Qiyu Qiu

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.

The Combi-Targeting Concept: In vitro and In vivo Fragmentation of a Stable Combi-Nitrosourea Engineered to Interact with the Epidermal Growth Factor Receptor while Remaining DNA Reactive

Purpose: JDA58 (NSC 741282), a “combi-molecule” optimized in the context of the “combi-targeting concept,” is a nitrosourea moiety tethered to an anilinoquinazoline. Here, we sought to show its binary epidermal growth factor receptor (EGFR)/DNA targeting property and to study its fragmentation in vitro and in vivo. Experimental Design: The fragmentation of JDA58 was detected in cells in vitro and in vivo by fluorescence microscopy and tandem mass spectrometry. EGFR phosphorylation and DNA damage were determined by Western blotting and comet assay, respectively. Tumor data were examined for statistical significance using the Students t test. Results: JDA58 inhibited EGFR tyrosine kinase (IC50, 0.2 μmol/L) and blocked EGFR phosphorylation in human DU145 prostate cancer cells. It induced significant levels of DNA damage in DU145 cells in vitro or in vivo and showed potent antiproliferative activity both in vitro and in a DU145 xenograft model. In cell-free medium, JDA58 was hydrolyzed to JDA35, a fluorescent amine that could be observed in tumor cells both in vitro and in vivo. In tumor cells in vitro or in vivo, or in plasma collected from mice, the denitrosated species JDA41 was the predominant metabolite. However, mass spectrometric analysis revealed detectable levels of the hydrolytic product JDA35 in tumor cells both in vitro and in vivo. Conclusions: The results in toto suggest that growth inhibition in vitro and in vivo may be sustained by the intact combi-molecule plus JDA35 plus JDA41, three inhibitors of EGFR, and the concomitantly released DNA-damaging species. This leads to a model wherein a single molecule carries a complex multitargeted-multidrug combination.

“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.

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 IC50 values in low micromolar range. Molecular modeling to predict the interactions of the molecule with the ATP binding site of EGFR suggests that the N‐ethylaminomorpholine side chain plays a binding role.

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

SummaryPrevious 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. Their half-lives were prolonged by ca 20–55 min when compared with their acetoxymethyltriazene counterparts. While they decomposed slowly in serum-containing medium, their intracellular decomposition was extremely rapid. They blocked EGFR tyrosine kinase in an isolated enzyme assay and in MDA-MB-468 breast cancer cells. Similarly, they all induced a dose-dependent DNA damage and G2/M cell cycle arrest in MDA-MB-468 cells, except the most stable compound ZRL2 (a 3-vinyl carbamate). ZRL4 (a chloromethyl carbamate) was the most potent and ZRL2 was the least active of the series against MDA-MB-468 cells. In selectivity assay with NIH-3T3 and NIH-3T3/HER-14, all compounds selectively blocked proliferation of NIH-3T3/HER-14. ZRS1 exerted the strongest growth inhibitory potency of the series. The results in toto suggest that ZRL2, despite being the most stable compound, could not hydrolyze at a rate that permitted the generation of DNA damaging species, thereby behaving primarily as an EGFR inhibitor. Thus the study permitted the definition of an optimized combi-molecule as one that decomposes at a rate that is slower than that of acetoxymethyltriazenes, but rapid enough to generate strong EGFR-DNA targeting potential and growth inhibition. Based on the latter criteria, ZRS1 and ZRL4 were tested in vivo and ZRS1 has proven the more effective. Graphical AbstractCombi-molecules of the triazenoquinazoline class are single molecules designed to damage DNA while capable of blocking EGFR-mediated signaling. Through a mechanism-based stability optimization study, a potent in vivo active molecule was discovered.

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. Further evaluation in 3D organ-like multilayer aggregates showed that it could block proliferation at submicromolar level. However, despite the superior potency of ZR2003 over Iressa in vitro, its activity xenograft models was not significantly different from that of Iressa. To rationalize these results, we determined the tumour concentration of both ZR2003 and Iressa in vivo and more importantly in vitro in multicellular aggregates. The results showed that in A431 and 4T1 xenografts, the level of ZR2003 absorbed in the tumours were consistently 2-fold less than those generated by Iressa. Likewise, in the multicellular aggregates model, the penetration of ZR2003 was consistently lower than Iressa. In serum containing media, the level of extractable or free ZR2003 was also inferior to those of Iressa. The results from this bioanalytical study, suggest that the discrepancy between the in vitro and in vivo potency of ZR2003 when compared with Iressa, may be imputed to its significantly lower tumour concentration.

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. The results in toto suggest that RB107 was stable enough to deliver the bioactive species to the tumour site and for optimal tumour distribution of the bioactive species, combi-molecules of the triazene class must be designed to be primarily degraded by hydrolytic cleavage and not by metabolic activation.

Abstract C92: DNA methylating combi‐molecule ZRS1 induces p53 and blocks the epidermal growth factor (EGFR)‐activated pathways in EGFR‐expressing human tumor cells: Relationship to O6‐methylguanine DNA methyltransferase (MGMT) status

The combi‐targeting concept seeks to design single molecules that combine an EGFR tyrosine kinase inhibitor with a DNA damaging agent. One such molecule, ZRS1 contains an EGFR tyrosine kinase targeting quinazoline moiety and a methyltriazene‐based DNA‐damaging function capped with an acetoxymethyloxy group that stabilizes the 1,2,3‐triazene chain. Here, we analyzed the DNA damage response, the MAPK and the PI3K pathways in response to the dual action of ZRS1. We also determined whether its mixed EGFR‐DNA targeting property could enhance its potency in tumor cells expressing the O6‐methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme that confers resistance to methylating agents. The growth inhibitory potency of ZRS1 was tested in a panel of 8 human tumor cell lines with varied levels of EGFR and MGMT. ZRS1 was more potent than the clinical methylating agent temozolomide (TEM) in all the cell lines tested, including the lung cancer (A549, A427 and Calu‐1), colon cancer [HT29, HCT116 and HCT116 (p53−/−)], and breast cancer cell lines (MDA‐MB‐468 and MDA‐MB‐231), regardless of their MGMT status. However, its potency was in the same range as or less than ZD1839, a single‐targeted EGFR inhibitor, against MGMT‐proficient cells. In the MGMT‐deficient cells or MGMT‐proficient cells in which MGMT levels were depleted with O6‐benzylguanine, its potency was superior to that of ZD1839 or TEM. ZRS1 exhibited IC50 of 0.5 µM in the A427 cell line, a value that was 25‐fold lower than that of TEM, 12‐fold lower that of ZD1839 and 15‐fold lower than that equi‐toxic and equimolar combinations of ZD1839+TEM. The two lung cancer cell lines A549 (MGMT+) and A427 (MGMT−) were selected for analyzing the cell signaling pathways underlying the growth inhibitory effect induced by the dual effects of ZRS1. The results showed that: 1. ZRS1 strongly inhibited EGFR phosphorylation in both cell lines and this translated into inhibition of ERK1/2, AKT, Bad S112 and Bad S136 phosphorylation in both cell lines 2. ZRS1 inflicted significant levels of genomic DNA damage and induced p53 in a dose‐dependent manner in both cell lines. Likewise Bax and GADD45 were also induced in both cell lines. The results in toto suggest that in the presence of MGMT, ZRS1 primarily acts as an EGFR inhibitor with no significant effect of its DNA damaging component. In contrast, in the absence of MGMT, its ability to (a) activate the p53 pathway (b) block the MAPK and PI3K pathways, translates into significant cell‐killing. Further studies with plasmid transfection either to knockdown endogenous MGMT or express exogenous MGMT are ongoing to elucidate the mechanism underlying the suppression of the potency of ZRS1 by MGMT. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C92.