Mechanism of action of Type I and Type II combi-molecules designed to target DNA and epidermal growth factor receptor in solid tomours

The overexpression of several receptors and dysfunction of signal transduction pathways characterize the heterogeneity of many solid tumours. Targeted therapies against epidermal growth factor receptor (EGFR)-overexpressing tumours involve well tolerated EGFR inhibitors. However, the potency of these inhibitors is mitigated by several factors including mutations in signaling pathways and receptor heterogeneity. To circumvent this problem, we recently designed a novel strategy that seeks to synthesize molecules "programmed" to release an EGFR inhibitor and a cytotoxic DNA-damaging agent. This led to mixed EGFR-DNA targeting agents, termed "type I combi-molecules". Previous work demonstrated that these compounds: (1) inhibited EGFR and (2) damaged DNA. However, the correlation of their degradation, localization and distribution in the cell with their mechanism of action remained elusive. In this thesis, we designed a fluorescence-labeled combi-molecule AL237 "programmed" to release a blue fluorescent EGFR inhibitor and a green DNA targeting species. The results showed that AL237 blocked EGFR phosphorylation and downstream MAPK pathway, subsequently leading to downregulation of XRCC1 and ERCC1, two DNA repair enzymes. Following demonstration of the ability of AL237 to fully block the MAPK pathway, we studied its localization in the tumour cells by fluorescence microscopy. The results showed that the released quinazoline colocalized with EGFR in the perinuclear region and the green fluorescence was detected in isolated nuclei. This led to a model whereby combi-molecule entered the cell, bind to the EGFR at the plasma membrane to prevent signal transduction and abundantly localized in the perinuclear region from where the released alkylating species could diffuse to the nucleus. This mechanism explained the significantly high levels of DNA damaging species in the nuclei of cells expressing EGFR. Further studies exploiting AL237 fluorescence properties demonstrated that its potency did not depend on the P-gp status of the cells. The P-gp independence of AL237 effect was explained by its intracellular degradation that prevented the efflux of the intact structure. Further studies on combi-molecules that do not require hydrolysis to generate the two targeting species (type II combi-molecules) showed that they exhibited potency in the submicromolar range. Studies designed to elucidate the mechanism underlying the exquisite potency of ZR2008 showed that it induced significant levels of apoptosis, independently of the AKT status of the cells. A constant in its potency was its ability to block cells in G1/S and to downregulate the antiapoptotic protein survivin. Our findings suggest that pathways leading to the inhibition of survivin could be a major molecular determinant for the cytotoxicity of ZR2008. The results from this work in toto contributed to the elucidation of the mechanism of action of two classes of combi-molecules: type I and type II.

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