Towards a ligand targeted enzyme prodrug therapy: single round panning of a beta-lactamase scaffold library on human cancer cells.

Abstract

A novel beta-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 (beta-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 beta-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound beta-lactamase activity. SKBR3 cell-binding beta-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected beta-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 beta-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.

• Authors: Girja S Shukla, Christopher J Murray, Melodie Estabrook, Guang-Ping Shen, Volker Schellenberger, David N Krag
• Journal: International journal of cancer (Int J Cancer) Vol. 120 Issue 10 Pg. 2233-42 (May 15 2007) ISSN: 0020-7136 [Print] United States
• PMID: 17285581 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: (c) 2007 Wiley-Liss, Inc.

Chemical References

• Ligands
• Oligopeptides
• Peptide Fragments
• Peptide Library
• Prodrugs
• Recombinant Proteins
• beta-Lactamases

Topics

• Amino Acid Sequence
• Breast Neoplasms (metabolism)
• Cell Line, Tumor
• Enterobacter cloacae (enzymology, genetics)
• Genetic Vectors (genetics)
• Humans
• Ligands
• Models, Molecular
• Oligopeptides (chemistry, metabolism)
• Peptide Fragments (chemistry, metabolism)
• Peptide Library
• Prodrugs (chemistry)
• Recombinant Proteins (chemistry, genetics)
• Substrate Specificity
• beta-Lactamases (chemistry, genetics, metabolism)