Identification of Breast Cancer Specific Proteolytic Activities for Targeted Prodrug Activation
Abstract
The underlying hypothesis of this proposal is that a breast tissue/cancer proteolytic activity can be identified by screening the extracellular fluid from human breast cancers with a fluorescence quenched random peptide library. The peptide substrate(s) identified from this screening could be used to produce prodrugs that are targeted for specific activation by proteolytic activity present in extracellular fluid of breast cancers while avoiding systemic toxicity. In the first year we developed methods to synthesize large fluorescently quenched peptide libraries as outlined in task 1. In preliminary studies, we incubated these libraries with a purified serine protease, human glandular kallikrein 2, and identified a series of putative substrates. These substrates were resynthesized as soluble peptides and peptides were then identified that were excellent hK2 substrates but unstable to non-specific hydrolysis in human plasma. Coupling these peptides to the thapsigargin analog, Ll2ADT, however, produced a prodrug that was readily hydrolyzed by hK2, stable in human plasma in vitro and mouse plasma both in vitro and in vivo, and was selectively cytotoxic to cancer cells in the presence of enzymatically active hK2. These studies demonstrated the feasibility of the approach to identification of protease substrates outlined in tasks 1-3 of the proposal. However, incubation of breast cancer homogenates or concentrated media from breast cancer cell lines did not yield any hydrolyzed peptides. This lack of hydrolys is most likely is due to a combination of ng/ml concentrations of protease in the extracellular fluid and the need to dilute samples to cover entire bead library (i.e. 10-40 mls). A new approach for combinatorial screening is required that would yield libraries of small physical volume to screen small volume of extracellular fluid from breast cancers without requiring substantial dilution.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2004
- Accession Number
- ADA426967
Entities
People
- Samuel Denmeade
Organizations
- Johns Hopkins Hospital