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 and screened for hydrolysis by human glandular kallikrein 2 shown to be present in 75% of breast cancers. A peptide substrate was identified and couple to the thapsigargin analog, L12ADT to produce 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 hydrolysis 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 m1s). New approaches are needed to identify breast cancer proteases that can concentrate proteases within breast cancer extracellular fluid or which can screen large libraries in much smaller volume. Two such methods, in vitro compartmentalization and macroglobulin complexation are being evaluated in the laboratory.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2005
- Accession Number
- ADA443732
Entities
People
- Samuel Denmeade
Organizations
- Johns Hopkins University