Here, we report on the first synthesis of fluorescent-labeled epidermal growth factor receptor-DNA targeting combi-molecules, and we studied the influence of P-glycoprotein status of human sarcoma MES-SA cells on their growth inhibitory effect and cellular uptake. The results showed that 6, bearing a longer spacer between the quinazoline ring and the dansyl group, was more stable and more cytotoxic than 4. In contrast to the latter, it induced significant levels of DNA damage in human tumor cells. Moreover, in contrast to doxorubicin, a drug known to be actively effluxed by P-gp, the more stable combi-molecule 6 induced almost identical levels of drug uptake and DNA damage in P-gp-proficient and -deficient cells. Likewise, in contrast to doxorubicin, 4 and 6 exerted equal levels of antiproliferative activity against the two cell types. The results in toto suggest that despite their size, the antiproliferative effects of 4 and 6 were independent of P-gp status of the cells.
Combi-molecules are agents designed to block receptors on their own and to further degrade to bioactive agents. Here we studied the fate of a novel combi-molecule of triazene class termed "ZRS1" in biological medium using multilayer aggregates and mouse tumour models. ZRS1 is a second generation derivative of RB107, a prodrug designed to release an EGFR inhibitor FD105 plus a methyl diazonium species. RB107 contains an acetoxymethyl function that is hydrolyzed too rapidly to generate BJ2000, a monoalkyltriazene that further degrades to FD105 and DNA alkylating methyldiazonium species. Recently, in order to prevent rapid hydrolysis of the acetoxymethylene function in the absence of cells and to delay the release of BJ2000, we designed ZRS1 that contains a more stable acetoxymethyl carbamate function. The results showed that ZRS1 was more stable than RB107 in cell culture medium supplemented with serum, with a rather long half life (>2 h). However, in an experiment where it was allowed to degrade in multilayer aggregates of ovarian cancer cells OV90, it rapidly released BJ2000 and its corresponding metabolite FD105, both in the medium and the multilayer aggregates. Interestingly, the intact ZRS1 could be detected in the multilayer aggregates with a T(max) around 10 min. Studies in vivo, in human DU145 prostate cancer xenograft model, revealed that ZRS1 blocked tumour growth and released FD105 and its acetylated metabolite FD105Ac, the latter being the major metabolite. Likewise, time course analysis in 4T1 mouse syngeneic breast cancer model showed a rapid release of FD105 and FD105Ac in the plasma and in the tumours. In summary, ZRS1 appeared as a good prodrug of the stable EGFR inhibitory metabolites FD105 and FD105Ac. Its ability to generate high concentrations of FD105Ac, a more potent EGFR inhibitor as is its major metabolite, is significant over previous methylating combi-molecules. Furthermore, this study showed that multilayer OV90 aggregates could be developed as an effective model to predict the stability and degradation of ZRS1 in vivo.
Solid tumors at the advanced stages are often characterized by the overexpression of tyrosine kinase receptors that stimulate growth through the MAP kinase pathway and activate antiapoptotic signaling through the PI3K kinase pathway. One such receptor is the epidermal growth factor receptor (EGFR) that is overexpressed in many tumors including brain, lung, breast, ovarian and prostate carcinomas. EGFR can synergize with other tyrosine kinases such as Src to promote invasion and metastasis. However, despite the implication of EGFR in multiple processes, inhibitors of its tyrosine kinase activity such as ZD1839 or erlotinib showed moderate antiproliferative activity against certain tumor types in the clinic. It has been reported that EGFR expressing cells that harbor dysfunctional PTEN, are moderately sensitive to EGFR inhibitors. Given the complexity of cell response the latter class of inhibitors, we thought it of interest to investigate agents that are not only directed at EGFR but also to divergent targets such as Src or DNA, with the purpose of producing single compounds with greater potency than their single inhibitor counterpart. These molecules termed “combi-molecules” were designed to remain small enough for their quinazoline moiety to be able to bind to the ATP site of EGFR and to block a divergent target such as Src or DNA. Using molecular modeling, a structure-based drug design program was used to identify a linker that could be placed between the quinazoline moiety required for binding in the ATP site and the appendage directed at the divergent target. A solvent exposed ASP residue near the opening of the EGFR ATP binding pocket was used to optimize interaction with ionizable linkers. This interaction was found to be tolerant of bulky substituents and was exploited to append other pharmacophores to the combi-molecules. The results showed that in the category of mixed EGFR-DNA targeting molecules, the EGFR inhibitory potency of EGFR was in the low micromolar to nM range and the compounds also retained significant DNA damaging potential. In the category of EGFR-Src targeting molecules, we identified SB163 that contained a quinazoline moiety, the ionizable spacer and an analogue of PP2. SB163 showed significant antiproliferative and antimetastatic property in a Boyden Chamber assay and its activity was superior to that of a combination of known Src inhibitor PP2 + ZD1839, a clinical inhibitor of EGFR. Furthermore, sub-cellular distribution studies, using a fluorescent probe containing the optimized ionizable spacer showed that their biodistribution is unique with preferential localization in the perinuclear region. The results in toto suggests that in the design of promiscuous molecules, an ionizable basic arm linked to the aminoquinazoline moiety preserves EGFR inhibitory potency and allow freedom to append other moiety directed at the cross- target.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A142.
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