Thimet oligopeptidase (EC 3.4.24.15) activates CPI-0004Na, an extracellularly tumour-activated prodrug of doxorubicin.

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.