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Toward an enzyme/prodrug strategy for cancer gene therapy: endogenous activation of carboxypeptidase A mutants by the PACE/Furin family of propeptidases.

Abstract
In an effort to develop a gene-dependent enzyme/prodrug therapy (GDEPT) for tumor-specific delivery of methotrexate (MTX) we have chosen to construct mutant forms of carboxypeptidase A1 (CPA) that circumvent the requirement for trypsin-dependent activation. The basis of this strategy is that methotrexate-alpha-peptides are inefficient substrates for the reduced folate carrier (RFC) and hence cannot be internalized by cells. However, the blocking amino acid can be cleaved by CPA to liberate MTX, which is then internalized by the RFC, resulting in inhibition of dihydrofolate reductase and cytotoxicity. A battery of mutant CPAs was generated, in which the putative trypsin cleavage sites in the propeptide were mutated to the consensus recognition sequence for mammalian subtilisin-like propeptidases. These mutant forms of CPA were evaluated for expression, activation, and catalytic activity by transiently transfecting them into COS-1 cells both in the absence and in the presence of cotransfected propeptidases. CPA95 was identified as the most efficiently cleaved mutant, and further studies of this mutant indicated that the endogenously activated enzyme had kinetic parameters identical to those of the trypsin-activated wild-type protein. In addition, endogenously activated CPA95 could effectively sensitize cells to MTX-Phe in culture, decreasing the IC50 of MTX-Phe from 25- to 250-fold in squamous cell carcinoma cells expressing active CPA as compared with the parental lines.
AuthorsD A Hamstra, A Rehemtulla
JournalHuman gene therapy (Hum Gene Ther) Vol. 10 Issue 2 Pg. 235-48 (Jan 20 1999) ISSN: 1043-0342 [Print] United States
PMID10022548 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • DNA Primers
  • Prodrugs
  • Recombinant Proteins
  • Carboxypeptidases
  • Carboxypeptidases A
  • Subtilisins
Topics
  • Animals
  • Base Sequence
  • COS Cells
  • Carboxypeptidases (genetics, metabolism)
  • Carboxypeptidases A
  • Catalytic Domain
  • Cloning, Molecular
  • DNA Primers
  • Enzyme Activation
  • Genetic Therapy
  • Mice
  • Mutagenesis
  • Neoplasms, Experimental (therapy)
  • Prodrugs (therapeutic use)
  • Protein Processing, Post-Translational
  • Rats
  • Recombinant Proteins (genetics, metabolism)
  • Subtilisins (metabolism)
  • Tumor Cells, Cultured

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