A Novel Exosite on Coagulation Factor VIIa and Its Molecular Interactions with a New Class of Peptide Inhibitors

Roberge, Martin; Santell, Lydia; Dennis, Mark S.; Eigenbrot, Charles; Dwyer, Mary A.; Lazarus, Robert A.

doi:10.1021/bi010592d

Cited by 49 publications

(62 citation statements)

References 45 publications

(90 reference statements)

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“…Under these conditions, the apparent velocity of FX hydrolysis by FVIIaWT was significantly higher than that of FVIIa303T. This finding suggests that the conformational equilibrium FVIIa « FVIIa*, where FVIIa is the zymogen-like form and FVIIa* is the functionally active form of the FVIIa molecule (Dennis et al, 2000Persson et al, 2001a;Roberge et al, 2001;Sichler et al, 2002), is severely shifted to the right. The perturbation of this conformational equilibrium is even more evident in the presence of TF, whose binding is unable to shift the conformational transition toward a fully active FVIIa form.…”

Section: Discussionmentioning

confidence: 87%

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The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

et al. 2004

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SummaryWe report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild‐type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (Kd from 4·4 nmol/l for FVIIaWT to 17·3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (kcat/Km from 2·3 × 107/mol/l s for FVIIaWT to 8·7 × 105/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen‐like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF‐catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the Km value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.

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Section: Discussionmentioning

confidence: 87%

“…Both these interactions inhibit, in a non-competitive way, FVIIa activity toward FX and synthetic amide substrates (Dennis et al, 2000Roberge et al, 2001). In contrast, recent studies showed that polyethylene glycol binds to a region located in proximity to the 60s loop, enhancing the enzyme function (Sichler et al, 2002).…”

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confidence: 99%

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

et al. 2004

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“…Examples of this class include peptide inhibitors of factor VIIa (67,68) and small molecule inhibitors of ␤-lactamase (69). Very recently, a novel allosteric peptide, pep419, which binds near the 90s helix in caspase-6 inducing a tetrameric state, has been reported to function via this second class of allosteric mechanism.…”

Section: Discussionmentioning

confidence: 99%

Zinc-mediated Allosteric Inhibition of Caspase-6

Velázquez-Delgado

Hardy

2012

Journal of Biological Chemistry

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Background: Caspase-6 is a critical factor in neurodegeneration, which is regulated by zinc binding. Results: Caspase-6 is inhibited by zinc and binds one zinc/monomer at an exosite distal from the active site. Conclusion: Zinc allosterically inhibits caspase-6 by locking it into a naturally occurring, inactive, and extended helical conformation. Significance: The allosteric inhibition observed in the presence of zinc may aid in the development of allosteric caspase-6 drugs.

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“…This was carried out by first combining two halves of the protease domain, one from the A-183⅐FVII zymogen structure (16,18) and the other from the TF⅐FVIIa structure (31). The rationale for this derives from the protease architecture (two distorted ␤-barrels) and the largely independent effects of A-183 in one barrel and the presentation of the S 1 subsite in the other barrel.…”

Section: Methodsmentioning

confidence: 99%

“…Here we have pursued a new strategy to obtain less complex peptide exosite inhibitors that are both specific and complete. The crystal structure of the protease domain of FVII in complex with A-183 revealed that its C terminus was in close proximity to the active site (16,18). This suggested that extending its C terminus into the active site region should result in a chimeric peptide having a high degree of specificity and potency due to its exosite interactions and more complete inhibition due to greater steric hindrance in the substrate binding cleft of the active site.…”

mentioning

confidence: 99%

Engineering Exosite Peptides for Complete Inhibition of Factor VIIa Using a Protease Switch with Substrate Phage

Maun

Lazarus²

2003

Journal of Biological Chemistry

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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 (EEWEVLCWTWET-CERGEGVEEELWEWR) with a C-terminal 12-mer extension containing an Arg in the P 1 position. A-183X was a more potent and complete inhibitor of FX activation, having a maximal extent of inhibition of ϳ99% with an IC 50 of 230 pM versus A-183 which maximally inhibited to 74% with an IC 50 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.The limitations of current anticoagulant therapies have stimulated the search for new alternatives based on selective inhibition of serine proteases in the coagulation pathway (1-4). Commonly used anticoagulants, such as coumarin and heparin, lack specificity and have a narrow therapeutic window, leading to undesirable side effects such as bleeding. The tightly regulated coagulation cascade consists of several highly homologous serine proteases and their cofactors and inhibitors (5, 6). The development of a specific inhibitor for a single coagulation factor could reduce side effects and improve the therapeutic profile. The architecture of the active site in all these proteases is very similar, which makes the development of a specific small molecule active site inhibitor challenging. Although relatively nonselective with respect to small chromogenic substrates, these proteases are highly specific for their natural macromolecular substrates. In order to achieve this, exosites on these enzymes play an important role in substrate recognition and catalysis (7-12). Blocking such important interactions could result in the specific inhibition of a single protease in this pathway.Factor VIIa (FVIIa) 1 is the initiator of the extrinsic blood coagulation pathway. Upon vascular damage, zymogen FVII binds to tissue factor (TF), a membrane-bound protein cofactor that is expressed in the surrounding tissue (13). This results in the formation of the active complex TF⅐FVIIa, which activates FX to FXa, FIX to FIXa, and FVII to FVIIa through specific proteolytic cleavages. Ultimately, this cascade leads to the generation of thrombin, resulting in the formation of fibrin and eventually a blood clot. Because FVII/FVIIa is present only in relatively low concentrations in blood (10 nM) and it initiates the coagulation cascade (13), this enzyme is an attractive drug target for the development of anticoagulants.Recent focus on the search for specific inhibitors has lead to ...

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A Novel Exosite on Coagulation Factor VIIa and Its Molecular Interactions with a New Class of Peptide Inhibitors

Cited by 49 publications

References 45 publications

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

Zinc-mediated Allosteric Inhibition of Caspase-6

Engineering Exosite Peptides for Complete Inhibition of Factor VIIa Using a Protease Switch with Substrate Phage

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