title: Thimet oligopeptidase (EC 3.4.24.15) activates CPI-0004Na, an extracellularly tumour-activated prodrug of doxorubicin.
abstract: CPI-0004Na is a tetrapeptidic extracellularly tumour-activated prodrug of doxorubicin. The tetrapeptide structure ensures blood stability and selective cleavage by unidentified peptidase(s) released by tumour cells. The purpose of this work was to identify the enzyme responsible for the first rate-limiting step of CPI-0004Na activation, initially attributed to a 70 kDa acidic (pI=5.2) metallopeptidase active at neutral pH that was subsequently purified from HeLa cell homogenates. Two electrophoretic bands were isolated and identified by matrix-assisted laser desorption ionisation-time of flight (MALDI-tof) and electrospray ionisation-quadrupole-time of flight (ESI-Q-tof) mass spectrometry as thimet oligopeptidase (TOP). The identity of the CPI-0004Na activating enzyme and TOP was further supported by the similar substrate specificity of the purified enzyme and recombinant TOP, by thiol stimulation of CPI-0004Na cleavage by cancer cell conditioned media (unique characteristic of TOP) and by the inhibition of CPI-0004Na activation by specific inhibitors or immunoprecipitation. Although other enzymes can be involved, TOP clearly appears to be a likely candidate for extracellular activation of the CPI-0004Na prodrug.
authors.affiliation: Université Catholique de Louvain, Laboratory of Cell Biology, Place Croix du Sud 5, 1348 Louvain-La-Neuve, Belgium. vdubois@diatos.com <vdubois@diatos.com>
authors.forename: V
authors.initials: V
authors.lastname: Dubois
authors.forename: M
authors.initials: M
authors.lastname: Nieder
authors.forename: F
authors.initials: F
authors.lastname: Collot
authors.forename: A
authors.initials: A
authors.lastname: Negrouk
authors.forename: T T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: S
authors.initials: S
authors.lastname: Gangwar
authors.forename: B
authors.initials: B
authors.lastname: Reitz
authors.forename: R
authors.initials: R
authors.lastname: Wattiez
authors.forename: L
authors.initials: L
authors.lastname: Dasnois
authors.forename: A
authors.initials: A
authors.lastname: Trouet
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000627:therapeutic use; D004347:Drug Interactions; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000494:pharmacology*; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism* / Q000627:therapeutic use; D011355:Prodrugs / Q000378:metabolism* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; C438951:N-(succinyl-beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin; D009842:Oligopeptides; D011355:Prodrugs; D004317:Doxorubicin; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase