Back to Search
Start Over
Protease-resistant peptide ligands from a knottin scaffold library.
- Source :
-
ACS chemical biology [ACS Chem Biol] 2011 Aug 19; Vol. 6 (8), pp. 837-44. Date of Electronic Publication: 2011 Jun 16. - Publication Year :
- 2011
-
Abstract
- Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 10(9) variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution and slow dissociation rates and were able to inhibit thrombin's enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a 2 h incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high-affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications.
- Subjects :
- Amino Acid Sequence
Antithrombins metabolism
Catalytic Domain
Cyclotides metabolism
Cystine Knot Motifs
Escherichia coli metabolism
Humans
Models, Molecular
Molecular Sequence Data
Peptide Hydrolases metabolism
Thrombin chemistry
Thrombin metabolism
Antithrombins chemistry
Antithrombins pharmacology
Cyclotides chemistry
Cyclotides pharmacology
Cystine-Knot Miniproteins chemistry
Peptide Library
Thrombin antagonists & inhibitors
Subjects
Details
- Language :
- English
- ISSN :
- 1554-8937
- Volume :
- 6
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- ACS chemical biology
- Publication Type :
- Academic Journal
- Accession number :
- 21615106
- Full Text :
- https://doi.org/10.1021/cb200039s