Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: UNIPROT:P15088 (
mast cell
)
14,925
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Methotrexate (MTX) is one of the most commonly used agents in the treatment of solid malignancies; however, the toxicities of MTX to bone marrow and gastrointestinal tract complicate this therapy. We, therefore, propose a gene-dependent enzyme prodrug therapy to limit these toxicities by localizing the production of MTX to the site of the tumor. The combination of MTX-alpha-peptide prodrugs, which cannot be internalized by the cellular reduced folate carrier, with
carboxypeptidase A
(
CPA
), which can remove the blocking peptide, has been demonstrated previously in vitro using antibody-dependent enzyme prodrug therapy.
CPA
is normally synthesized as a zymogen that is inactive without proteolytic removal of its propeptide by trypsin. Therefore, to adapt this system to gene-dependent enzyme prodrug therapy, a mutant form of
CPA
was engineered,
CPA
(
ST3
), that does not require trypsin-dependent zymogen cleavage but is instead activated by ubiquitously expressed intracellular propeptidases. Purification, peptide sequencing, and kinetic analysis indicated that mature
CPA
(
ST3
) is structurally and functionally similar to the trypsin-activated, wild-type enzyme. In addition,
CPA
(
ST3
)-expressing tumors cells were sensitized to MTX prodrugs in a dose- and time-dependent manner. To limit diffusion of
CPA
, a cell surface localized form was generated by constructing a fusion protein between
CPA
(
ST3
) and the phosphatidylinositol linkage domain from decay accelerating factor. SDS-PAGE and flow cytometric analysis of infected tumor cells indicated that
CPA
(DAF) was cell surface localized. Finally, after retroviral transduction, this enzyme/prodrug strategy exhibited a potent bystander effect, even when <10% of the cells were transduced, because extracellular production of MTX sensitized both transduced and nontransduced cells.
...
PMID:Expression of endogenously activated secreted or cell surface carboxypeptidase A sensitizes tumor cells to methotrexate-alpha-peptide prodrugs. 1067 50
