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Resistance to chemotherapy in advanced cancers can be mediated by different factors such as epidermal growth factor receptor (EGFR) overexpression and DNA repair enzymes. Therefore, current standards of care usually involve combinations of multiple treatments. Here, to reduce the adverse effects of multiple drug combinations and improve outcome, we proposed a single drug approach to block multiple overlapping effects that characterize chemoresistance. Thus, we designed a new linker that allows assembly of multiple functions (e.g., inhibition of EGFR phosphorylation, induction of DNA lesions, and blockade of their repair) into a single molecule. This led to the successful synthesis of a novel and potent combi-molecule JS230. Here, we demonstrated that in resistant prostate cancer cells overexpressing EGFR, it was capable of (a) inhibiting EGFR in a dose-dependent manner, (b) damaging DNA, and (c) sustaining the damage by inhibiting the DNA repair protein poly(ADP-ribose) polymerase (PARP). The triple mechanism of action of JS230 cumulated into growth inhibitory potency superior to that of classical two- or three-drug combinations.

Disordered expression of the epidermal growth factor receptor (EGFR) has been associated with induction of DNA repair genes (e.g. XRCC1, ERCC1) and resistance to radiation and genotoxic drugs. However, our previous work showed that EGFR inhibition did not affect O6-methylguanine-DNA methyltransferase (MGMT)-mediated resistance. In order to block uncoupled events associated with EGFR and MGMT, we designed MR30, a single molecule termed "combi-molecule" that contains a quinazoline arm targeted to EGFR and an O6-benzylguanine (O6-BG) moiety to block MGMT. Molecular analysis of the mechanism of action of its two arms showed that: (a) it could block EGFR phosphorylation, (b) down-regulate the RAF-MAPK and the PI3K-AKT pathways, and (c) covalently modify MGMT through S-benzylation, as confirmed by MALDI analysis of a direct binding assay with isolated MGMT, (d) it induced a dose-dependent down-regulation of MGMT in lung and melanoma cells. The pleiotropic mechanism of action of MR30 culminated into strong growth inhibition (IC50: 0.018-6.02 μM), with superior activity when compared with an equimolar combination of gefitinib (a clinical EGFR inhibitor) and O6-BG (a known MGMT inhibitor). Pulse exposure experiments were required to attenuate the contribution of EGFR inhibition to the strong potency of MR30, thereby allowing to achieve the dose level required to sensitize cells to temozolomide (TMZ). Indeed, MR30 significantly sensitized EGFR-MGMT co-expressing cells to TMZ (p

Bio-electrochemical systems have been applied successfully for electroreduction of waste carbon dioxide to chemicals. Despite the recent advances in biocathode design and performance, a fundamental understanding of how support electrode materials affect growth, organization and electroactivity of biofilms, and electron transfer across the electrode/bacterium interface is still lacking. Our study demonstrates that surface nanostructuring with semiconducting single-walled carbon nanotubes (SWCNTs) is beneficial for interfacial charge transfer and CO2 reducing activities of Clostridium ljungdahlii biofilms by mimicking the biological functions of conductive bio-wires, not by the commonly suggested mechanism of simply increasing biofilm coverage. We also show that applying a negative potential during biofilm growth is essential for production of electroactive biofilms. 13C isotope labeling experiments conclusively demonstrate that biocathodes can simultaneously utilize ethanol while reducing CO2. Deuterium isotope labeling experiments confirmed that the availability of electrochemically produced H2 as a redox mediator does not limit the efficiency of extracellular electron transfer (EET) and CO2 electro-reduction. These results provide important mechanistic information about EET across the bacterium/material interface in a model biohybrid system.

In order to enhance the cytotoxic potential of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a PARP targeting scaffold. A cell-based selectivity assay involving a BRCA2-deficient Chinese hamster cell line and its corresponding BRCA2 wild type transfectant, was used to predict the PARP targeting potential of the latter agents. The results showed that adding a DNA damaging function to the PARP inhibitors decreased but did not abrogate the selective targeting of the BRCA2-deficient cells. The DNA damaging moiety augmented the potency in BRCA2 deficient cells by 2-20 fold. The most selective dual PARP-DNA targeting agent 14b was found to possess dual DNA and PARP targeting properties.

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.

Background Ovarian cancer is the leading cause of death for gynecological cancers and the 6th cause of women cancer death in developed countries. The late stage detection, the peritoneal dissemination and the acquisition of resistance against carboplatin are the main reasons to explain this poor prognosis and strengthen the need of alternative treatments to improve the management of ovarian cancer and/or to sensitize tumors to platinum salts. Epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (Met) and cellular Src kinase (c-Src) are crucial kinases implied in ovarian tumor growth, survival, invasion and resistance to carboplatin. Their expression is increased in advanced ovarian cancers and is correlated with poor prognosis. Despite a clear potential in inhibiting these proteins in ovarian cancer, as a single agent or in combination with a carboplatin treatment, we need to target kinases in tandem because of their capacity to trigger compensatory pathways that synergize to promote drug resistance. Methods Here we target EGFR, c-Src and Met individually or in combination with carboplatin, using Gefitinib, Dasatinib and Crizotinib respectively, in a panel of carboplatin-sensitive (OVCAR-3, IGROV-1 and A2780) and carboplatin-resistant cells (SKOV-3 and EFO-21). We studied the ability of the most potent combination to induce apoptosis, regulate migration, invasion and to modulate the activation of proliferation and survival proteins. Results Crizotinib, Dasatinib and Gefitinib, alone or in combination with carboplatin, showed a cell-specific cytotoxic synergy in ovarian cancer cells. The Dasatinib plus Gefitinib combination was synergistic in OVCAR-3, SKOV-3 and, in IGROV-1 cells (high concentrations). This combination was unable to induce apoptosis but suppressed cell migration, invasion and the activation of EGFR, Erk, c-Src and Akt compared to single treatments. Conclusions Combining carboplatin with kinase inhibitors lead to synergistic interactions in a cell-specific manner. Unlike platinum-based combinations, mixing Dasatinib with Gefitinib led to cytotoxic activity, inhibition of cell migration and invasion. Thus, the Dasatinib + Gefitinib combination presents anti-tumour properties that are superior to those of platinum-based combinations, indicating that it may well represent a promising new treatment modality to be tested in the clinic. Electronic supplementary material The online version of this article (doi:10.1186/s13048-017-0319-2) contains supplementary material, which is available to authorized users.

Purpose : The hormonally active form of vitamin D [1,25(OH)2D3], has several antitumor effects including antiproliferative, prodifferentiative and proapoptotic functions in tissues expressing and binding the vitamin D receptor. 1,25(OH)2D3 is synthesized from its precursor 25-hydroxyvitamin D [25(OH)D] via the catalytic action of the mitochondria cytochrome P450 enzyme 25(OH)D-1α-hydroxylase (CYP27B1). In the current study we investigated the role of CYP27B1 ablation on vitamin D signaling using the mouse mammary tumor virus promoter-driven polyoma middle T oncoprotein (MMTV-PyMT) mouse, an oncogene-driven model of highly aggressive spontaneous mammary tumors that closely mimics the estrogen receptor negative breast cancer in human disease. Methods: Tumors from animals were digested and primary cells were obtained from both CYP27B1 ablated AOH93Cre+ cells and wild type MT1107 Cre- control cells. The cells were next grown in complete DMEM medium, transfected with either a GFP-tagged human VDR or RXRα and treated with 1,25(OH)2D3 (10−7 mol/L) or 25(OH)D3 (10−7 mol/L) before data acquisition. Results: Using live and fixed cell fluorescence imaging, in situ proximity ligation assay and immunoblotting techniques, we show that the nuclear localization of both endogenous and exogenously tagged VDR and RXR were impaired in CYP27B1 ablated cells when compared to non-ablated cells. Furthermore, the intranuclear shuttling of VDR and its interaction with RXR were significantly reduced in CYP27B1 ablated cells as shown by both Florescence Recovery After Photobleaching and Fluorescence Resonance Energy Transfer techniques. Our results also show that CYP27B1 ablation disrupts VDR function by significantly impairing its nuclear localization, intranuclear transport, interaction with adaptor proteins, hVDR/hRXR, and this correlated with impaired binding to DNA and chromatin. Lastly, we demonstrate impaired induction of 53BP1, p65 and γ-H2AX DNA double-strand breaks in the CYP27B1 ablated cells compared to non-ablated cells. Conclusion: These results suggest that ablation of CYP27B1 results in dysregulation in vitamin D signaling which may impair its anticancer functions. Citation Format: Sylvester Jusu, John Presley, Bertrand Jean-Claude, Ursula Stochaj, Richard Kremer. Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-024.

Background Mutations of the DNA repair proteins BRCA1/2 are synthetically lethal with the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), which when inhibited, leads to cell death due to the absence of compensatory DNA repair mechanism. The potency of PARP inhibitors has now been clinically proven. However, disappointingly, acquired resistance mediated by the reactivation of wild type BRCA1/2 has been reported. In order to improve their efficacy, trials are ongoing to explore their combinations with temozolomide (TMZ). Here, in order to enhance potency in BRCA1/2-mutant cells, we report on the design of single molecules termed “combi-molecules” capable of not only inhibiting PARP but also damaging DNA like TMZ, which is known to induce a large number of DNA adducts. The majority of these lesions are processed through PARP-dependent base-excision repair machinery. Paradoxically, the least abundant lesion, the O6-methylguanine adduct is the most cytotoxic. Its repair by the O6-methylguanine DNA methyl transferase (MGMT) confers robust resistance to TMZ. Thus, we surmise that a combi-molecule designed to generate the same DNA adducts as TMZ, with an additional ability to block PARP, could induce BRCA1/2 mutant selective potency and a growth inhibitory profile independent of MGMT status. Methods The hydrolysis of EG22 and its stabilized form ZSM02 was analyzed by HPLC and fluorescence spectroscopy. Growth inhibitory potency was determined by SRB assay. PARP inhibition was determined by an enzyme assay and DNA damage by the comet assay. Subcellular distribution was visualized by confocal microscopy. Results Studies on EG22 showed that: (a) it inflicted anomalously higher levels of DNA damage than TMZ (b) it induced PARP inhibitory potency in the same range as ANI, a known PARP inhibitor (IC50 = 0.10 μM) (c) it showed strong potency in both BRCA1/2 wild type and mutated cells with 6-fold selectivity for the mutants and it was 65–303-fold more potent than TMZ and 4–63-fold than ANI alone and 3–47-fold than their corresponding equimolar combinations and (d) its potency was independent of MGMT expression. Conclusion The results in toto suggest that a combi-molecular approach directed at blocking PARP and damaging DNA can lead to single molecules with selective and enhanced potency against BRCA1/2 mutant and with activity independent of MGMT, the major predictive biomarker for resistance to TMZ.

In breast cancer cells expressing c-Src and EGFR, a control of one of the two oncogenes over proliferation and invasion is observed, whereas in others, the synergistic interaction between them is required for tumor progression. With the purpose of developing molecules with the highest probability for blocking the adverse effects of these two oncogenes, we designed AL622, which contains a quinazoline head targeted to EGFR and a linker that bridges it to the PP2-like structure for targeting c-Src. In case the entire molecule would not be capable of blocking c-Src, we designed AL622 to hydrolyze to an intact c-Src-targeting PP2 molecule. After confirming its binary c-Src-EGFR targeting potency of AL622, we analyzed its potency in isogenic NIH3T3 cells transfected with EGFR and HER2 and human breast cancer cells known to be dominated by c-Src function. The results showed that in EGFR/HER-2-driven cells, it was more potent than PP2 and its activity was in the same range as the latter in more c-Src-driven cells. Its ability to block motility and invasion was comparable with that of PP2 and corresponding combinations, indicating that AL622 could be a better antitumor agent in cells where c-Src and/or EGFR play a role.

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. EXPERIMENTAL DESIGN To analyze its mixed EGFR-DNA targeting potential, we performed an enzyme linked immunosorbent assay (ELISA) and western blotting analysis of EGFR phosphorylation in cells stimulated with EGF. DNA damage was analyzed using the comet assay, and apoptosis quantitated by annexin V binding assay. Growth inhibition in vitro was determined by the sulforhodamine B (SRB) assay and in vivo efficacy analyzed in male CD-1 nude mice. RESULTS The results showed that: Under physiological conditions, JDF12 was hydrolyzed to JDF04R and both agents were capable of inhibiting isolated EGFR tyrosine kinase (TK) and EGFR phosphorylation in EGF-stimulated cells. JDF12 significantly damaged DNA, induced apoptosis in DU145 cells and was up to 2–10-fold more potent than equieffective combinations of MTZ and JDF04R or Iressa in a panel that also included LNCaP and its EGFR and ErbB2 transfectants. In vivo, it induced significant antitumor activity in a DU145 xenograft model. CONCLUSIONS The results suggest that the superior cytotoxicity of JDF12 when compared with MTZ and JDF04R may be imputed to its potent EGFR-DNA targeting properties and confirm the ability of this novel strategy to confer EGFR targeting properties to a classical alkylator. Prostate 72:1273–1285, 2012. © 2012 Wiley Periodicals, Inc.

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). Prior work in head and neck cancer showed that cell treatment with an inhibitor of the epidermal growth receptor (EGFR) gefitinib led to an increase in TP expression and sensitized them to 5'-DFUR. This work seeks to investigate the factors influencing the potency of gefitinib + 5'-DFUR combination. Here, we studied these factors in a panel of six human breast cancer cell lines, with varied levels of sensitivity to gefitinib. Our results first confirmed that 5'-DFUR potency linearly correlates with TP basal levels in the panel of cell lines. In contrast, the strength of the synergistic effect of the gefitinib + 5'-DFUR combination, as measured by their combination indices (CI) correlates with pEGFR percent inhibition and with the modulation of TP expression by gefitinib (as quantitated by TP fold change) rather than TP basal levels. The results, in toto, suggest that the extent of modulation of TP by gefitinib may be used as a predictor of tumour sensitivity to gefitinib + capecitabine/5'-DFUR combinations.

In order to induce a tandem targeting of EGFR, DNA, and MEK, we built complex combi-molecules containing an EGFR targeting quinazoline and an aminoethyltriazene moiety linking the entire molecule to PD98059. Two complex molecules were synthesized: one with a short aminoethyl spacer, AL232, and the other AL414 with a longer aminoethylaminoethyl spacer. AL414 was a more potent inhibitor of EGFR tyrosine kinase than AL232. Both combi-molecules blocked EGFR phosphorylation in whole cells and downregulated extracellular signaling-regulated kinases (ERK1,2). However, only AL414 was capable of inducing DNA damage. Thus, it was taken in vivo for metabolic analysis. The results showed that 3 h after injection, AL414 was hydrolyzed to an EGFR inhibitor FD105, which was further acetylated to FD105Ac, a more potent inhibitor of EGFR. The detected flavone derivative was PD98059 linked to the hydroxyalkyl moiety resulting from the decomposition of the alkyldiazonium species. Independent synthesis of the latter metabolite and further in vitro analysis showed that it was deprived of antiproliferative activity. The results in toto suggest that while AL414 is a three-compartment combi-molecule, only the EGFR and DNA targeting species can be released and the cleavage to the intact MEK inhibitor PD98059 was mitigated by the stability of the carbamate.

Poly (ADP-ribose) polymerase (PARP) inhibitors are a novel therapeutic approach for the selective targeting of tumours with BRCA1/2 hereditary deficiencies. BRCA1/2 are two key proteins involved in homologous recombination (HR) repair. Although the therapeutic benefit of PARP inhibitors has now been proven in the clinic in patients with BRCA1/2 mutations, much effort has been deployed to expand the use of PARP inhibitors beyond tumours harbouring inherited deficiencies in HR- mediated DNA repair. Several combination modalities with agents that can induce HR deficiency and exploit the concept of “contextual synthetic lethality” have been reported for PARP inhibitors. Here we exploited the contextual synthetic lethality between PARP and the epidermal growth factor receptor (EGFR) to design and synthesize a novel dual EGFR targeted PARP inhibitor, termed ZSMR06. The results showed that: a) ZSMR06 is capable of inhibiting EGFR and PARP in whole cells in the nanomolar range (36-63 nM); b) it was selectively potent against BRCA2 mutant and EGFR expressing isogenic cells; c) as monotherapy, ZSMR06 was extremely potent with growth inhibitory activities superior to that of an equimolar combination of olaparib (a clinical PARP inhibitor) and gefitinib (a clinical EGFR inhibitor) in a large panel of tumour cell lines; d) ZSMR06 strongly potentiated the effect of temozolomide (TMZ) and induced significantly stronger (p Citation Format: Martin Rupp, Zhor Senhaji Mouhri, Ursula Stochaj, Bertrand Jean-Claude. Tandem targeting of poly (ADP-ribose) polymerase (PARP) and epidermal growth factor receptor (EGFR) as a novel strategy for enhancing radio- and chemosensitivity of refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2925.

To monitor the subcellular distribution of mixed epidermal growth factor (EGF) receptor (EGFR)–DNA targeting drugs termed combi-molecules, we designed AL237, a fluorescent prototype, to degrade into a green fluorescent DNA damaging species and FD105, a blue fluorescent EGFR inhibitor. Here we showed that AL237 damaged DNA in the 12.5 to 50 μmol/L range. Despite its size, it blocked EGFR phosphorylation in an enzyme assay (IC50 = 0.27 μmol/L) and in MDA-MB468 breast cancer cells in the same concentration range as for DNA damage. This translated into inhibition of extracellular signal-regulated kinase 1/2 or BAD phosphorylation and downregulation of DNA repair proteins (XRCC1, ERCC1). Having shown that AL237 was a balanced EGFR-DNA targeting molecule, it was used as an imaging probe to show that (a) green and blue colors were primarily colocalized in the perinuclear and partially in the nucleus in EGFR- or ErbB2-expressing cells, (b) the blue fluorescence associated with FD105, but not the green, was colocalized with anti-EGFR red-labeled antibody, (c) the green fluorescence of nuclei was significantly more intense in NIH 3T3 cells expressing EGFR or ErbB2 than in their wild-type counterparts (P < 0.05). Similarly, the growth inhibitory potency of AL237 was selectively stronger in the transfectants. In summary, the results suggest that AL237 diffuses into the cells and localizes abundantly in the perinuclear region and partially in the nucleus where it degrades into EGFR and DNA targeting species. This bystander-like effect translates into high levels of DNA damage in the nucleus. Sufficient quinazoline levels are released in the cells to block EGF-induced activation of downstream signaling. Mol Cancer Ther; 9(4); 869–82. ©2010 AACR.

Within the context of a new tumor-targeting strategy termed "combi-targeting," we designed RB24 to inhibit epidermal growth factor receptor (EGFR) or Her2 phosphorylation and to further degrade upon hydrolysis to 4-(3'-bromophenylamino)-6-aminoquinazoline (RB10; another EGFR/Her2 inhibitor) plus a strong DNA-alkylating species. 6-(3-Acetoxymethyl-3-methyltriazenyl)-4-(3'-bromophenylamino)quinazoline (RB24) showed significant antiproliferative activity against human breast cancer cells, and transfection of one such cell line, MDA-MB-435, with ErbB1 or ErbB2 (Her2) dramatically enhanced cell death by apoptosis. RB24 was capable of releasing 2- to 3-fold higher levels of RB10 in the transfectants than in their wild-type counterparts. More importantly, RB10 was abundantly distributed in the perinuclear region of the cells, and its elevated levels in the ErbB transfectants were concomitant with increased levels of DNA lesions in the latter cells. This selectivity could be abolished by coincubation of the cells with exogenous RB10, suggesting that the entire combi-molecule may bind primarily to its cognate perinuclear sites before degradation. This localization may exert a bystander effect, allowing the alkylating species to be abundantly propagated into the nucleus. Cell response to this novel targeting mechanism was mediated by 1) activation of c-Jun NH(2)-terminal kinase in response to DNA damage and 2) down-regulation of Bad through blockade of EGFR tyrosine kinase activity: two events that cooperatively converged into enhancement of apoptosis in the oncogene-transfected cells.

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.

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 1.34 could be observed at survival fraction of 0.01. Flow cytometric analysis showed significant enhancement of cell cycle arrest in G2/M (P

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. Combi-molecule 6a blocked EGFR phosphorylation in an irreversible manner, induced DNA-cross-links, and arrested the cells in mid-S.

We recently designed molecules termed "type II combi-molecules" to block the epidermal growth factor receptor and to damage DNA without the requirement for hydrolytic cleavage. Here, we studied two such combi-molecules (JDD36 and JDE05), containing a novel quinazoline-linked chloroethyltriazolinium system. The epidermal growth factor receptor-targeting potential of these novel structures was studied by ELISA for isolated epidermal growth factor receptor and by Western blotting for whole-cell assays. DNA damage was analyzed using the single-cell microelectrophoresis comet assay. Antiproliferative effects were determined by the sulforhodamine B assay. JDD36 showed an IC50 of 0.6 micromol/l in the ELISA for epidermal growth factor receptor tyrosine kinase, a dose-dependent inhibition of epidermal growth factor receptor phosphorylation and significant levels of DNA damage in the human DU145 prostate cancer cell line. JDD36 was an overall 2- to 15-fold stronger antiproliferative agent than JDE05 that showed potent epidermal growth factor receptor inhibitory activity (IC50 epidermal growth factor receptor, 0.035 micromol/l) but weak DNA-damaging potential. In a panel of LNCaP erbB transfectants, in contrast to JDE05, JDD36 showed remarkable and selective potency against the LNCaPerbB2 transfectant. The results in toto suggest that the overall superior potency of JDD36 when compared with JDE05 may be imputed to its balanced binary epidermal growth factor receptor-DNA-targeting properties that may induce a tandem blockade of epidermal growth factor receptor-mediated mitogenic signaling while depleting alternative survival mechanism by damaging DNA.

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 (IC50 = 14.01 μM) and (c) it induced dose-dependent levels of DNA strand breaks. In contrast, temozolomide (TEM), a clinical DNA damaging triazene capable of generating, like ZRCM5, a methyldiazonium species, could neither block bcr-abl tyrosine kinase activity in isolated enzyme nor in whole cell autophosphorylation assays. In cells expressing varied levels of bcr-abl, ZRCM5 was consistently more potent than TEM. The significant potency of ZRCM5 against the leukemia cells was attributed to its ability to simultaneously to block bcr-abl and related DNA repair activity while inducing significant DNA lesions in bcr-abl expressing leukemia cells. Further studies are ongoing to increase the affinity of ZRCM5 with the purpose of further enhancing its potency in bcr-abl expressing cells.

Kinin B(1) receptors (B(1)R) are involved in many pathophysiological processes, and its expression is up-regulated in inflammatory pulmonary disease. Although bacteria can generate kinin peptides, the molecular signaling mechanisms regulating B(1)R during infection by intact pathogens is unknown. The serious opportunistic clinical isolate Burkholderia cenocepacia (B. cen.) belongs to the important B. cepacia complex (Bcc) of gram-negative pathogens that rapidly causes fatal pulmonary disease in hospitalized and immunocompromised patients and those with cystic fibrosis. We demonstrate here that B. cen. infection induced a rapid increase in B(1)R mRNA (1 h) proceeded by an increase in B(1)R protein expression (2 h), without affecting B(2) receptor expression in human pulmonary fibroblasts. The B(1)R response was dose-dependent and maximal by 6 to 8 h (3- to 4-fold increase), however, brief B. cen. infection could sustain B(1)R up-regulation. In contrast, nonclinical Bcc phytopathogens were much less B(1)R inducive. The protein synthesis inhibitor cycloheximide and transcriptional inhibitor actinomycin D abrogated the B(1) response to B. cen. indicating de novo B(1)R synthesis. B. cen. activated p38 mitogen-activated protein kinase (MAPK), and blocking p38 MAPK with the specific inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) dramatically reduced B. cen.-induced B(1)R. Furthermore, B. cen. regulation of B(1)R was diminished by the anti-inflammatory glucocorticoid dexamethasone. In conclusion, this study is the first demonstration that infection with intact pulmonary pathogens like B. cen. positively modulates the selective expression of B(1)R. Thus, providing evidence that B(1)R regulation may be an important and novel mechanism in the inflammatory cascade in response to chronic pulmonary infection and disease.

We have previously reported the synthesis of SMA41, a unimolecular combination of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of the quinazoline class and a DNA-damaging monomethyltriazene termed “combimolecule”. Hydrolysis of 1-[4-( m -tolylamino)-6-quinazolinyl]-3-methyltriazene (SMA41) gives rise to an intact TKI [6-amino-4-(3-methylanilino)quinazoline; SMA52] capable of inhibiting epidermal growth factor (EGF)-induced EGFR autophosphorylation and a DNA-targeting methyldiazonium species. Herein, we showed that SMA41 blocked EGF-induced EGFR autophosphorylation by an irreversible mechanism, suggesting that it may covalently damage the receptor in these cells. More importantly, this was associated with significant inhibition of mitogen-activated protein kinase activation in A431 cells. In cells treated with [14C]SMA41, radio-high-performance liquid chromatography detection of both N 7- and O 6-methylguanine revealed an almost complete repair of the O 6-methylguanine lesions and a greater tolerance of the N 7-methylguanine adducts 24 h post-treatment. In contrast to temozolomide (a cyclic triazene used in the clinic) and the reversible inhibitor SMA52, SMA41 induced significant cell cycle arrest in S, G2, and M phases 24 h after a 2-h drug exposure. Furthermore, in vivo studies demonstrated that SMA41 was well tolerated. At 200 mg/kg, it showed approximately 2-fold greater antiproliferative activity than SMA52 in A431 cells implanted in immunocompromised SCID mice. These results suggest that the binary targeting properties of SMA41 are associated with a binary cascade of events in the cells that seem to culminate into significant growth inhibition in vitro and in vivo.

The synthesis of a novel acetoxymethyltriazene designed to be a prodrug of multiple inhibitors of the epidermal growth factor receptor (EGFR) and a methyldiazonium species is described. Studies with each of the expected metabolites demonstrated significant EGFR tyrosine kinase inhibitory activities and the released methyldiazonium was trapped with p-nitrobenzylpyridine. Their ability to damage genomic DNA in whole cells was demonstrated by using the single cell microelectrophoresis (comet) assay. The results suggest that this approach may well represent a novel drug combination strategy involving single molecules masking multiple bioactive agents.

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.

The combi-targeting concept postulates that a molecule termed a "combi-molecule" designed to interact with an oncoreceptor on its own and allowed to further degrade to another more stable inhibitor of the latter receptor + a DNA-damaging species should be more potent than the individual combination of the same inhibitor with a DNA-damaging agent in cells expressing the targeted receptor. Recently, using the epidermal growth factor receptor (EGFR) as a target, we demonstrated the feasibility of combi-molecules with dual EGFR/DNA-targeting properties and with the ability to degrade to another potent inhibitor of EGFR. However, despite a clear demonstration of their superior potency when compared with classical combinations in EGFR-expressing cells, the true contribution of each fragment of the combi-molecules to their overall antiproliferative activity remained elusive. Here, we report a structure-function approach whereby a series of quinazoline-based "combi-triazenes" were altered to either abrogate the affinity of the EGFR-targeting quinazoline head or to suppress the DNA-damaging property of the triazene tail. The results showed that (a) inactivation of the quinazoline head by appending an N-methylaniline group to its 4-position reduced EGFR tyrosine kinase (TK) inhibitory activity by ca. 200-fold and decreased the ability of the combi-molecule to block serum-induced growth stimulation in c-erbB2 transfected NIH3T3 cells by ca. 10-fold, (b) abrogation of the alkylating activity or the DNA-damaging potential of the triazene tail by forming 3,3-dimethyltriazenes did not suppress EGFR TK inhibitory affinity but decreased the antiproliferative activity in basal growth assays, and (c) the antiproliferative activities of the monoalkyltriazenes that possessed binary EGFR TK inhibitory and alkylating activities were superior to those of their monotargeted counterparts. The results in toto suggest that each component of the dual targeting property of combi-triazenes plays a critical role in their overall antiproliferative activity.

The synthesis of 1-[4-(m-tolyl)amino-6-quinazolinyl]-3-[14C]-methyl triazene (SMA41) is described. This triazene was designed to be hydrolyzed under physiological conditions to N4-m-tolyl-quinazoline-4,6-diamine (SMA52), a moderate inhibitor of the epidermal growth factor receptor (EGFR) and the DNA alkylating species [14C]-methyldiazonium. A radiolabeled probe was needed to test the hypothesis that in situ hydrolysis of SMA41 may induce alkylation of the ATP binding site of EGFR. 14C-SMA41 was obtained with a radiochemical yield of 21% and a specific activity of 54.6 mCi/mmol, as determined by HPLC quantitation and scintillation counting. Radio-TLC analyses showed 98% radiochemical purity. Copyright © 2003 John Wiley & Sons, Ltd.

Purpose. Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Methods. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Results. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC50 values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the EGFR transfectant and delayed cell entry to G2/M in both cell types. When the cells were starved and growth-stimulated with FBS (conditions under which both cell types were growth-stimulated), in contrast to TEM, SMA41 and its hydrolytic metabolite SMA52 exhibited approximately nine- and threefold stronger inhibition of growth of the EGFR transfectant. Conclusions. These results suggest that, in addition to its ability to induce DNA damage and cell cycle perturbations, SMA41 is capable of selectively targeting the cells with a growth advantage conferred by EGFR transfection. When compared with the monoalkyltriazene prodrug TEM, its potency may be further enhanced by its ability to hydrolyze to another signal transduction inhibitor (SMA52) plus a DNA alkylating agent that may further contribute to chemosensitivity. Thus, our new "combi-targeting" strategy may well represent a tandem approach to selectively blocking receptor tyrosine kinase-mediated growth signaling while inducing significant levels of cytotoxic DNA lesions in refractory tumors.

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”.

The Combi-Targeting concept postulates that a molecule termed combi-molecule (C-molecule) with binary epidermal growth factor receptor (EGFR) targeting/DNA-damaging properties and with the ability to be hydrolyzed to another EGFR inhibitor should induce sustained antiproliferative activity in cells overexpressing EGFR. Because we postulate that the EGFR affinity of the C-molecule and that of its hydrolytic metabolites are critical parameters for sustained potency against EGFR-overexpressing cells, we synthesized BJ2000 (IC(50) = 0.1 microM, competitive binding at ATP site), a novel C-molecule that can decompose into a 6-amino-4-anilinoquinazoline FD105 (IC(50) = 0.2 microM). Studies using the EGFR-overexpressing A431 cells revealed that BJ2000 could damage DNA and block epidermal growth factor-stimulated EGFR autophosphorylation by a partially irreversible mechanism. Blockade of EGFR autophosphorylation subsequently induced inhibition of mitogen-activated protein kinase activation and c-fos gene expression. Enzyme-linked immunosorbent assay and growth factor-mediated stimulation of proliferation assays in the EGFR-expressing NIH3T3HER14 demonstrated the preferential EGFR-targeting properties of BJ2000, and more importantly suggest that blockade of EGFR phosphorylation by this drug translate into significant growth inhibitory effects. These properties culminated into irreversible antiproliferative effects as confirmed by a sulforhodamine B assay. Five days after a 2-h treatment, BJ2000 retained significant antiproliferative effect in A431 cells, whereas its reversible metabolite FD105 almost completely lost its activity. This result in toto lend support to the Combi-Targeting concept according to which a molecular conjugate kept small enough to interact with EGFR and designed to degrade into another inhibitor of the same target plus a DNA-damaging species may induce sustained growth inhibitory effect in EGFR-overexpressing cells.

Temozolomide (TMZ) is an oral alkylating agent commonly used as a first-line chemotherapeutic agent for the treatment of high-grade gliomas and melanoma. Alkylation of the O6-position of guanine in DNA is mainly responsible for the antitumor effect of TMZ. The primary mechanism of chemoresistance to TMZ has been shown to be the overexpression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) that specifically removes the methyl lesions from the O6-position of guanine. While O6-benzylguanine (O6-BG), a potent inhibitor of MGMT, was shown to sensitize tumor cells to TMZ in vitro and in vivo, the O6-BG+TMZ combination failed in clinical trial due to acute hematologic toxicity. To circumvent this problem, we wish to target O6-BG to the epidermal growth factor receptor (EGFR), a receptor that is not generally expressed in the hematological cells but overexpressed in many solid tumors. We have now successfully designed and synthesized one such molecule termed MR30 and showed that: 1) MR30 is capable of blocking EGFR and depleting MGMT levels in whole cells; 2) MR30 is a unique molecule capable of inducing stronger growth inhibition than the 2-drug combination involving clinical EGFR and MGMT inhibitors in a panel of melanoma, lung, prostate and ovarian cancer cell lines; 3) MR30 in vitro potentiated the effect of TMZ on MGMT positive cell lines; 4) its kinase inhibitory profiling over 25 different kinases demonstrated selectivity for EGFR, HER2 and EGFR mutant forms; 5) an isogenic model showed more than 70-fold selectivity towards EGFR expressing cells in growth inhibitory assay; 6) MR30 showed good cell penetration with distribution in the perinuclear region. The results in toto suggest that MR30 has the potential to be developed as a tumor selective potentiator of TMZ. Citation Format: Martin Rupp, Bertrand J. Jean-Claude. Design of a novel MGMT inhibitor targeted to EGFR overexpressing tumor cells: a new approach to selectively potentiate temozolomide in refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4043. doi:10.1158/1538-7445.AM2017-4043

Over the past decade, PARP inhibition has been actively pursued as a novel approach for the selective therapy of tumors with BRCA1/2 mutations. The therapeutic benefits of PARP inhibitors have now been proven in the clinic against BRCA1/2 mutant ovarian cancers. This is hitherto limited to BRCA1/2 mutations, which only accounts for 5-10% of all cancers with hereditary mutations in the homologous recombination pathway. Therefore, new strategies are not only required to enhance the potency of PARP inhibitors but also to expand their use beyond BRCA mutation. While several combination modalities have been reported for PARP inhibitors, the concept of targeted PARP inhibitor has not yet been explored. Here using our novel combi-targeting approach, we report on the design of PARP inhibitors targeted to EGFR, a tyrosine kinase receptor overexpressed in several solid tumors. Recently, reports on the relationship between EGFR, PARP and BRCA have begun to emerge, one of which described a contextual synthetic lethality between EGFR and PARP (PloS one 7.10 (2012)). Here we report on the design and synthesis of novel PARP-EGFR combi-molecule based on structural modification of olaparib as a PARP inhibitor warhead and the quinazoline moiety for targeting EGFR. The results showed that: (a) it is capable of inducing a dose-dependent inhibition of PARP in isolated enzyme assay, (b) it induced a dose-dependent inhibition of EGFR in an isolated kinase assay, (c) it showed a dose-dependent inhibition of EGFR phosphorylation and downstream signaling in whole-cell assay, (d) it was selectively potent towards BRCA2 mutant and also EGFR-overexpressing cell lines, (d) it was extremely potent with activities superior to that of olaparib or gefitinib alone and their corresponding equimolar combination in three established triple negative breast cancer cell lines, (e) subcellular distribution analysis showed that it was abundantly localized in the perinuclear region. These results in toto suggest that this new combi-molecule could be developed as a single drug modality emulating the combination of PARP and EGFR inhibitors with the added benefit of being targeted to EGFR-expressing tumor cells. Citation Format: Zhor Senhaji Mouhri, Martin Rupp, Bertrand J. Jean-Claude. A novel combi-molecule engineered to target the putative synthetic lethal interactions between the epidermal growth factor receptor (EGFR) and poly(ADP-ribose)polymerase (PARP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4035. doi:10.1158/1538-7445.AM2017-4035

Cytotoxicity, reduction of macromolecule synthesis and cell cycle perturbations by two novel 3-(2-chloroethyl)-tetrazepinones, PYRCL and QUINCL were compared with those produced by the structurally related 3-(2-chloroethyl)-tetrazinone, mitozolomide, in the OVCAR-3 cell line. Methods: Macromolecule synthesis was determined by incorporation of 3H-thymidine, 3H-uridine and 3H-leucine into acid-precipitable fractions of OVCAR-3 cell extracts. Maxam-Gilbert sequencing was used to compare the DNA alkylating sites induced by the tetrazepinones, with those created by mitozolomide. Alkaline sucrose-density sedimentation was employed to detect genomic DNA damage. Also, the effects of the tetrazepinones on the cell cycle were determined by univariate flow cytometry. Results: At 3 h post-treatment, mitozolomide appeared as a selective inhibitor of DNA synthesis, while both tetrazepinones inhibited the synthesis of all three macromolecules. At 24 h post-treatment, the inhibition of DNA synthesis was observed to increase in cells treated with mitozolomide, while it decreased in those previously exposed to the tetrazepinones. Also at 24 h post-treatment, mitozolomide induced accumulation of cells in S(late)/G2M at low concentrations and in S-middle at high concentrations. In contrast, at the same recovery time, cells treated with the tetrazepinones accumulated specifically in G2M, the strength of the block being dose-dependent. At an equimolar concentration, the tetrazepinones induced weaker guanine N-7 alkylation than mitozolomide. By 24 h after treatment, cells exposed to the tetrazepinones showed significantly greater DNA fragmentation than those previously treated with mitozolomide. Conclusion: In summary, based on (a) their effects on DNA, RNA, protein synthesis and on the cell cycle, (b) their alkylating power and (c) their interactions with DNA, the 3-(2-chloroethyl)tetrazepinones appeared to kill tumor cells by a novel mechanism which may significantly differ from that of their 3-(2-chloroethyl)-tetrazinone counterpart, mitozolomide.

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. The results showed that (i) ZR2009 was 10-fold less potent than its positional isomer ZR2003 in EGFR tyrosine kinase inhibition, (ii) it consistently exhibited significantly weaker antiproliferative potency than ZR2003, (iii) in reversibility assays, while ZR2003 induced sustained inhibition of EGFR phosphorylation, ZR2009 inhibitory activity was partially reversed, and (iv) likewise, ZR2009 significantly lost its activity in short exposure growth inhibitory assays and induced lower levels of DNA damage than ZR2003. Molecular modeling suggested that while the chloroethylamino group in ZR2003 was at 3.5 A away from Cys773, that of ZR2009 was at 6.3 A. The results in toto suggest that, while the chloroethyl is a strong alkylating group, its appendage to the 6-position is optimal for DNA damage, sustained EGFR, and growth inhibition.

The mechanism of action of the novel heterocycle 3,5-dimethyl-pyrido-1,2,3,5-tetrazepin-4-one (PYRZ), structurally related to temozolomide, was studied in the human ovarian tumour cell line OVCAR-3. Our results showed that, despite its marked structural similarities to temozolomide, PYRZ presents properties that are atypical of 1,2,3-triazene-containing alkylating agents. In a Maxam-Gilbert DNA sequencing assay, PYRZ showed background levels of DNA alkylation, in contrast to temozolomide which strongly alkylated DNA preferentially at guanine residues. At high concentrations, PYRZ inhibited the synthesis of DNA, RNA and protein 3 h after treatment, in contrast to temozolomide which, in previous work, was found to preferentially inhibit DNA synthesis in OVCAR-3 cells. In cells exposed to PYRZ, alkaline sucrose density-gradient centrifugation showed a dose-dependent increase in DNA fragmentation only 12 and 24 h after treatment. PYRZ induced increasing accumulation of cells in late S and G2+M 6-24 h after treatment. This also contrasts with previous work that showed delayed cell cycle arrest induced by temozolomide in OVCAR-3 cells and in the murine leukaemia L1210 cells. Cell-killing kinetics by PYRZ showed a series of sigmoidal dose-response curves with 50-90% cell killing attained as early as 24 h after treatment in the 25-100 microM dose range. (IC50 clonogenic assay 18 microM). The results suggest that the mechanism of cell killing by PYRZ may be different from that of its parent drug temozolomide, and other alkyl-triazene-containing molecules of the same class. Images Figure 6

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. We used three cell lines expressing low levels (U87MG) or transfected to overexpress wildtype (U87/EGFR) or a variant (U87/EGFRvIII) of ErbB1. The results showed that Iressa±HN2 and the combi-molecules, ZRBA4 and ZR2003, significantly blocked ErbB1 phosphorylation in U87MG cells. Addition of OCT significantly altered cell cycle distribution. Analysis of the DNA damage response pathway revealed strong upregulation of p53 by HN2 and the combi-molecules. Apoptosis was only induced by a 48 h exposure to HN2. All other treatments resulted in cell necrosis. This is in agreement with Akt-Bad pathway activation and survivin upregulation. Despite strong DNA damaging properties and downregulation of ErbB1 phosphorylation by these molecules, the strongest effect of SSTR activation was on cell cycle distribution. Therefore, any enhanced antiproliferative effects of combining ErbB1 inhibition with SSTR activation must be addressed in the context of cell cycle arrest.

ZRF4, a combi-molecule with increased efficacy as compared with the individual components in chronic lymphocytic leukemia lymphocytes in vitro

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.

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.

RB24 (NSC 741279), a 3-methyltriazene termed “combi-molecule” designed to possess mixed epidermal growth factor receptor (EGFR) targeting and DNA methylating properties showed over a 100-fold greater antiproliferative activity than Temodal® (TEM), a 4-fold greater potency than gefitinib and a 5-fold stronger activity than an equi-effective combination of gefitinib + TEM against the O6-alkylguanine transferase (AGT)-proficient DU145 cell line that co-expresses EGFR. Investigation of the mechanisms underlying the unique potency of RB24 revealed that cell exposure to TEM was accompanied by activation of p38MAPK and concomitant elevation of the levels of X-ray repair cross-complementing group 1 (XRCC1) protein. Levels of phospho-p38MAPK and XRCC1 were increased by 2-fold in EGF-stimulated cells. In contrast, EGF-stimulation did not alter the status of these proteins in RB24-treated cells and this translated into a 2-fold lower level of XRCC1 when compared with those exposed to TEM + EGF. These effects correlated with significantly delayed DNA repair activity in combi-molecule-treated cells when compared with TEM-exposed ones. Further analysis demonstrated that in contrast to TEM, RB24 could block Bad phosphorylation at serine 136 in a dose-dependent manner and induced significantly higher levels of apoptosis than the former molecule. Tandem depletion of XRCC1 and Bad activation through alternative pathways using the MEK1 inhibitor, PD98059, led to substantial levels of apoptosis in RB24-treated cells. The results in toto indicate that the superior activity of the combi-molecule may be attributed to its ability to down-regulate DNA repair proteins such as XRCC1 and to alleviate anti-apoptotic signaling through blockade of EGFR-mediated signaling while inflicting high levels of DNA lesions to the cells.

The antiproliferative effect of tandem somatostatin receptor (SSTR) activation, epidermal growth factor receptor (EGFR) inhibition, and induction of DNA damage was analyzed using octreotide (OCT), a SSTR agonist, the clinical DNA methylating agent temozolomide (TMZ), Iressa, an EGFR inhibitor, and dual EGFR-DNA targeting agents termed “combi-molecules”. Using SSTR-expressing glioma cells harbouring low levels of EGFR (U87MG) or transfected to overexpress EGFR (U87/EGFR) or a variant (U87/EGFRvIII), we showed that Iressa, alone or in combination with the DNA damaging agent TMZ, and combi-molecules RA2 and RA5 inhibited EGF-induced phosphorylation of EGFR in U87MG and more moderately in U87/EGFR and U87/EGFRvIII transfected cells. This translated into equivalent levels of Erk 1/2 inhibition. Activation of SSTRs with OCT did not modulate the effects of the various treatments on Erk 1/2 phosphorylation. Likewise, SSTR activation did not alter TMZ- or DNA-damaging combi-molecules, RA2 and RA5, induced p53 activation nor upregulation. However, SSTR activation significantly shifted TMZ-, RA2- and RA5-induced cell-cycle arrest to earlier phases (i.e., G2/M to late S, late S to S, S to G1). Further analysis showed that apoptosis was not induced. This was in agreement with the fact that p53 activation did not induce Bax upregulation nor did EGFR inhibition promote Bad dephosphorylation. Moreover, enhancement of survivin, an anti-apoptotic protein, expression was observed. The results in toto suggest that the combination of SSTR activation with EGFR inhibition and DNA damage affects cell-cycle progression but a disconnection between the targeted signalling pathways in these brain tumour cells precludes synergistic cell-killing by the triple growth inhibitory events.

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.

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. 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.

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the “molar tooth sign” on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G(2)/M phase arrest, and NU7026 increases this CLB-induced G(2)/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as gammaH2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.

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 Student's 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.

The concept of synthetic lethality arises when the alteration of a single gene A or B alone does not affect cell survival but that of both genes lead to cell death. More importantly, if A is mutated, inhibition of the functions of the gene B product should also lead to cell death. This is achievable in some tumours, predominantly in breast and ovarian cancers, where the mutation of the DNA repair proteins BRCA1/2 depletes the DNA repair capacity of the cells. The compensatory DNA repair mechanism in the cells is supported by a DNA repair enzyme poly(ADP-ribose) polymerase (PARP), which when inhibited, leaves the cells with no compensatory DNA repair mechanisms. Hence DNA lesions accumulate in the cells and ultimately cause their death. The ability of PARP inhibitors to induce cell death in BRCA1/2 mutated tumours has been demonstrated in both preclinical models and in the clinic. While the PARP inhibitor olaparib met the requirements for recent FDA approval for the treatment of ovarian cancer, the overall survival obtained with many PARP inhibitors as single agents in patients with BRCA1,2 mutated tumours has been disappointing,. Also, acquired resistance caused by the reactivation of wild type BRCA1/2 is an emerging drawback in BRCA1/2 related therapies. Our hypothesis is that molecules termed “combi-molecules” capable of not only inhibiting PARP but also inducing DNA damage, could significantly enhance cell-killing in BRCA1/2 deficient tumours and perhaps be indicated at the earlier stages of the diseases. Here, we designed and studied the potency and mechanism of action of our first prototype EG22. The results showed that: (a) it inflicted higher levels of DNA damage than temozolomide (a clinical DNA damaging agent) and >100-fold stronger growth inhibitory potency than the latter in our cell panel, regardless of the BRCA1/2 status of the cells, (b) it showed more than 5-fold stronger growth inhibitory potency than 4-ANI, a known PARP inhibitor, in BRCA1/2 deficient cells and more than 8-fold in wild type expressing cells (c) a PARP assay showed that it induced a dose-dependent inhibition of PARP with an IC50 value almost equal to that of 4-ANI and (d) in cell-based selectivity assay involving a pair of isogenic Chinese hamster cell line (engineered to express BRCA2 wild type), it induced 20-fold selectivity for the mutant form. The results in toto suggest that EG22 is a new molecular entity with a novel and dual mechanism of action. Furthermore, reactivation of BRCA1/2 being one of the mechanisms of resistance to DNA damage-based therapy of patients with mutant BRCA1/2, the combi-molecular approach has the potential to be developed as an alternative treatment, when the tumours heterogeneously express BRCA1/2. Citation Format: Zhor Senhaji Mouhri, Elliot Goodfellow, Bertrand Jean-Claude. Targeting PARP and DNA as a novel unimolecular combination model for the enhancement of chemosensitivity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C185.

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

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 chromatographymass 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 (C max = 50 μmol/l, after 30 min), a 6-aminoquinazoline that was N-acetylated to give FD105Ac (IAc) (C max =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 C max .