Dubois V, Nieder M, Collot F, Negrouk A, Nguyen T T, Gangwar S, Reitz B, Wattiez R, Dasnois L, Trouet A
Université Catholique de Louvain, Laboratory of Cell Biology, Place Croix du Sud 5, 1348 Louvain-La-Neuve, Belgium.
Eur J Cancer. 2006 Nov;42(17):3049-56. doi: 10.1016/j.ejca.2005.10.030. Epub 2006 Apr 27.
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.
CPI-0004Na是一种四肽类细胞外肿瘤激活的阿霉素前药。四肽结构确保了血液稳定性以及被肿瘤细胞释放的未知肽酶进行选择性切割。这项工作的目的是鉴定负责CPI-0004Na激活第一步限速步骤的酶，最初认为是一种70 kDa的酸性（pI = 5.2）金属肽酶，在中性pH下具有活性，随后从HeLa细胞匀浆中纯化得到。通过基质辅助激光解吸电离飞行时间（MALDI-tof）和电喷雾电离四极杆飞行时间（ESI-Q-tof）质谱法分离并鉴定出两条电泳带为硫醇寡肽酶（TOP）。纯化酶和重组TOP具有相似的底物特异性、癌细胞条件培养基对CPI-0004Na切割的硫醇刺激作用（TOP的独特特征）以及特异性抑制剂或免疫沉淀对CPI-0004Na激活的抑制作用，进一步支持了CPI-0004Na激活酶与TOP的一致性。尽管可能涉及其他酶，但TOP显然是CPI-0004Na前药细胞外激活的一个可能候选者。
