Cancer cells are characterized by a complex network of interrelated and compensatory signaling driven by multiple kinases that reduce their sensitivity to targeted therapy. Therefore, strategies directed at inhibiting two or more kinases are required to robustly block the growth of refractory tumour cells. Here we report on a novel strategy to promote sustained inhibition of two oncogenic kinases (Kin-1 and Kin-2) by designing a molecule K1-K2, termed “combi-molecule”, to induce a tandem blockade of Kin-1 and Kin-2, as an intact structure and to be further hydrolyzed to two inhibitors K1 and K2 directed at Kin-1 and Kin-2, respectively. We chose to target EGFR (Kin-1) and c-Src (Kin-2), two tyrosine kinases known to synergize to promote tumour growth and progression. Variation of K1-K2 linkers led to AL776, our first optimized EGFR-c-Src targeting prototype. Here we showed that: (a) AL776 blocked EGFR and c-Src as an intact structure using an in vitro kinase assay (IC50 EGFR = 0.12 μM and IC50 c-Src = 3 nM), (b) it could release K1 (AL621, a nanomolar EGFR inhibitor) and K2 (dasatinib, a clinically approved Abl/c-Src inhibitor) by hydrolytic cleavage both in vitro and in vivo, (c) it could robustly inhibit phosphorylation of EGFR and c-Src (0.25–1 μM) in cells, (d) it induced 2–4 fold stronger growth inhibition than gefitinib or dasatinib and apoptosis at concentrations as low as 1 μM, and, (e) blocked motility and invasion at sub-micromolar doses in the highly invasive 4T1 and MDA-MB-231 cells. Despite its size (MW = 1032), AL776 blocked phosphorylation of EGFR and c-Src in 4T1 tumours in vivo. We now term this new targeting model consisting of designing a kinase inhibitor K1-K2 to target Kin-1 and Kin-2, and to further release two inhibitors K1 and K2 of the latter kinases, “type III combi-targeting”.

Capecitabine, an orally available prodrug of 5-FU, requires activation by carboxylesterase (CES) enzymes present in the liver to generate 5'-deoxy-5-flurocytidine ribose (5'-DFCR). The deamination of the latter by cytidine deaminase gives 5'-deoxy-5-fluorouridine ribose (5'-DFUR). Finally, the conversion of 5'-DFUR to the cytotoxic drug 5-FU, occurs primarily in the tumour and is catalyzed by thymidine phosphorylase (TP). Accordingly, it was surmised that events associated with an increase of TP levels should enhance the potency of capecitabine and its metabolites. EGFR inhibition was found to be one such event. The observed synergy between gefitinib and 5'-DFUR has inspired the design of single molecules capable of acting as prodrugs of both an EGFR inhibitor and 5-FU. Here, we report on the synthesis and characterization of one such molecule, ZRX1, that consists of an acetylated 5'-DFCR moiety linked to a quinazoline inhibitor of EGFR through an alkyl dicarbamate spacer that requires CES activation to generate the two active metabolites. Our results showed that ZRX1 was ineffective as an intact molecule. However, when CES was present, ZRX1 induced an increase in EGFR inhibition, TP expression, DNA damage and apoptosis. ZRX1 was, at least, 3-fold more potent than capecitabine and 5'-DFUR and recapitulated the effects of the combination treatments. LC-MS analysis showed that in the presence of CES, ZRX1 is metabolized into a mixture of bioactive quinazoline derivatives and 5'-DFCR derived metabolites. Our results in toto, suggest that capecitabine-based EGFR targeting combi-molecules of the same type than ZRX1, have the potential to induce stronger growth inhibitory potency than capecitabine, 5'-DFUR or single EGFR inhibitors and equivalent potency when compared with combinations of EGFR inhibitors + 5'-DFUR.

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.