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.

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.

Positional isomerization of a non-cleavable combi-molecule dramatically altered tumor cell response profile.

To potentiate the quinazoline-based inhibitor of the epidermal growth factor receptor (EGFR), a chloroethyl alkylating moiety was appended to its 6-position. This led to molecules with extremely strong EGFR inhibitory potency and anomalously strong DNA-damaging potential. To assess the role of the chloroethyl group on potency, we designed a molecule in which it is shifted to the 7-position where it would be less reactive and away from the cys773 of the EGFR ATP site.

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

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).

The Concept of Divergent Targeting through the Activation and Inhibition of Receptors as a Novel Chemotherapeutic Strategy: Signaling Responses to Strong DNA-Reactive Combinatorial Mimicries.

Recently, we reported the combination of multitargeted ErbB1 inhibitor-DNA damage combi-molecules with OCT in order to downregulate ErbB1 and activate SSTRs. Absence of translation to cell kill was believed to be partially due to insufficient ErbB1 blockage and DNA damage. In this study, we evaluated cell response to molecules that damage DNA more aggressively and induce stronger attenuation of ErbB1 phosphorylation.

"Combi-targeting" mitozolomide: conferring novel signaling inhibitory properties to an abandoned DNA alkylating agent in the treatment of advanced prostate cancer.

Purpose: 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.

Inhibition of EGFR phosphorylation in a panel of human breast cancer cells correlates with synergistic interactions between gefitinib and 5'-DFUR, the bioactive metabolite of Xeloda.

Capecitabine (Xeloda) is a prodrug of 5-FU used in the clinical management of advanced breast cancer. It is metabolized first in the liver by carboxylesterases to generate 5'-deoxy-5-flurocytidine ribose (5'-DFCR), which is subsequently converted to 5'-deoxy-5-fluorouridine ribose (5'-DFUR) by cytidine deaminase in tumour and normal tissues. The conversion of 5'-DFUR to the cytotoxic 5-FU, occurs primarily in the tumour and is catalyzed by thymidine phosphorylase (TP).

A bioanalytical investigation on the exquisitely strong in vitro potency of the EGFR-DNA targeting type II combi-molecule ZR2003 and its mitigated in vivo antitumour activity.

ZR2003 is a type II of combi-molecule designed to target DNA and the epidermal growth factor receptor (EGFR) without requirement for hydrolysis. In human tumour cell lines cultured as monolayers, it showed 6.5-35 fold greater activity than Iressa.

In vitro and in vivo biodistribution of ZRS1, a stabilized type I N-acetoxymethyl carbamate-containing combi-molecule.

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.

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).

Characterization of the potency of epidermal growth factor (EGFR)-DNA targeting combi-molecules containing a hydrolabile carbamate at the 3-position of the triazene chain.

Previous strategies for stabilizing combi-triazenes were based on masking the 1,2,3-triazene chain with a 3-acetoxymethylene group. The half-lives of the latter molecules were only ca 5 min longer than those of their parent 1,2,3-triazenes. The novel combi-molecules described herein contain a hydrolysable carbamate group that modulates their kinetics of degradation.

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.

Interaction of ionizing radiation and ZRBA1, a mixed EGFR/DNA-targeting molecule.

ZRBA1 is a molecule termed 'combi-molecule' designed to induce DNA-alkylating lesions and to block epidermal growth factor receptor (EGFR) tyrosine kinase. Owing to its ability to downregulate the EGFR tyrosine kinase-mediated antiapoptotic signaling and DNA repair proteins, we inferred that it could significantly sensitize cells to ionizing radiation. Using the MDA-MB-468 human breast cancer cell line in which ZRBA1 has already been reported to induce significant EGFR/DNA-targeting potency, the results showed that: (i) concurrent administration of ZRBA1 and 4 Gy radiation led to a significant decrease in cell viability, (ii) the greater efficacy of the combination was sequential, being limited to conditions wherein the drug was administered concurrently with radiation or before radiation, and (iii) the efficacy enhancement of the combination was further confirmed by clonogenic assays from which a dose enhancement factor of 1.

Molecular analysis of the in vivo metabolism and biodistribution of metabolically and non-metabolically activated combi-molecules of the triazene class.

Combi-molecules are novel agents designed to be hydrolyzed into two bioactive species: an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor + a DNA alkylating agent. With the purpose of enhancing the tumour concentration of the bioactive species, we synthesized and compared the activities of RB107, a quinazolinotriazene designed to generate the bioactive BJ2000 upon hydrolysis, ZRDM and RB107ZR that require metabolic activation to generate BJ2000. The results showed that RB107 released the highest level of BJ2000 and its degradation product FD105 in vivo and high levels of the DNA alkylating methyl diazonium ion in the brain, kidney, liver and the DU145 tumours as confirmed by (14)C-labeling.

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.

Synthesis of water soluble bis-triazenoquinazolines: an unusual predicted mode of binding to the epidermal growth factor receptor tyrosine kinase.

A novel type of 3,3-disubstituted bis-triazenes containing an ethylaminoethyl linker flanked by two identical anilinoquinazoline ring was synthesized. These model molecules contained an N-ethylaminomorpholine moiety designed to enhance water solubility. Despite their significant bulkiness, they blocked epidermal growth factor receptor (EGFR) tyrosine kinase in a dose-dependent manner with IC(50) values in low micromolar range.

Optimization of novel combi-molecules: identification of balanced and mixed bcr-abl/DNA targeting properties.

Steps toward the identification of combi-molecules with strong abl tyrosine kinase (TK) inhibitory property and significant DNA damaging potential are described. The optimized combi-molecule 13a was shown to induce approximately twofold stronger abl TK inhibitory activity than Gleevec and high levels of DNA damage in chronic myelogenous leukemic cells.

Novel nitrogen mustard-armed combi-molecules for the selective targeting of epidermal growth factor receptor overexperessing solid tumors: discovery of an unusual structure-activity relationship.

To enhance the potency of "combi-molecules", we designed 6a-d and 18 to release an inhibitor of EGFR TK and a bifunctional alkylator. The combi-molecules blocked EGFR TK with potency increasing with the basicity of the mustard moiety. They selectively killed cells transfected with EGFR and were potent against the DU145 prostate cancer cells.

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.

Mechanism of action of a novel "combi-triazene" engineered to possess a polar functional group on the alkylating moiety: evidence for enhancement of potency.

Previous studies showed that SMA41, a 3-methyltriazene termed "combi-molecules" possessing a dual epidermal growth factor receptor (EGFR)/DNA targeting properties induced potent antiproliferative activity against alkylating-agent-resistant cells expressing EGFR in vitro. However, despite its marked potency, its antitumour activity in vivo was significantly hampered by its poor hydrosolubility and the moderate reactivity of its alkylating moiety. To circumvent this problem, we designed the quinazolinotriazene ZRBA1 to contain a N,N-dimethylaminoethyl group grafted to the 3-position of the triazene chain where it could serve both as a water soluble and a more potent alkylating moiety.

Engineering 3-alkyltriazenes to block bcr-abl kinase: a novel strategy for the therapy of advanced bcr-abl expressing leukemias.

Recently, within the framework of a new strategy termed "combi-targeting," we designed ZRCM5 to contain a 2-phenylaminopyrimidopyridine moiety targeted to bcr-abl kinase and a triazene tail capable of generating a methyldiazonium species upon hydrolysis. The ability of ZRCM5 to block tyrosine kinase activity was tested in a short 10 min exposure ELISA involving isolated bcr-abl kinase and Western blotting assays. The results showed that: (a) ZRCM5 was hydrolyzed with a half-life of 27 min in cell culture media, (b) it blocked bcr-abl autophosphorylation in promyeloblastic leukemia K562 cells in a dose-dependent manner (IC(50)=14.

Cytokinetics and mechanism of action of AKO4: a novel nitrogen mustard targeted to bcr-abl.

The "combi-targeting" concept seeks to design molecules to not only block tyrosine kinase (TK) activity but also to induce DNA damage. Here we design AK04, a molecule that combines the pharmacophore chlorambucil with that of STI-571 (Gleevec). The results showed that although a less potent abl TK inhibitor than STI571, AK04 was capable of significantly blocking bcr-abl phosphorylation not only in a purified abl assay but also in the bcr-abl+ K562 cells.

Synthesis of half-mustard combi-molecules with fluorescence properties: correlation with EGFR status.

The synthesis of 6-(2-chloroethylamino)-4-anilinoquinazolines ZR2002 and ZR2003 designed to block EGFR tyrosine kinase and to damage genomic DNA is described. These compounds present fluorescence properties that permitted the quantitation of their subcellular uptake by flow cytometry. Fluorescence intensities increased with increasing levels of EGFR in a panel of isogenic and established cell lines.

[Combi-molecules: a global approach towards better chemoselectivity and chemosensitivity].

It is now known that tumour cells possess many signaling pathways to repair damage inflicted by alkylating agents. However, most of these cytotoxic agents only target DNA and this does not suffice to induce sustained antiproliferative activity. Furthermore, the efficacy of antitumour alkylating agents is hampered by a lack of selectivity for tumour tissues.

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 TGFalpha-induced cell growth over PDGF and serum.