RESUMEN
The synthesis of two series of novel substituted pyrimidine derivatives bearing a sulfamide group have been described and their in vitro cancer growth inhibition activities have been evaluated against three human tumour cell lines (HT-29, M21, and MCF7). In general, growth inhibition activity has been enhanced by the introduction of a bulky substituent on the aromatic ring with the best compound having GI50<6µM for all the human tumour cell lines. The MCF7 selective compounds were evaluated on four additional human invasive breast ductal carcinoma cell lines (MDA-MB-231, MDA-MB-468, SKBR3, and T47D) and were selective against T47D cell line in all cases except one, suggesting a potential antiestrogen activity.
Asunto(s)
Antineoplásicos/farmacología , Pirimidinas/farmacología , Sulfonamidas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
Islet amyloid polypeptide (IAPP) has been shown to form amyloid deposits in pancreatic islets, thereby furthering type 2 diabetes disease progression. Further discovery of new molecules is needed to create a diverse set of molecules that impede pancreatic amyloidosis. We have recently designed and synthesized N-phenyl-N'-(2-ethyl)ureas (EU) that are non-cytotoxic small molecules, to evaluate the role of the aryl-substituted moiety on the inhibition of hIAPP fibrillization. Several EUs were tested in vitro for their anti-amyloidogenic activity using the fluorometric ThT assay, the photo-induced cross-linking (PIUCP) assay, and cell survival assay in pancreatic MIN-6 cells. EU-362 and EU-418 were able to significantly inhibit the formation of hIAPP fibrils and protected cells from amyloid cytotoxic effects. Our results suggest that increasing the nucleophilic potency of the aryl moiety significantly enhances the anti-amyloidogenic activity of the molecules.
Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/antagonistas & inhibidores , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Urea/análogos & derivados , Amiloide/metabolismo , Amiloidosis/tratamiento farmacológico , Amiloidosis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/efectos adversos , Islotes Pancreáticos/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Urea/farmacologíaRESUMEN
Phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) are a new family of antimitotic prodrugs bioactivated in breast cancer cells expressing CYP1A1. In this study, we report that the 14C-labeled prototypical PAIB-SO [14C]CEU-818 and its antimitotic counterpart [14C]CEU-602 are distributed in whole mouse body and they show a short half-life in mice. To circumvent this limitation, we evaluated the effect of the homologation of the alkyl side chain of the imidazolidin-2-one moiety of PAIB-SOs. Our studies evidence that PAIB-SOs bearing an n-pentyl side chain exhibit antiproliferative activity in the nanomolar-to-low-micromolar range and a high selectivity toward CYP1A1-positive breast cancer cells. Moreover, the most potent n-pentyl PAIB-SOs were significantly more stable toward rodent liver microsomes. In addition, PAIB-SOs 10 and 14 show significant antitumor activity and low toxicity in chorioallantoic membrane (CAM) assay. Our study confirms that homologation is a suitable approach to improve the rodent hepatic stability of PAIB-SOs.
Asunto(s)
Antimitóticos , Neoplasias , Profármacos , Ratones , Animales , Antimitóticos/química , Profármacos/química , Citocromo P-450 CYP1A1 , Roedores , Microsomas Hepáticos , Bencenosulfonatos/químicaRESUMEN
We recently discovered a new family of prodrugs deriving from phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) bioactivatable by cytochrome P450 1A1 (CYP1A1) into potent antimitotics referred to as phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs). PAIB-SOs display significant selectivity toward human breast cancer cells based on the N-dealkylation of PAIB-SOs into their corresponding PIB-SOs by CYP1A1. In this study, we have evaluated the molecular mechanism of the bioactivation of PAIB-SOs into PIB-SOs by branching the linear alkyl chain on the imidazolidin-2-one (IMZ) moiety of PAIB-SOs by branched alkyl groups such as isopropyl, isobutyl and sec-butyl. Our results show that PAIB-SOs bearing an isobutyl group on the IMZ moiety and either a methoxy, a chloro or a bromo group at positions 3, 3,5 or 3,4,5 on the aromatic ring B exhibit antiproliferative activity ranging from 0.13 to 6.9 µM and selectivity toward MCF7 and MDA-MB-468 mammary cancer cells comparatively to other cell lines tested. Moreover, the most potent and selective PAIB-SOs bearing an isobutyl group and either a 3,5-Cl (44), 3,5-Br (45) or a 3,4,5-OMe (46) on the IMZ moiety exhibit antiproliferative activity in the sub-micromolar range and high selectivity ratios toward mammary cancer cells. They stop the cell cycle of MCF7 cells in the G2/M phase and disrupt their cytoskeleton. Furthermore, our studies evidenced that PAIB-SOs bearing either an isopropyl, a sec-butyl or an isobutyl group are hydroxylated on the carbon atom adjacent to the IMZ (Cα-OH) but only PAIB-SOs bearing an isobutyl group are bioactivated into PIB-SOs. Finally, PAIB-SOs 45 and 46 exhibit low toxicity toward normal cells and chick embryos and are thus promising antimitotic prodrugs highly selective toward CYP1A1-expressing breast cancer cells.
Asunto(s)
Antimitóticos/química , Bencenosulfonatos/química , Citocromo P-450 CYP1A1/metabolismo , Profármacos/química , Animales , Antimitóticos/síntesis química , Antimitóticos/farmacología , Bencenosulfonatos/síntesis química , Bencenosulfonatos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Pollos , Citocromo P-450 CYP1A1/química , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Semivida , Humanos , Microsomas Hepáticos/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Profármacos/farmacocinética , Profármacos/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) are antimicrotubule agents interacting covalently with ß-tubulin near the colchicine-binding site (C-BS). Glutamyl 198 residue in ß-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic residues, Cys239 and Cys354, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). By use of mass spectrometry, we have now identified residues in ß-tubulin that have become modified irreversibly by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU), and N,N'-ethylenebis(iodoacetamide) (EBI). The binding of HPCEU and 4ZCombCEU to ß-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs then were used as molecular probes to assess, in mouse B16F0 and human MDA-MB-231 cells, the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU, and 4ZCombCEU on the acetylation of Lys40 on α-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation, as observed also with other microtubule depolymerizing agents. Therefore, CEU affects the stability and the dynamics of microtubule, likewise a E198G mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular cross-link between Cys239 and Cys354 of ß-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/function and the biological activity of several cellular proteins under physiological conditions.
Asunto(s)
Antineoplásicos/metabolismo , Microtúbulos/química , Moduladores de Tubulina/metabolismo , Tubulina (Proteína)/metabolismo , Urea/análogos & derivados , Acetilación , Acilación , Animales , Células Cultivadas , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Relación Estructura-Actividad , Tubulina (Proteína)/químicaRESUMEN
Triple-negative breast cancer (TNBC) is a major concern among the different subtypes of breast cancer (BC) due to the lack of effective treatment. In a previous study by our group aimed at understanding the difference between TNBC and non-TNBC tumors, we identified the gene TBC1 domain family member 9 (TBC1D9), the expression of which was lower in TNBC as compared to non-TNBC tumors. In the present study, analysis of TBC1D9 expression in TNBC (n = 58) and non-TNBC (n = 25) patient tumor samples validated that TBC1D9 expression can differentiate TNBC (low) from non-TNBC (high) samples and that expression of TBC1D9 was inversely correlated with grade and proliferative index. Moreover, we found that downregulation of the TBC1D9 gene decreases the proliferation marginally in non-TNBC and was associated with increased migratory and tumorigenic potential in both TNBC and luminal BC cell lines. This increase was mediated by the upregulation of ARL8A, ARL8B, PLK1, HIF1α, STAT3, and SPP1 expression in TBC1D9 knockdown cells. Our results suggest that TBC1D9 expression might limit tumor aggressiveness and that it has a differential expression in TNBC vs. non-TNBC tumors.
RESUMEN
Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide) (EBI) to crosslink in living cells the cysteine residues at position 239 and 354 of ß-tubulin, residues which are involved in the colchicine-binding site. The ß-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of ß-tubulin that migrates faster than ß-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: ß-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.
RESUMEN
PURPOSE: We investigated the involvement of MAPK signaling in the cell death mechanisms of classical microtubule interfering agents (MIA) and aryl-3-(2-chloroethyl)ureas (CEU) acting as antimitotics, along with CEU that don't affect directly microtubules (non-MIA CEU). METHODS: To ascertain the activated signaling pathway profile of MIA and non-MIA CEU, Western blot, immunoprecipitation and transfection experiments were performed. RESULTS: Non-MIA CEU do not activate p38, as opposed to MIA, and the extent of ERK and JNK activation is lower than in response to MIA. The effect of MIA and non-MIA CEU on focal adhesion associated protein was also studied; MIA were shown to induce focal adhesion dismantlement associated with a sustained increase in paxillin phosphorylation and FAK cleavage, as opposed to non-MIA CEU. In addition, bcl-2 phosphorylation and AKT cleavage, induced by all MIA tested, was not observed in response to non-MIA CEU further emphasizing the differential cell death mechanisms induced by MIA and non-MIA CEU. Pharmacologic and genetic approaches emphasize that the ASK1-p38 pathway activation contributes to the cytotoxic mechanism of MIA, in contrast to non-MIA CEU. ASK1-p38 is important for increased paxillin phosphorylation and FAK cleavage, suggesting that ASK-1-p38 is an upstream event of FA structure dismantlement induced by MIA. Moreover, the endogen inhibitor of ASK-1, thioredoxin, is released from ASK-1 in response to MIA as opposed to non-MIA CEU. CONCLUSION: Our study supports that ASK1-p38 activation is an important signaling event, induced by MIA, which impairs focal adhesion structure and induces anchorage-dependent apoptosis or anoikis.
Asunto(s)
Antineoplásicos/farmacología , MAP Quinasa Quinasa Quinasa 5/metabolismo , Moduladores de Tubulina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anoicis/efectos de los fármacos , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Línea Celular Tumoral , Adhesiones Focales/metabolismo , Humanos , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transfección , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Aryl chloroethyl ureas (CEUs) are new protein alkylating agents exhibiting anticancer activity both in vitro and in vivo. We report herein that 14C-labeled CEU derivatives, designated CEU-025 and CEU-027, covalently bind to thioredoxin-1 (TRX1). Covalent binding of these molecules slightly decreases the disulfide-reducing activity of recombinant TRX1, when compared with the effect of strong thioalkylating agents such as N-ethylmaleimide. Moreover, site-directed mutagenesis and diamide competition assays demonstrated that TRX1 cysteinyl residues are not the prime targets of CEUs. CEU-025 abrogates the nuclear translocation of TRX1 in human cancer cells. In addition, we show that CEU-025 can block TRX1 nuclear translocation induced by cisplatin. Unexpectedly, pretreatment with sublethal CEU-025 concentrations that block TRX1 nuclear translocation protected the cells against cisplatin cytotoxicity. Overexpression of TRX1 in HT1080 fibrosarcoma cells attenuated CEU-025 cytotoxicity, while its suppression using TRX1-specific siRNA increased the effects of CEU-025, suggesting that loss of function of TRX1 is involved, at least in part, in the cytotoxic activity of CEU-025. These results suggest that CEU-025 and CEU-027 exhibit anticancer activity through a novel, unique mechanism of action. The importance of TRX1 and the dependence of the cytotoxicity of CEU-025 and CEU-027 on TRX1 intracellular localization are also discussed.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Tiorredoxinas/metabolismo , Urea/análogos & derivados , Urea/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Catálisis , Línea Celular Tumoral , Cisplatino/farmacología , Humanos , Transporte de Proteínas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
OBJECTIVES: In this study, the antiproliferative activity of 3 phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) was assessed in a time-dependent manner together with their hepatic stability and metabolism using human, mouse and rat liver microsomes. METHODS: CEU-818, -820 and -913 were selected as promising hit compounds. Their antiproliferative activity on human breast carcinoma MCF-7 cells was evaluated using escalating concentrations of drugs at 24, 36 and 48 h and the sulforhodamine B assay. Their hepatic stability was evaluated by HPLC-UV of extracts obtained from human, mouse and rat liver microsomes. KEY FINDINGS: The antiproliferative activity of PAIB-SOs is concentration and time-dependent and requires between 24 and 36 h of contact with MCF-7 cells to detect a significant antiproliferative activity. PAIB-SOs stability in microsomes usually decreases following this order: human ≈ (rat > mouse). The CEU-913 exhibits the longest half-life in rat and human liver microsomes while the CEU-820 exhibits the longest half-life in mouse liver microsomes. CONCLUSIONS: Our in vitro results suggest that PAIB-SOs should have a minimum contact time of 24 h with the tumour to trigger significant antitumoural activity. The activity of mouse liver microsomes towards PAIB-SOs is higher than rat microsomes and tends to be higher than human liver microsomes.
Asunto(s)
Antineoplásicos/farmacología , Bencenosulfonatos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Microsomas Hepáticos/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Bencenosulfonatos/administración & dosificación , Bencenosulfonatos/química , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Femenino , Semivida , Humanos , Células MCF-7 , Ratones , Profármacos , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Factores de TiempoRESUMEN
Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure-activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N'-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on beta(II)-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The FlexiDock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-beta198 rather than Cys-beta239. In addition, these studies suggest that Cys-beta239 together with Asn-alpha99, Ser-alpha176, Thr-alpha177, Leu-beta246, Asn-beta247, Ala-beta248, Lys-beta252 and Asn-beta256 are implicated in the stabilization of a C-BS-CEU complex prior to the acylation of Glu-beta198 by CEU. Our molecular models propose the formation of a stabilized C-BS-CEU complex before the completion of the Glu-beta198 acylation; acylation triggering conformational changes of beta-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin.
Asunto(s)
Colchicina/metabolismo , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Urea/análogos & derivados , Sitios de Unión , Línea Celular Tumoral , Colchicina/química , Simulación por Computador , Humanos , Modelos Químicos , Modelos Moleculares , Relación Estructura-Actividad , Tubulina (Proteína)/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacología , Urea/química , Urea/farmacologíaRESUMEN
Chloroethylureas (CEU) are soft alkylating agents that covalently bind to beta-tubulin (betaTAC) and affect microtubule polymerization dynamics. Herein, we report the identification of a CEU subset and its corresponding oxazolines, which induce cell growth inhibition, apoptosis, and microtubule disruption without alkylating beta-tubulin (N-betaTAC). Both betaTAC and N-betaTAC trigger the collapse of mitochondrial potential (DeltaPsi(m)) and modulate reactive oxygen species levels, following activation of intrinsic caspase-8 and caspase-9. Experiments using human fibrosarcoma HT1080 respiratory-deficient cells (rho(0)) and uncoupler of the mitochondrial respiratory chain (MRC) showed that betaTAC and N-betaTAC impaired the MRC. rho(0) cells displayed an increased sensitivity toward N-betaTAC as compared with rho(+) cells but, in contrast, were resistant to betaTAC or classic chemotherapeutics, such as paclitaxel. Oxazoline-195 (OXA-195), an N-betaTAC derivative, triggered massive swelling of isolated mitochondria. This effect was insensitive to cyclosporin A and to Bcl-2 addition. In contrast, adenine nucleotide translocator (ANT) antagonists, bongkrekic acid or atractyloside, diminished swelling induced by OXA-195. The antiproliferative activities of the N-betaTACs CEU-025 and OXA-152 were markedly decreased in the presence of atractyloside. Conversely, pretreatment with cyclosporin A enhanced growth inhibition induced by betaTAC and N-betaTAC. One of the proteins alkylated by N-betaTAC was identified as the voltage-dependent anion channel isoform-1, an ANT partner. Our results suggest that betaTAC and N-betaTAC, despite their common ability to affect the microtubule network, trigger different cytotoxic mechanisms in cancer cells. The role of mitochondria in these mechanisms and the potential of N-betaTAC as a new therapeutic approach for targeting hypoxia-resistant cells are discussed.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Hipoxia de la Célula , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Compuestos de Fenilurea/uso terapéutico , Animales , Antineoplásicos Alquilantes/química , Células CHO , Cricetinae , Cricetulus , Ensayos de Selección de Medicamentos Antitumorales , Transporte de Electrón/efectos de los fármacos , Células HT29 , Humanos , Microtúbulos/efectos de los fármacos , Modelos Biológicos , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Fase G1/efectos de los fármacos , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Tiorredoxinas/metabolismo , Urea/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Estereoisomerismo , Urea/análogos & derivados , Urea/químicaRESUMEN
Recently, a subset of N-phenyl-N'-(2-chloroethyl)ureas (CEU) was found abrogating the nuclear translocation of thioredoxin-1 and arresting the cell cycle in G(0)/G(1) phase. Several derivatives were prepared to assess their effect on cell cycle progression and on the intracellular location of Trx-1. Compounds 1-20, 21-40, and 41-60 exhibited GI(50) between 1 and 80 microM. Immunocytochemistry analysis showed compounds 4, 6, 8, 10, 11, 23, 24, 26-31, 34, 37, 41, 44, 46-51, 53, 56, and 57 inhibiting the nuclear translocation of Trx-1. Our results suggest that increasing the electrophilic character of these molecules might enhance the antiproliferative activity at the expense of the selectivity toward thioredoxin-1 and the G(0)/G(1) phase arrest.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Antineoplásicos/química , Antineoplásicos/farmacología , Tiorredoxinas/metabolismo , Urea/análogos & derivados , Urea/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Estructura Molecular , Relación Estructura-Actividad , Urea/químicaRESUMEN
In the course of the development of N-phenyl-N'-(2-chloroethyl)ureas (CEUs) as potential antineoplastic agents, we investigated the effect of carbonylated substituting chains of the aromatic ring of CEU on their covalent binding to the colchicine-binding site (C-BS). In this study, we found that CEU, 5e, 5f, 8e, and 8f substituted by either a methyl ester or a methyl ketyl group at the omega-position exhibited a significant antiproliferative activity on HT-29, M21, and MCF-7 tumor cells. SDS-PAGE assays and cell cycle analysis confirmed that 5e, 5f, 8e, and 8f covalently bind to the C-BS and arrest the cell division in G(2)/M phase. Surprisingly, the presence of omega-carboxyl, omega-ethyl esters or omega-amides decreased significantly both the antiproliferative activity and the specificity toward beta-tubulin.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Ácido Carbónico/química , Compuestos de Cloro/química , Colchicina/química , Urea/análogos & derivados , Urea/farmacología , Alquilación , Antineoplásicos/síntesis química , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Compuestos de Cloro/síntesis química , Compuestos de Cloro/farmacología , ADN/genética , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismo , Urea/síntesis química , Urea/químicaRESUMEN
To decipher the mechanism underlying the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas (CEU) to the colchicine-binding site on beta(II)-tubulin and to design new and selective antimitotic drugs, we developed 3D quantitative structure-activity relationships (3D-QSAR) models using CoMFA and CoMSIA analyses. The present study correlates the cell growth inhibition activities of 56 structurally related CEU derivatives to several physicochemical parameters representing steric, electrostatic, and hydrophobic fields. Both CoMFA and CoMSIA models using two different optimum numbers of components (ONC) 10 and 4, respectively, gave good internal predictions and their cross-validated r2 values were between 0.639 and 0.743. These comprehensive CoMFA and CoMSIA models are useful in understanding the structure-activity relationships of CEU. The two models were compared to the X-ray crystal structure of the complex of tubulin-colchicine and analyzed for similarities between the two modes of analysis. These models will inspire the design of new CEU derivatives with enhanced inhibition of tumor cell growth and targeting specificity of beta(II)-tubulin and the cytoskeleton.
Asunto(s)
Antimitóticos/química , Relación Estructura-Actividad Cuantitativa , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Urea/análogos & derivados , Sitios de Unión , Colchicina , Diseño de Fármacos , Humanos , Modelos MolecularesRESUMEN
Aryl chloroethylureas (CEUs) are potent anti-neoplastic agents alkylating specific intracellular proteins such as beta(II)-tubulin. Recently we have identified a new subset of CEU derived from compound 36 that alkylates thioredoxin isoform 1 (Trx-1), inhibits the nuclear translocation of Trx-1, and favors the accumulation of cells in G(0)/G(1) phase. We have evaluated the effects of various substituents and their position on the aromatic ring of a series of derivatives of 36 on (i) the anti-proliferative activity, (ii) the cell cycle progression, (iii) the nuclear translocation of Trx-1, and (iv) their covalent binding to beta-tubulin. The same experiments were performed on representative CEU derivatives where the 2-chloroethyl amino moiety is replaced by either an ethyl, a 2-aminooxazolinyl or a 2-chloroacetyl group. On one hand, our results suggest that CEUs substituted on the phenyl ring at position 3 or 4 by cycloalkyl and substituted cycloalkyl or cycloalkoxy groups inhibit the nuclear translocation of Trx-1 and arrest the cell cycle progression in G(0)/G(1). On the other hand, CEUs substituted by a fused aromatic ring, an aliphatic chain, or a fused aliphatic ring are alkylating beta(II)-tubulin but not Trx-1. Beside the expected inactivity of the ethylurea derivatives, none of the modification to the electrophilic moiety led to cross-selectivity of the drugs toward beta-tubulin but increased the anti-proliferative activity and resulted in mitigated effects on Trx-1 translocation.
Asunto(s)
Microtúbulos/metabolismo , Tiorredoxinas/metabolismo , Tubulina (Proteína)/metabolismo , Urea/análogos & derivados , Urea/química , Alquilación , Antineoplásicos/química , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Estructura Molecular , Oxidación-Reducción , Relación Estructura-Actividad , Tubulina (Proteína)/química , Urea/farmacologíaRESUMEN
We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylureas (CEU) with three different model membranes by (31)P and (2)H solid-state NMR spectroscopy. First, we have prepared model membranes that mimic the mitochondrial inner (Mito IM) and outer (Mito OM) membranes and the endoplasmic reticulum membrane (End Ret). Our results indicate that the effects of the CEU derivatives on lipid bilayers are related to their cytotoxic activity. More specifically, a strong correlation is observed between the drug location in both the mitochondrial inner and outer membranes and its cytotoxicity. In addition, the results indicate that the lipid composition of the model membrane has a very important influence on the effects of CEUs. More specifically, a high proportion of cardiolipin in the mitochondrial inner membrane gives this system the highest fluidity and consequently, this model membrane is more rigidified by the presence of CEUs compared to the mitochondrial outer and endoplasmic reticulum membranes. Finally, the results propound a hypothesis for the location of CEUs in membranes.
Asunto(s)
Antineoplásicos/química , Membrana Dobles de Lípidos , Espectroscopía de Resonancia Magnética/métodos , Urea/químicaRESUMEN
Prodrug-mediated utilization of the cytochrome P450 (CYP) 1A1 to obtain the selective release of potent anticancer products within cancer tissues is a promising approach in chemotherapy. We herein report the rationale, preparation, biological evaluation, and mechanism of action of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) that are antimicrotubule prodrugs activated by CYP1A1. Although PAIB-SOs are inert in most cells tested, they are highly cytocidal toward several human breast cancer cells, including hormone-independent and chemoresistant types. PAIB-SOs are N-dealkylated into cytotoxic phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) in CYP1A1-positive cancer cells, both in vitro and in vivo. In conclusion, PAIB-SOs are novel chemotherapeutic prodrugs with no equivalent among current antineoplastics and whose selective action toward breast cancer is tailored to the characteristic pattern of CYP1A1 expression observed in a large percentage of human breast tumors.
Asunto(s)
Antimitóticos/farmacología , Bencenosulfonatos/química , Neoplasias de la Mama/tratamiento farmacológico , Citocromo P-450 CYP1A1/metabolismo , Profármacos/farmacología , Animales , Antimitóticos/farmacocinética , Bencenosulfonatos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Embrión de Pollo , Citocromo P-450 CYP1A1/genética , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Profármacos/farmacocinéticaRESUMEN
The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community. Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules. Here, we present results supporting our assumption that 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-[3-(2-chloroethyl)ureido]phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo. They covalently bind to beta-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology. Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner. It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay. In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs. On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.