Selection and Characterization of a New Class of Peptide Exosite Inhibitors of Coagulation Factor VIIa

Dennis, Mark S.; Roberge, Martin; Quan, Cliff; Lazarus, Robert A.

doi:10.1021/bi010591l

Cited by 40 publications

(64 citation statements)

References 33 publications

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“…A similar trend was observed with an allosteric peptide inhibitor of factor VIIa, which binds near the factor VIIa 60-loop (33). A possible explanation is that synthetic substrates (occupying only S1-S3 subsites) are inadequate surrogates of natural protein substrates, which require precise alignment of the scissile peptide and interactions beyond the S1-S3 sites.…”

Section: Discussionsupporting

confidence: 59%

Structural insight into distinct mechanisms of protease inhibition by antibodies

Wu¹,

Eigenbrot

Liang³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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To better understand how the relatively flat antigen-combining sites of antibodies interact with the concave shaped substrate-binding clefts of proteases, we determined the structures of two antibodies in complex with the trypsin-like hepatocyte growth-factor activator (HGFA). The two inhibitory antibodies, Ab58 and Ab75, were generated from a human Fab phage display library with synthetic diversity in the three complementarity determining regions (H1, H2, and H3) of the heavy chain, mimicking the natural diversity of the human Ig repertoire. Biochemical studies and the structures of the Fab58:HGFA (3.5-Å resolution) and the Fab75:HGFA (2.2-Å resolution) complexes revealed that Ab58 obstructed substrate access to the active site, whereas Ab75 allosterically inhibited substrate hydrolysis. In both cases, the antibodies interacted with the same protruding element (99-loop), which forms part of the substrate-binding cleft. Ab58 inserted its H1 and H2 loops in the cleft to occupy important substrate interaction sites (S3 and S2). In contrast, Ab75 bound at the backside of the cleft to a region corresponding to thrombin exosite II, which is known to interact with allosteric effector molecules. In agreement with the structural analysis, binding assays with active site inhibitors and enzymatic assays showed that Ab58 is a competitive inhibitor, and Ab75 is a partial competitive inhibitor. These results provide structural insight into antibody-mediated protease inhibition. They suggest that unlike canonical inhibitors, antibodies may preferentially target protruding loops at the rim of the substrate-binding cleft to interfere with the catalytic machinery of proteases without requiring long insertion loops.catalysis ͉ enzyme ͉ phage display

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Section: Discussionsupporting

confidence: 59%

Structural insight into distinct mechanisms of protease inhibition by antibodies

Wu¹,

Eigenbrot

Liang³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The challenge of this approach is to bind A-183 phage libraries to FVIIa under conditions when it is inactive and then activate the enzyme in the presence of the bound phage libraries to select for only those phage that are released due to enzymatic cleavage. The latter condition becomes even more challenging since A-183 inhibits TF⅐FVIIa activity, albeit incompletely (15). The amidolytic activity of FVIIa has been studied under a variety of different conditions such as changes in pH, different salts at various concentrations, and the effect of TF (40).…”

Section: Resultsmentioning

confidence: 99%

“…The significance of the standard deviation was calculated essentially as described (35). 15 , respectively, where D stands for nucleotides A, G or T; N, S, and R nucleotides are defined above. This consensus library was sorted as described above, and individual clones were sequenced.…”

Section: Figmentioning

confidence: 99%

“…The extension plasmids pZCT-A-183X, pZCT-A-183-[GGS] 3 GGR, pZCT-A-183-[GGS] 4 , pZCT-A-183X-R12A, and pZCT-A-183X-⌬R12 were constructed by Kunkel mutagenesis using pZCT-A183 as template by inserting DNA sequence encoding GEGVEEELWEWR, GGSGGSGGSGGR, GGSGGSGGSGGS, GEGVEEELWEWA, and GEGVEEELWEW, respectively, between the C terminus of A-183 and the stop codon. All constructs were transformed into E. coli strain 27C7 and grown in low phosphate minimal media (15). All Z-fusion peptides were secreted into the media and purified using IgG-Sepharose (Amersham Biosciences) as described previously (38) followed by size exclusion on Superdex 75 (Amersham Biosciences) at 0.5 ml/min flow rate using Tris buffer, pH 7.5, and 200 mM NaCl.…”

Section: Construction and Panning Of A Partially Randomized Consensusmentioning

confidence: 99%

“…The extent of inhibition of the A-series peptides correlated with the degree of anticoagulant activity determined by prolongation of the TF-dependent prothrombin time (PT) (15).…”

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confidence: 99%

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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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Application of Recombinant DNA Technology in Medicinal Chemistry and Drug Discovery

Basu

Oyelere

2003

Burger's Medicinal Chemistry and Drug Discovery

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Drug discovery is usually a long and arduous process. However, pharmaceutical industries are making a significant progress in simplifying certain aspects of drug discovery process, especially in the area of target discovery, validation, and lead‐compounds discovery. Advances in recombinant DNA technology are intimately connected with the explosion of newly identified pharmaceutically important targets, and an understanding of how these targets function to regulate biological processes of interest. This is providing new and exciting opportunities for therapeutic intervention in diseases including those diseases that were thought to be untreatable a few years back. In this chapter we describe how each stage of drug discovery, from target identification to lead generation and even clinical trials, have benefited from the tools of recombinant DNA technology.

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Selection and Characterization of a New Class of Peptide Exosite Inhibitors of Coagulation Factor VIIa

Cited by 40 publications

References 33 publications

Structural insight into distinct mechanisms of protease inhibition by antibodies

Structural insight into distinct mechanisms of protease inhibition by antibodies

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

Application of Recombinant DNA Technology in Medicinal Chemistry and Drug Discovery

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