Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.

Roberts, Bruce; Markland, William; Ley, Arthur C.; Kent, Rachel B.; White, Derek W. R.; Guterman, Sonia K.; Ladner, Robert C.

doi:10.1073/pnas.89.6.2429

Cited by 174 publications

(78 citation statements)

References 26 publications

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“…EPI-hNE4 (DX-890) is a low molecular mass (6237 Da) inhibitor of HNE that was discovered and patented by Dyax Corp (Cambridge, MA). It was engineered using phage display (Roberts et al, 1992) and is expressed in Pichia pastoris. It is derived from the second Kü nitz domain of the light chain (bikunin) of the naturally occurring human protease inhibitor, inter-␣-trypsin inhibitor (residues 285-340; Swiss-Prot accession number PO2760), from which it differs at five positions (Becher et al, 2006).…”

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confidence: 99%

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

Attucci¹,

Gauthier²,

Korkmaz³

et al. 2006

J Pharmacol Exp Ther

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EPI-hNE4 (depelstat) is a potent inhibitor of human neutrophil elastase derived from human inter-␣-trypsin inhibitor and designed to control the excess proteolytic activity in the sputum of cystic fibrosis patients. We analyzed its resistance to the proteolysis it is likely to encounter at inflammatory sites in vivo. EPI-hNE4 resisted hydrolysis by neutrophil matrix metalloproteases (MMPs) and serine proteases that are released from activated neutrophils in inflammatory lung secretions, including MMP-8 and MMP-9, and the elastase-related protease 3 and cathepsin G. It also resisted degradation by epithelial lung cell MMP-7 but was broken down by the Pseudomonas aeruginosa metalloelastase pseudolysin, when used in a purified system, but not when this protease competed with equimolar amounts of neutrophil elastase. We also investigated the inhibitory properties of EPI-hNE4 at the surface of purified blood neutrophils and in the sputum of cystic fibrosis patients where neutrophil elastase is in both a soluble and a gel phase. The elastase at the neutrophil surface was fully inhibited by EPI-hNE4 and formed soluble complexes. The elastase in cystic fibrosis sputum supernatants was inhibited by stoichiometric amounts of EPIhNE4, allowing titration of the protease. But the percentage of inhibition in whole sputum homogenates varied from 50 to 100%, depending on the sample tested. EPI-hNE4 was rapidly cleaved by the digestive protease pepsin in vitro. Therefore, EPI-hNE4 seems to be an elastase inhibitor suitable for use in aerosols to treat patients with cystic fibrosis.

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EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

Attucci¹,

Gauthier²,

Korkmaz³

et al. 2006

J Pharmacol Exp Ther

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“…So far, several studies have shown that through semisynthetic (14,15) or recombinant approaches (16 -18) it is possible to change or enhance the BPTI inhibition spectrum. Additionally, phage display technology has been applied to generate potent inhibitors directed toward different serine proteinases based on either the BPTI sequence (19) or sequences of homologous inhibitors: amyloid ␤-protein precursor (20 -22) and the first domain of TFPI (23,24). Stassen et al converted BPTI into a potent inhibitor of factors VII a -TF, X a , XI a and plasma kallikrein through grafting into BPTI binding loop sequences of individual TFPI domains (17).…”

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Inhibition of Six Serine Proteinases of the Human Coagulation System by Mutants of Bovine Pancreatic Trypsin Inhibitor

Grzesiak¹,

Krokoszynska²,

Krowarsch³

et al. 2000

Journal of Biological Chemistry

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A series of 12 bovine pancreatic trypsin inhibitor variants mutated in the P 4 and P 3 positions of the canonical binding loop containing additional K15R and M52L mutations were used to probe the role of single amino acid substitutions on binding to bovine trypsin and to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X a and XII a , thrombin, and protein C. The mutants were expressed in Escherichia coli as fusion proteins with the LE1413 hydrophobic polypeptide and purified from inclusion bodies; these steps were followed by CNBr cleavage and oxidative refolding. The mutants inhibited the blood-clotting proteinases with association constants in the range of 10 3 -10 10 M ؊1 . Inhibition of plasma kallikrein, factors X a and XII a , thrombin, and protein C could be improved by up to 2 orders of magnitude by the K15R substitution. The highest increase in the association constant for P 3 mutant was measured for factor XII a ; P13S substitution increased the K a value 58-fold. Several other substitutions at P 3 resulted in about 10-fold increase for factor X a , thrombin, and protein C. The cumulative P 3 and P 1 effects on K a values for the strongest mutant compared with the wild type bovine pancreatic trypsin inhibitor were in the range of 2.2-(plasmin) to 4,000-fold (factors XII a and X a ). The substitutions at the P 4 site always caused negative effects (a decrease in the range from over 1,000-to 1.3-fold) on binding to all studied enzymes, including trypsin. Thermal stability studies showed a very large decrease of the denaturation temperature (about 22°C) for all P 4 mutants, suggesting that substitution of the wild type Gly-12 residue leads to a change in the binding loop conformation manifesting itself in non-optimal binding to the proteinase active site.Blood coagulation is a series of proteolytic events resulting in clot formation. Equally important are the processes of anticoagulation and fibrinolysis that are also mediated by proteolytic enzymes. Research during the past decade has resulted in the determination of spatial structures for many of these enzymes, including thrombin, factors VII a , IX a , and X a , protein C, tissue plasminogen activator, and plasmin (1-6). The specificity of a particular enzyme toward its cognate sequence results from well defined subsites on the enzyme surface recognizing not only the scissile peptide bond but often also more extended regions. These structural studies nicely explain earlier data on sequence-specific cleavage of natural substrates and provide a framework for drug design efforts.Because the coagulation/fibrinolysis processes are of vital importance, they are precisely controlled. Protein inhibitors are one of the most essential regulating factors. Interestingly, the scaffold of Kunitz-type inhibitors is used both to control the natural coagulation process in human blood and to prevent blood clotting in a blood-sucking organism, the soft tick. In the former case, tissue factor pathway inhibitor (TFPI) 1 compo...

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“…In comparison, the presence of Ala 39 and Phe 60 in FVIIa resulted in the selection of Ile at the P 3 Ј position, whereas in human leukocyte elastase, Phe was selected to fit in the area flanked by Gly 39 and Ala 60 (14,15,28). At the P 2 Ј position (residue 17), the preference for Ala at position 17 is consistent with a relatively small hydrophobic pocket, which likely involves Ile 151 in plasma kallikrein.…”

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confidence: 99%

Potent and Selective Kunitz Domain Inhibitors of Plasma Kallikrein Designed by Phage Display

Dennis¹,

Herzka²,

Lazarus³

1995

Journal of Biological Chemistry

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Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (K i *) ranging from ϳ75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a K i * ‫؍‬ 15 ؎ 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with K i * values ranging from ϳ0.3 to 15 nM; KALI-DY inhibited Factor XIa with a K i * ‫؍‬ 8.2 ؎ 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor⅐Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay >3.5-fold at 1 M.

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Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.

Cited by 174 publications

References 26 publications

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

Inhibition of Six Serine Proteinases of the Human Coagulation System by Mutants of Bovine Pancreatic Trypsin Inhibitor

Potent and Selective Kunitz Domain Inhibitors of Plasma Kallikrein Designed by Phage Display

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