar rat. Promising oral bioavailabilities were also observed for these compounds.
Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold
1 as a new and selective integrin α5β1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin α5β1 inhibitor scaffolds for potential systemic treatment is described.
Correlation of structural features of selected
N-phenyl piperidine derivatives
3 with integrin α5β1 binding affinities and selectivities as well as pharmacokinetic properties are described. The best ...compounds were compared in pharmacokinetic studies in rat.
Recently, a new class of selective integrin α5β1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of
N-phenyl piperidine derivatives are described.
Abstract 24
Tissue factor pathway inhibitor (TFPI) is an activated (a) factor X (FXa)-dependent inhibitor of the extrinsic factor X (FX) activation complex and efficiently regulates the extrinsic ...pathway of coagulation. We are developing peptide inhibitors of TFPI with a view to improving hemostasis in hemophilia. The goal is to inhibit the interaction of TFPI with the factor Xa (FXa)-tissue factor (TF)-factor VIIa (FVIIa) complex, and thereby enhance thrombin formation to the extent that a stable clot is formed. Successful development of these peptides could allow treatment of hemophilia via a non-intravenous route of administration.
By screening mRNA display libraries, we identified a de novo peptide which binds to and efficiently inhibit TFPI. The peptide was optimized by iterative amino acid substitution resulting in affinity-improved peptide with a well-characterized structure-activity relation. The affinity of the peptide was analyzed by Biacore and ELISA experiments. One of our optimized peptides bound to immobilized TFPI with an affinity below 1 nM. Kunitz domain 1 of TFPI was identified as the interaction site by NMR studies.
We characterized the inhibitory activity of this TFPI binding peptide using in vitro model assay systems including inhibition of FXa and of the extrinsic tenase (TF-FVIIa-PL-Ca2+ complex). Inhibition of cell-associated TFPI was verified in a TF- and TFPI-dependent cell-based assay using TNFalpha-stimulated human umbilical vein endothelial cells (HUVECs) as a source of TF and TFPI. Inhibition of plasma TFPI was probed by thrombin generation experiments using FVIII-deficient patient plasma and ROTEM experiments using FVIII-inhibited whole blood. Furthermore, a cross-reactivity of the peptide to murine TFPI was demonstrated in thrombin generation experiments and in murine FXa inhibition model assays.
Conjugation of a 40-kDa polyethyleneglycol (PEG) to the peptide base structure resulted in a peptide with significantly reduced renal clearance. The in vivo terminal half-life of this 40kD-PEG modified peptide was 21.5h and 19.8h after 1 mg/kg i.v and s.c. administration, respectively, with 73% bioavailability after s.c. administration.
In a tail-tip bleeding model, administration of an anti-murine-TFPI antibody and 40kD-PEG-peptide in combination with sub-therapeutical doses of recombinant coagulation factors (10 IU/kg of rFVIII in FVIII ko mice and rFIX in FIX ko mice) led to a marked reduction of blood loss compared with buffer-treated or rFVIII/rFIX-treated mice.
In addition, we established a murine nail-cut model using C57Bl6 wild-type mice for testing in vivo efficacy in a normal FVIII background as well. The TFPI sensitivity of the nail-cut model was verified by an anti-murine TFPI antibody. 40kD-PEG-peptide significantly reduced blood loss after i.v. and s.c. administration in all treatment groups compared with vehicle-treated animals. Furthermore, the i.v. administration of the peptide was well tolerated in all animals across all treatment groups without any signs of acute toxicity.
In conclusion, we identified a low-molecular-weight peptide which efficiently inhibit all forms of naturally occurring TFPI proteins, including plasma TFPI and TFPI in vivo. Our results demonstrate that targeting TFPI efficiently improves hemostasis in hemophilia and provide an in vivo proof of concept for a non i.v. route of administration. Thus, the TFPI inhibitory peptides could be useful to prevent bleeding in hemophilia patients. Moreover, this new approach would probably allow treatment of inhibitor patients.
Dockal:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Polakowski:3B Pharmaceuticals GmbH: Employment. Frank:3B Pharmaceuticals GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Reinecke:3B Pharmaceuticals GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.
The authors have developed a class of potent inhibitors against the phosphate specific prolyl isomerase hPin1, which induced apoptosis in transformed cell lines.
Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin a5b1 inhibitor class which was designed for local administration. Herein the ...discovery of new orally available integrin a5b1 inhibitor scaffolds for potential systemic treatment is described.
Recently, a new class of selective integrin a5b1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of N-phenyl piperidine derivatives ...are described.
Starting from the structure of integrin alphavbeta3 in a complex with a peptidic ligand plus SAR data on nonpeptidic ligands, we derived a new class of integrin alpha5beta1 antagonists (1). Several ...synthesis strategies were applied to evaluate the chemical space around the essential pharmacophore groups R1 to R3 to obtain highly active and selective pyrrolidine derivatives as integrin alpha5beta1 antagonists. Integrin selectivity was controlled by switching from a sulfonamide moiety to a mesitylene amide moiety for R3. This finding represents a general feature for modulating selectivity toward other related integrin receptors. On the basis of the encouraging results from various in vitro studies, the most active compounds were selected for further in vivo studies in animal models and preclinical development.
The synthesis of a series of RGD mimetics is described. All compounds consist of a central 2,5‐disubstituted tetrahydrofuran core, a variable linker to a guanidino group, and a β‐amino alanine unit ...to mimic the carboxylic acid. Three types of linkers were investigated: a simple four‐atom methylene chain (type A, compounds 14, 15, 16, and 17), a four‐atom methylene chain with an additional chiral center, and a nitrogen substituent (type B, compounds 38, 39, and 40), and an amide linker of different length with an additional chiral center (type C, compounds 59, 60, 61, and 62). A variety of compounds were tested as potential integrin antagonists in a receptor binding assay (αIIbβ3, αvβ3, and αvβ5). The relative and absolute configuration of the chiral centers at the THF ring had a pronounced effect on the binding activity and selectivity. Compound 14 proved to be a selective inhibitor of αIIbβ3 (IC50=20 nM), whereas compound 40 exhibited high activity for binding of αIIbβ3 (IC50=67 nM) and αvβ3 (IC50=52 nM).
RGD mimetics containing a THF core unit such as 1 have been synthesized and biologically evaluated for integrin receptor binding (αIIbβ3‐, αvβ3‐, and αvβ5‐type). Compound 1 shows a high activity (IC50 = 20 nM) and selectivity in binding at αIIbβ3.
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