Zakaria Rachid

Multiple mechanisms of action of ZR2002 in human breast cancer cells: A novel combi‐molecule designed to block signaling mediated by the ERB family of oncogenes and to damage genomic DNA

The mechanism of action of ZR2002, a chimeric amino quinazoline designed to possess mixed EGFR tyrosine kinase (TK) inhibitory and DNA targeting properties, was compared to those of ZR01, a reversible inhibitor of the same class and PD168393, a known irreversible inhibitor of EGFR. ZR2002 exhibited 4‐fold stronger EGFR TK inhibitory activity than its structural homologue ZR01 but was approximately 3‐fold less active than the 6‐acrylamidoquinazoline PD168393. It preferentially blocked EGF and TGFα‐induced cell growth over PDGF and serum. It also inhibited signal transduction in heregulin‐stimulated breast tumour cells, indicating that it does not only block EGFR but also its closely related erbB2 gene product. In contrast to its structural homologues, ZR2002 was capable of inducing significant levels of DNA strand breaks in MDA‐MB‐468 cells after a short 2 hr drug exposure at a concentration as low as 10 μM. Reversibility studies using whole cell autophosphorylation and growth assays in human breast cell lines showed that in contrast to its reversible inhibitor counterpart ZR01, ZR2002 induced irreversible inhibition of EGF‐stimulated autophosphorylation in MDA‐MB‐468 cells and irreversible inhibition of cell growth. Moreover despite possessing a weaker binding affinity than PD168393, it induced a significantly more sustained antiproliferative effect than the latter after a pulse 2 hr exposure. More importantly, in contrast to ZR01 and PD168393, ZR2002 was capable of inducing significant levels of cell death by apoptosis in MDA‐MB‐468 cells. The results in toto suggest that the superior antiproliferative potency of ZR2002 may be due to its ability to induce a protracted blockade of receptor tyrosine kinase‐mediated signaling while damaging cellular DNA, a combination of events that may trigger cell‐killing by apoptosis.

Design and synthesis of new stabilized combi-triazenes for targeting solid tumors expressing the epidermal growth factor receptor (EGFR) or its closest homologue HER2.

The monoalkyltriazene moiety lends itself well to the design of combi-molecules. However, due to its instability under physiological conditions, efforts were directed towards stabilizing it by grafting a hydrolysable carbamate onto the 3-position. The synthesis and biological activities of these novel N-carbamyl triazenes are described.

Synthesis of pyrimidinopyridine-triazene conjugates targeted to abl tyrosine kinase.

The synthesis and abl tyrosine kinase inhibitory activities of alkyltriazenes conjugated to phenylaminopyrimidines are described. Significant abl inhibitory activities were observed only when a benzamido spacer was inserted between the 1,2,3-triazene chain and the 2-phenyaminopyridopyrimidine moiety.

Combi-targeting concept: an optimized single-molecule dual-targeting model for the treatment of chronic myelogenous leukemia

Blockade of Bcr-Abl by the inhibitor Imatinib has proven efficacious in the therapy of chronic myelogenous leukemia (CML). However resistance to the drug emerges at the advanced phases of the disease. Therefore, novel therapy models remained to be designed. We have developed a novel dual targeted agent termed “combi-molecule” designed to not only block Bcr-Abl but also damage DNA. ZRF1, the first optimized prototype of the approach, was “programmed” to degrade into another inhibitor ZRF0 plus a methyl diazonium species. It was ∼2-fold stronger Abl tyrosine kinase inhibitor than Imatinib and a more potent DNA-damaging agent than Temodal. In the p53 wild-type Mo7p210 cells, the potency of ZRF1 was ∼1,000-fold superior to that of the equieffective combinations of Imatinib plus Temodal. More importantly, its superior potency over Imatinib was more pronounced in Bcr-Abl-positive cells coexpressing wild-type p53. Studies to rationalize these results showed that, through its Bcr-Abl inhibitory function, it down-regulated p53. However, sufficient level of the latter protein was available for transactivating p21 and Bax, which are required for cell cycle arrest and apoptosis. The results suggest that, in p53 wild-type cells, apoptosis is induced not only through Bcr-Abl inhibition but also through the p53-controlled DNA-damaging pathway, leading to an additive effect that translates into enhanced cell death. The study conclusively showed that p53 is a major determinant for the cytotoxic advantages of the novel combi-molecular approach in CML, a disease in which 70% to 85% of all the cases express wild-type p53. [Mol Cancer Ther 2008;7(5):1033–43]

ZRX1, the first EGFR inhibitor-capecitabine based combi-molecule, requires carboxylesterase-mediated hydrolysis for optimal activity

SummaryCapecitabine, 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.

“Combi-targeting” mitozolomide: Conferring novel signaling inhibitory properties to an abandoned DNA alkylating agent in the treatment of advanced prostate cancer

At the preclinical stage, mitozolomide (MTZ) showed exciting preclinical activity but failed later in clinical trial due to toxic side effects. We surmised that by targeting MTZ to epidermal growth factor receptor (EGFR), we may not only alter its toxicity profile, but also enhance its potency in EGFR‐overexpressing tumors. To test this hypothesis, we designed JDF12, studied its mechanism of action in human prostate cancer (PCa) cells and determined its potency in vivo.

The combi-targeting concept : Evidence for the formation of a novel inhibitor in vivo

With the purpose of developing drugs that can block multiple targets in tumor cells, molecules termed combi-molecules or TZ–I have been designed to be hydrolyzed in vitro to TZ+I, where TZ is a DNA-damaging species and I is an inhibitor of the epidermal growth factor receptor (EGFR). Using HPLC and liquid chromatography–mass spectrometry (LC–MS), we investigated the mechanism of in vivo degradation of a prototype of one such combi-molecule, ZRBA1, which when administered i.p. rapidly degraded into FD105 (Cmax=50 μmol/l, after 30 min), a 6-aminoquinazoline that was N-acetylated to give FD105Ac (IAc) (Cmax=18 μmol/l, after 4 h). A similar rate of acetylation was observed when independently synthesized FD105 was administered i.p. More importantly, the EGFR binding affinity of IAc was 3-fold greater than that of I, indicating that the latter is converted in vivo into an even more potent EGFR inhibitor. The results in toto suggest that while in vitro TZ–I is only hydrolyzed to I+TZ, further acetylation of I in vivo leads to a third component – a highly potent EGFR inhibitor with a delayed Cmax.

Target modulation by a kinase inhibitor engineered to induce a tandem blockade of the epidermal growth factor receptor (EGFR) and c-Src: the concept of type III combi-targeting.

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”.

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model.

PURPOSE ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. METHODS AND MATERIALS The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Western blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. RESULTS Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. CONCLUSIONS Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.

MGMT is a molecular determinant for potency of the DNA-EGFR-combi-molecule ZRS1.

To enhance the potency of current EGFR inhibitors, we developed a novel strategy that seeks to confer them an additional DNA damaging function, leading to the design of drugs termed combi-molecules. ZRS1 is a novel combi-molecule that contains an EGFR tyrosine kinase targeting quinazoline arm and a methyltriazene-based DNA damaging one. We examined its effect on human tumor cell lines with varied levels of EGFR and O6-methylguanine DNA methyltransferase (MGMT). ZRS1 was more potent than the clinical methylating agent temozolomide in all cell lines, regardless of their MGMT status. However, its potency was in the same range as or less than that of Iressa, an EGFR inhibitor, against MGMT-proficient cells. In the MGMT-deficient or in MGMT-proficient cells exposed to the MGMT inhibitor O6-benzylguanine, its potency was superior to that of Iressa and temozolomide or a temozolomide+Iressa combination. Cell signaling analysis in A549 (MGMT+) and A427 (MGMT−) showed that ZRS1 strongly inhibited EGFR phosphorylation and related signaling pathways. In addition, the p53 pathway was activated by DNA damage in both cell lines, but apoptosis was significantly more pronounced in A427 cells. Using MGMT shRNA to block endogenous MGMT protein expression in A549 resulted in significant sensitization to ZRS1. Furthermore, transfection of MGMT into A427 greatly decreased the potency of ZRS1. These results conclusively show that MGMT is a critical molecular determinant for the full-blown potency of the dual EGFR-DNA targeting combi-molecule. Mol Cancer Res; 9(3); 320–31. ©2011 AACR.