Towards a ligand targeted enzyme prodrug therapy: Single round panning of a β-lactamase scaffold library on human cancer cells
| Autor: | Girja S. Shukla, Guang-Ping Shen, Christopher J. Murray, Melodie Estabrook, Volker Schellenberger, David N. Krag |
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| Rok vydání: | 2007 |
| Předmět: |
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Molecular Cancer Research Phage display medicine.medical_treatment Genetic Vectors Breast Neoplasms Peptide Biology Ligands beta-Lactamases Substrate Specificity Targeted therapy chemistry.chemical_compound Peptide Library Cell Line Tumor Enterobacter cloacae medicine Humans Prodrugs Amino Acid Sequence Panning (camera) chemistry.chemical_classification Prodrug Molecular biology Peptide Fragments Recombinant Proteins Amino acid Oncology chemistry Rolling circle replication Oligopeptides DNA |
| Zdroj: | International Journal of Cancer. 120:2233-2242 |
| ISSN: | 0020-7136 |
| DOI: | 10.1002/ijc.22138 |
| Popis: | A novel β-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (β-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual β-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound β-lactamase activity. SKBR3 cell-binding β-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected β-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor cell-lines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted β-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy. © 2007 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
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