We have designed a novel
tumor targeting strategy that consists of designing molecules termed "combi-molecules" or TZ-I to be masked forms of multiple
antitumor agents. One such molecule
SMA41, the TZ-I prototype, has been shown to target the
epidermal growth factor receptor (EGFR) and to degrade under physiological conditions to give SMA52 (I) (an inhibitor of EGFR) and methyldiazonium (TZ) (
a DNA alkylating species). While the antiproliferative advantages of this novel binary targeting strategy have now been demonstrated, the exact subcellular localization of the degradation products released from
SMA41 remained elusive. Here we exploited the fluorescence properties of SMA52 to study its release from
SMA41 and its subcellular distribution. Further, using 14C-labeled
SMA41, we determined the distribution of the methydiazonium within subcellular macromolecules (
DNA,
RNA,
protein). The results showed that
SMA41 degraded to SMA52 in the
carcinoma of the vulva cell line A431 with a half-life of 11min. The latter compound was primarily distributed in the perinuclear region. At equimolar concentrations, higher levels of SMA52 were observed when released from SM41 than when the cells were directly exposed to SMA52, indicating that the combi-molecular approach may offer a transport advantage to the released bioactive species. Radioactivity associated with
SMA41 3-[ 14C]-methyl group was distributed throughout
DNA,
RNA, and
protein, the latter macromolecule being the most alkylated. The results suggest that
SMA41 (TZ-I) may diffuse into the cells, break down into two species: SMA52 (I) concentrated in the perinuclear region and methyldiazonium (TZ) that diffuses in all intracellular organelles and unspecifically alkylates
RNA,
protein, and nuclear
DNA.