Engineering exosite peptides for complete inhibition of factor VIIa using a protease switch with substrate phage

Limitations of current anticoagulant therapies have led us to develop two distinct classes of exosite peptide inhibitors for the initiator of the clotting process, the tissue factor-factor VIIa (TF.FVIIa) complex (Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531). Although both peptide classes are potent and selective inhibitors of TF.FVIIa, neither showed 100% inhibition at saturating concentrations. Crystal structures of these peptides in complex with the FVII/FVIIa protease domain revealed their distinct binding sites and close proximity to the active site. The favorable orientation of the 15-mer A-site peptide A-183 (EEWEVLCWTWETCER) suggested that a C-terminal extension into the FVIIa active site could yield a chimeric inhibitor that was not only potent and selective but complete as well. A novel two-step "protease switch" approach using substrate phage display was developed by first binding all phage containing A-183 and C-terminal extension libraries to immobilized and inactive FVIIa. Upon altering pH and adding TF to switch on FVIIa enzymatic activity, only those phage released by proteolytic cleavage within the extension were propagated. This process selected for both preferred sequence and length in the extension, leading to a 27-mer peptide A-183X (EEWEVLCWTWETCERGEGVEEELWEWR) with a C-terminal 12-mer extension containing an Arg in the P1 position. A-183X was a more potent and complete inhibitor of FX activation, having a maximal extent of inhibition of approximately 99% with an IC50 of 230 pm versus A-183 which maximally inhibited to 74% with an IC50 of 1.5 nm. A-183X also had a maximal prolongation of the prothrombin time of 7.6- versus 1.9-fold for A-183, making it a more effective anticoagulant.     

Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The gamma-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF.PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF.PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF.PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.     

Structure-based design of P3 moieties in the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. Structure-based designs of the P3 moiety in the peptide mimetic factor VIIa inhibitor successfully lead to novel inhibitors with selectivity for FVIIa/TF and extrinsic coagulation the same as or even higher than those of previously reported peptide mimetic factor VIIa inhibitors. X-ray crystal structure analysis reveals that one of the novel inhibitors shows improved selectivity by forming interactions between the inhibitor and FVIIa as expected. Another of the novel inhibitors achieves improved selectivity through an unexpected hydrogen bond with Gln217, with a unique bent conformation in FVIIa/TF accompanied by conformational changes of the inhibitor and the protein.     

A novel exosite on coagulation factor VIIa and its molecular interactions with a new class of peptide inhibitors

A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa (FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides, identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that is distinct from both the active site and the exosite of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-4701. Peptide A-183 prolonged TF-dependent clotting in human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit sequence differences in the protease domain, localized the binding site to residues in the 60s loop and the C-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showed that proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium binding constants for zymogen FVII, FVIIa and TF x FVIIa were determined using immobilized N-terminal biotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteases was observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa.     

The discovery of fluoropyridine-based inhibitors of the factor VIIa/TF complex--Part 2

The activated factor VII/tissue factor complex (FVIIa/TF) is known to play a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. A fluoropyridine-based series of FVIIa/TF inhibitors was discovered which utilized a diisopropylamino group for binding in the S2 and S3 binding pockets of the active site of the enzyme complex. In this series, an enhancement in binding affinity was observed by substitution at the 5-position of the hydroxybenzoic acid sidechain. An X-ray crystal structure indicates that amides at this position may increase inhibitor binding affinity through interactions with the S1'/S2' pocket.     

Macromolecular substrate affinity for free factor VIIa is independent of a buried protease domain N-terminus

The initial recognition and binding of macromolecular substrates by factor VIIa (FVIIa) in complex with tissue factor has been shown to be mediated by areas distinct from the active site (so-called exosites). The present aim was to shed light on whether the N-terminal tail of the protease domain of FVIIa influences factor X (FX) binding, and whether the zymogen-like conformation of free FVIIa has a decreased affinity for FX compared to the active conformation. Two derivatives of FVIIa, one (FFR-FVIIa) with a stably buried N-terminus representing the active conformation of FVIIa and one (V154G-FVIIa) with a fully exposed N-terminus representing the zymogen-like conformation, were used as inhibitors of FVIIa-catalyzed FX activation. Their inhibitory capacities were very similar, with K(i) values not significantly different from the K(m) for FX. This indicates that the conformational state of the N-terminus does not affect FX binding or, alternatively, that the activation domain including the N-terminal insertion site is easily shifted to the stable conformation ensuing FX docking to the zymogen-like conformation. The net outcome is that FX binding to the zymogen-like form of FVIIa does not appear to be impaired.     

Spectroscopic probing of the influence of calcium and the gla domain on the interaction between the first EGF domain in factor VIIa and tissue factor.

The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor-like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4-carboxyglutamic acid (Gla)-domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla-domainless and full-length FVIIa.     

Activation and active site occupation alter conformation in the region of the first epidermal growth factor-like domain of human factor VII

The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.     

VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis,p70, and p90 S6 kinase phosphorylation

FVIIa binding to tissue factor (TF) and subsequent signal transduction have now been implicated in a variety of pathophysiological processes, including cytokine production during sepsis, tumor angiogenesis and neoangiogenesis, and leukocyte diapedesis. The molecular details, however, by which FVIIa/TF affects gene expression and cellular physiology, remain obscure. Here we show that FVIIa induces a transient phosphorylation of p70/p85(S6K) and p90(RSK) in BHK cells stably transfected with either full-length TF or with a cytoplasmic domain-truncated TF but not in wild type BHK cells. Phosphorylation of these kinases was also observed in HaCaT cells, expressing endogenous TF. Phosphorylation of p70/p85(S6K) coincided with protein kinase B and GSK-3beta phosphorylation. Activation of p70/p85(S6K) was sensitive to inhibitors of phosphatidylinositol 3-kinase and to rapamycin, whereas phosphorylation of p90(RSK) was sensitive to PD98059. FVIIa stimulation of p70/p85(S6K) and p90(RSK) correlated with phosphorylation of the eukaryotic initiation factor eIF-4E, up-regulation of protein levels of eEF1alpha and eEF2, and enhanced [(35)S]methionine incorporation. These effects were not influenced by inhibitors of thrombin or FXa generation and were strictly dependent on the presence of the extracellular domain of TF, but they did not require the intracellular portion of TF. We propose that a TF cytoplasmic domain-independent stimulation of protein synthesis via activation of S6 kinase contributes to FVIIa effects in pathophysiology.     

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.     

Novel interactions of large P3 moiety and small P4 moiety in the binding of the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.     

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-d-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K_i = 8 μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.     

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.     

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF_1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF_1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.     

Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium

Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. (125)I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time- and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively (125)I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in (125)I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR.     

Design,parallel synthesis,and crystal structures of biphenyl antithrombotics as selective inhibitors of tissue factor FVIIa complex. Part 1: Exploration of S2 pocket pharmacophores

Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF·FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF·FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC₅₀ value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF·FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure–activity relationship.     

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.     

The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor.

Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa*TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa*TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIX(WT)), FIX(Q50P), FIX(PCEGF1) (EGF1 domain replaced with that of protein C), FIX(DeltaEGF1) (EGF1 domain deleted), FX(WT), and FX(PCEGF1). Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa*TF or FVIIa*sTF activated each mutant significantly more slowly than the FIX(WT) or FX(WT). Importantly, in ligand blot assays, FIX(WT) and FX(WT) bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K(i) of approximately 60 microm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.