Small peptides blocking inhibition of factor Xa and tissue factor-factor VIIa by tissue factor pathway inhibitor (TFPI).

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 ß-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.

C5a receptor targeting in neointima formation after arterial injury in atherosclerosis-prone mice.

BACKGROUND: Receptor binding of complement C5a leads to proinflammatory activation of many cell types, but the role of receptor-mediated action during arterial remodeling after injury has not been studied. In the present study, we examined the contribution of the C5a receptor (C5aR) to neointima formation in apolipoprotein E-deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody. METHODS AND RESULTS: Mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C5aRA and anti-C5aR-blocking monoclonal antibody or vehicle control. Compared with controls, neointima formation was significantly reduced in mice receiving C5aRA or anti-C5aR-blocking monoclonal antibody for 1 week but not for 3 weeks, attributable to an increased content of vascular smooth muscle cells, whereas a marked decrease in monocyte and neutrophil content was associated with reduced vascular cell adhesion molecule-1. As assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry, C5aR was expressed in lesional and cultured vascular smooth muscle cells, upregulated by injury or tumor necrosis factor-alpha, and reduced by C5aRA. Plasma levels and neointimal plasminogen activator inhibitor-1 peaked 1 week after injury and were downregulated in C5aRA-treated mice. In vitro, C5a induced plasminogen activator inhibitor-1 expression in endothelial cells and vascular smooth muscle cells in a C5aRA-dependent manner, possibly accounting for higher vascular smooth muscle cell immigration. CONCLUSIONS: One-week treatment with C5aRA or anti-C5aR-blocking monoclonal antibody limited neointimal hyperplasia and inflammatory cell content and was associated with reduced vascular cell adhesion molecule-1 expression. However, treatment for 3 weeks failed to reduce but rather stabilized plaques, likely by reducing vascular plasminogen activator inhibitor-1 and increasing vascular smooth muscle cell migration.

Complement 5a receptor inhibition improves renal allograft survival.

Complement activation plays a key role in mediating apoptosis, inflammation, and transplant rejection. In this study, the role of the complement 5a receptor (C5aR) was examined in human renal allografts and in an allogenic mouse model of renal transplant rejection. In human kidney transplants with acute rejection, C5aR expression was increased in renal tissue and in cells infiltrating the tubulointerstitium. Similar findings were observed in mice. When recipient mice were treated once daily with a C5aR antagonist before transplantation, long-term renal allograft survival was markedly improved compared with vehicle-treatment (75 versus 0%), and apoptosis was reduced. Furthermore, treatment with a C5aR antagonist significantly attenuated monocyte/macrophage infiltration, perhaps a result of reduced levels of monocyte chemoattractant protein 1 and the intercellular adhesion molecule 1. In vitro, C5aR antagonism inhibited intercellular adhesion molecule 1 upregulation in primary mouse aortic endothelial cells and reduced adhesion of peripheral blood mononuclear cells. Furthermore, C5aR blockade markedly reduced alloreactive T cell priming. These results demonstrate that C5aR plays an important role in mediating acute kidney allograft rejection, suggesting that pharmaceutical targeting of C5aR may have potential in transplantation medicine.

Complement C5 mediates experimental tubulointerstitial fibrosis.

Renal fibrosis is the final common pathway of most progressive renal diseases. C5 was recently identified as a risk factor for liver fibrosis. This study investigated the role of C5 in the development of renal tubulointerstitial fibrosis by (1) induction of renal fibrosis in wild-type and C5(-/-) mice by unilateral ureteral ligation (UUO) and (2) investigation of the effects of a C5a receptor antagonist (C5aRA) in UUO. In C5(-/-) mice, when compared with wild-type controls, markers of renal fibrosis (Sirius Red, type I collagen, fibronectin, alpha-smooth muscle actin, vimentin, and infiltrating macrophages) were significantly reduced on day 5 of UUO. On day 10, fibronectin mRNA and protein expression were still reduced in the C5(-/-) mice. Cortical mRNA of all PDGF isoforms and of TGF-beta(1) (i.e., central mediators of renal disease) were significantly reduced in C5(-/-) mice when compared with controls. Renal tubular cell expression of the C5aR was sparse in normal cortex but markedly upregulated after UUO. Treatment of wild-type UUO mice with C5aRA also led to a significant reduction of cortical Sirius Red staining, fibronectin protein expression, and PDGF-B mRNA expression on day 5. Neither genetic C5 deficiency nor C5aRA treatment caused any histologic changes in the nonobstructed kidneys. In cultured murine cortical tubular cells, C5a stimulated production of TGF-beta(1), and this was inhibited by C5aRA. Using a combined genetic and pharmacologic approach, C5, in particular C5a, is identified as a novel profibrotic factor in renal disease and as a potential new therapeutic target.

Transformation of a biologically active Peptide into peptoid analogs while retaining biological activity.

We report the stepwise transformation of a linear peptide epitope recognized by the anti-transforming growth factor alpha monoclonal antibody Tab2 into peptomers and finally into peptoid analogs. The key experiment in this study is the substitution analysis in which each position of the peptide is exchanged by a set of different peptoid building blocks resulting in a peptidomimetic array. After probing the array toward antibody binding, the best binding peptomer spots were selected and subjected to a successive transformation. The best peptoid found in this study has a K(D) of 200 nM when binding to Tab2, which is only 8-fold higher than the starting peptide. Moreover, this approach permits to ask directly questions about the transformation of peptide lead structures into non-peptidic compounds in the context of protein recognition.

Peptidomimetic C5a receptor antagonists with hydrophobic substitutions at the C-terminus: increased receptor specificity and in vivo activity.

A new class of peptidomimetic C5a receptor antagonists characterized by C-terminal amino acids with hydrophobic side chains is presented. Systematic optimization of the first hits led to JPE1375 (36), which was intensively characterized in vitro and in vivo. Compound 36 exhibits high microsomal stability and receptor specificity and is highly active in an immune complex mediated peritonitis model (reverse passive Arthus reaction) in mice.