Tissue factor derived β-hairpin peptides that bind and inhibit FVII activity

The formation of the Tissue Factor(TF):Factor VII(FVII) complex is a pivotal event that initiates coagulation; targeting this early step allows for the prevention of the subsequent cascade amplification driven by positive feedback loops. For this reason, the TF:FVII complex is attracting increasing interest as a potential therapeutic target for regulating the coagulation cascade in a specific and timely manner. In order to generate TF-mimics capable of inhibiting this protein-protein interaction, we have designed four small cyclic peptides that simulate a TF region containing the two antiparallel β-strands: 106-110 (RVFSY) and 123-128 (EPLYEN). These strands are known to interact with FVII at well-known hot spots surrounding residues 365-369. With the aim of obtaining structures as similar as possible to the corresponding region of TF and therefore able to interact better with FVII, the four combinations of proline-proline dipeptides resulting from the four permutations of D-Pro and L-Pro have been introduced between the two strands. These strands have been connected on the opposite side by a disulphide bond in order to stabilise the resulting structures and also make them more resistant to protease action. The two cyclopeptides with the D-Pro-L-Pro and D-Pro-D-Pro moieties adopt β-hairpin-like conformations that recapitulate the structure of the two strands, as demonstrated by CD, NMR and molecular simulation studies. They also bind FVII and inhibit its activity in a Factor X-generating chromogenic assay. The other two peptides are significantly more disordered and are inactive in the same tests. Overall, the data validate the peptide design and confirm the region 365-369 of FVII as a target site for the design of coagulation inhibitors.