RESUMEN
Here, we report on the in vitro binding properties of the known pyridine dicarboxamide G-quadruplex ligand 360A and a new dimeric analogue (360A)2A to human telomeric DNA higher-order G-quadruplex (G4) structures. This study points to original binding features never reported for G4 ligands, and reveals a greater efficiency for the dimeric ligand to displace RPA (a ssDNA binding protein involved in telomere replication) from telomeric DNA.
RESUMEN
The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC(50) values of 18-100 nM in a standard TRAP assay.
Asunto(s)
ADN/química , Etidio/química , Conformación de Ácido Nucleico , Telomerasa/antagonistas & inhibidores , ADN/genética , Colorantes Fluorescentes/química , Guanina/química , Humanos , Oligonucleótidos/química , Oligonucleótidos/genética , Espectrometría de Fluorescencia , Telomerasa/genética , Telomerasa/metabolismo , Telómero/enzimología , Telómero/genéticaRESUMEN
Telomerase offers the potential opportunity to control cell proliferation by interfering with a totally new and unique biological process which is cell senescence. The aim of this review is to impartially present the state of the art in telomerase with the pros and the cons of the current scientific situation of this fast-growing and fascinating topic for answering the key question asked by experimental and medical oncologists: Will telomerase be a therapeutic target for the third millenium? The most convincing argument (which is a scientifically documented one) for going ahead with this target is obviously the strong correlation existing between the level and frequency of telomerase expression and the malignant properties of tumors. This has been now largely documented in established tumor cell lines and fresh tumor samples obtained from patients. Noteworthy is the very important difference of telomerase expression between malignant and normal tissues. This difference is much higher than those observed for classical enzymatic targets of chemotherapy such as thymidylate synthetase, dihydrofolate reductase and topoisomerases. If this translates to the clinical situation, telomerase inhibitors might display a good selectivity for tumor cells with a minimal toxicity for normal tissues. The most appealing criticism (which is still purely speculative) is obviously the clinical relevance of inhibiting telomerase in cancer patients. According to the paradigm currently proposed for telomeres and telomerases, it can be predicted that telomerase inhibition will not affect a tumor until its telomeres reach the critical size for entering senescence. This means that during anti-telomerase therapy, the tumor cells will continue grow undergoing 20-30 divisions until the telomeres reach a critical size leading to tumor senescence. Does this make sense, especially in patients with advanced tumors at the beginning of the therapy? Ultimately, the definitive answer to the question will not come from intellectual speculation but from the properties of telomerase inhibitors, first in tumor bearing animals, then finally in cancer patients! Several institutions are very active in the development of telomerase inhibitors. Different stategies are used: direct inhibition of telomerase, interference with telomeres (G quartets), interaction with other proteins involved in the regulation of telomerase and telomeres.
Asunto(s)
Antineoplásicos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Telomerasa/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Predicción , Humanos , Estructura Molecular , Telomerasa/genética , Telomerasa/metabolismo , Telómero/química , Telómero/metabolismoRESUMEN
To test the Kirsten-Ras (Ki-Ras) alternative prenylation hypothesis in malignant transformation, we used a novel farnesyltransferase inhibitor competitive to farnesyl-pyrophosphate, RPR130401, and a CaaX peptidomimetic geranylgeranyltransferase-1 inhibitor GGTI-298. In Ki-Ras-overexpressing transformed adrenocortical cells, RPR130401 at 1-10 microM inhibited very efficiently the [(3)H]farnesyl but not [(3)H]geranylgeranyl transfer to Ras. However, proliferation of these cells was only slightly sensitive to RPR130401 (IC(50)=30 microM). GGTI-298 inhibited the growth of these cells with an IC(50) of 11 microM but cell lysis was observed at 15 microM. The combination of 10 microM RPR130401 and 10 microM GGTI-298 inhibited efficiently (80%) cell proliferation. These combined inhibitors but not each inhibitor alone blocked the cell cycle in G(0)/G(1) and disrupted MAP kinase activation. Thus, combination of two inhibitors, at non-cytotoxic concentrations, acting on the farnesyl-pyrophosphate binding site of the farnesyltransferase and the CaaX binding site of the geranylgeranyltransferase-1 respectively is an efficient strategy for disrupting Ki-Ras tumorigenic cell proliferation.
Asunto(s)
Benzamidas/farmacología , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Genes ras/efectos de los fármacos , Indoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fase S/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Transferasas Alquil y Aril/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Línea Celular Transformada , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Farnesiltransferasa , Citometría de Flujo , Lovastatina/farmacología , Prenilación de Proteína/efectos de los fármacos , Ratas , Simvastatina/farmacología , Factores de Tiempo , Proteínas ras/metabolismoRESUMEN
We have investigated the combined use of partial least squares (PLS) and statistical design principles in principal property space (PP-space), derived from principal component analysis (PCA), to analyze farnesyltransferase inhibitors in order to identify "activity trends" (an approach we call a "directional" approach) and quantitative structure-activity relationships (QSAR) for a congeneric series of inhibitors: the benzo[f]perhydroisoindole (BPHI) series. Trends observed in the PCA showed that the descriptors used were relevant to describe our structural data set by clearly identifying two well-defined structural subclasses of inhibitors. D-Optimal design techniques allowed us to define a training set for PLS study in PP-space. Models were derived for each biological assay under evaluation: the in vitro Ki-Ras and cellular HCT116 tests. Each of these assay-based sets was subdivided once more into two subsets according to two structural classes in this BPHI series as revealed by the PCA model. The response surface modeling (RSM) methodology was used for each subset, and the corresponding RSM plots helped us identify "activity trends" exploited to guide further analogue design. For more precise activity predictions more refined PLS models on constrained PP-spaces were developed for each subset. This approach was validated with predicted sets and demonstrates that useful information can be extracted from just a few very informative and representative compounds. Finally, we also showed the potential use of such a strategy at an early stage of an optimization process to extract the first "activity trends" that might support decision making and guide medicinal chemists in the initial design of new analogues and/or lead followup libraries.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Transferasas Alquil y Aril/química , Inteligencia Artificial , Simulación por Computador , Bases de Datos como Asunto , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Análisis de los Mínimos Cuadrados , Modelos Químicos , Análisis Multivariante , Relación Estructura-Actividad , Proteínas ras/químicaRESUMEN
With the aim of obtaining new antitumoral agents, a series of 5,8-quinazolinediones was prepared. 5-Amino-6-methoxyquinazoline was oxidized by Fremy's salt to give 6-methoxy-5,8-quinazolinedione. Nucleophilic substitution reaction at C6, electrophilic substitution at C7, and synthesis of 7-amino-6-methoxy-5,8-quinazolinedione, the parent compound of streptonigrin, were studied. These compounds were tested for cytotoxic properties on L1210 leukemia cells in vitro. One of them, 6,7-bis(1-aziridinyl)-5,8-quinazolinedione, which exhibits a high cytotoxic activity (ID50 = 0.08 microM), was further screened in standard antitumor systems, including L1210 leukemia, P388 lymphocytic leukemia, sarcoma 180, and B16 melanocarcinoma. This drug gives a significant antitumoral effect on P388 leukemia but is inactive on other experimental models. Moreover, this compound was found to be highly mutagenic for Salmonella typhimurium TA98 and TA100 strains (Ames test), suggesting that DNA damage could be responsible for its cytotoxicity.
Asunto(s)
Antineoplásicos/síntesis química , Quinazolinas/síntesis química , Animales , Antineoplásicos/uso terapéutico , Leucemia L1210/tratamiento farmacológico , Leucemia P388/tratamiento farmacológico , Ratones , Pruebas de Mutagenicidad , Quinazolinas/uso terapéutico , Sarcoma 180/tratamiento farmacológicoRESUMEN
Potent inhibitors of EGF-dependent protein tyrosine kinase (PTK) activity were synthesized in a series of 5-[(2,5-dihydroxybenzyl)amino]salicylates. Several of these compounds inhibited EGF-dependent DNA synthesis in ER 22 cells with IC50 < 1 microM. In this series of PTK inhibitors, the role of the salicylate moiety as a potential divalent ion chelator was tested and found to be nonessential in all cases. The length and ramification of the substituting carboxyl group were investigated to improve cellular bioavailability, and this analysis provided compounds with increased inhibitory effect on EGF-induced DNA synthesis. Salicylates esterified with long hydrophobic chains were shown to be noncompetitive inhibitors of ATP, in contrast to the free acid and methyl salicylate. Moreover, all the tested inhibitors were shown to be noncompetitive inhibitors of the peptide substrate. Structure-activity relationships allowed us to suspect a hydrophobic pocket in the tyrosine kinase domain, preferentially interacting with aromatic rings. Finally, the selectivity of the best inhibitors was tested against other kinases, and they were found to be selective for tyrosine kinase. They were also shown to be good inhibitors of EGF-receptor autophosphorylation.
Asunto(s)
Compuestos de Bencilo/síntesis química , Compuestos de Bencilo/farmacología , Receptores ErbB/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Salicilatos/síntesis química , Salicilatos/farmacología , Animales , Compuestos de Bencilo/química , Ratas , Salicilatos/química , Relación Estructura-ActividadRESUMEN
Two synthetic routes to new oxygen-bridged analogues of farnesyltransferase inhibitors are described that follow either a [3 + 2]/[4 + 2] or a [4 + 2]/[3 + 2] sequence of reactions. The first approach has been achieved by reacting the in situ generated phenylisobenzofuran (PIBF) 4 with pyrroline 5a and has led stereoselectively to racemic 18, which was transformed in a few steps into the target molecule 2. The second pathway relies on a key intermediate 6, obtained either by condensation of PIBF with methyl acrylate, followed by a deprotonation/selenation and an oxidation/elimination sequence, or by cycloaddition between PIBF and alpha-phenylselenoacrylate 11, followed by the same oxidation/elimination sequence. The reaction of 6 with amino dipole 7 gives diastereoselective access to pyrrolidine 25, a precursor of the second target 3, an epimer of 2.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Hidrocarburos Aromáticos con Puentes/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Indoles , Hidrocarburos Aromáticos con Puentes/farmacología , Ciclización , Farnesiltransferasa , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Difracción de Rayos XRESUMEN
The cycloaddition of 1-phenylisobenzofuran (PIBF) with methyl acrylate yields, in a moderate endo/exo ratio, the expected oxa-bridged adduct, which can be deprotonated and condensed on diphenyl diselenide to provide, in a stereoconvergent step, the "endo" alpha-selenoester. Its "exo" epimer is obtained by reacting PIBF and methyl alpha-phenylselenoacrylate. These adducts can be oxidized to give a common unsaturated bridged ester that can react with an imminium ylide to provide the expected pyrrolidine stereoselectively.
RESUMEN
Human telomeres, which consist of repeated TTAGGG sequences, have recently become the focus of intense and highly competitive biological research. This scientific interest lies in their unique biological functions: telomeres are essential for genome integrity and appear to play an important role in cellular aging and cancer. As telomerase appears to be selectively expressed in tumors versus normal cells, this enzyme represents a good target for inhibition. Different types of telomerase inhibitors have recently been described. We will present briefly the different strategies that have been proposed to achieve efficient telomerase inhibition, with a special emphasis on G-quartet ligands.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Telomerasa/antagonistas & inhibidores , Guanina/química , Modelos Moleculares , Oligonucleótidos Antisentido/farmacología , ARN sin Sentido/farmacología , Moldes GenéticosRESUMEN
Telomeres of human chromosomes contain a G-rich 3'-overhang that adopts an intramolecular G-quadruplex structure in vitro which blocks the catalytic reaction of telomerase. Agents that stabilize G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalyzed by telomerase and can therefore act as antitumor agents. We have identified by Fluorescence Resonance Energy Transfer a new series of quinoline-based G-quadruplex ligands that also exhibit potent and specific anti-telomerase activity with IC50 in the nanomolar concentration range. Long term treatment of tumor cells at subapoptotic dosage induces a delayed growth arrest that depends on the initial telomere length. This growth arrest is associated with telomere erosion and the appearance of the senescent cell phenotype (large size and expression of beta-galactosidase activity). Our data show that a G-quadruplex interacting agent is able to impair telomerase function in a tumor cell thus providing a basis for the development of new anticancer agents.
Asunto(s)
Apoptosis , ADN , Telómero/efectos de los fármacos , Triazinas/farmacología , Línea Celular Transformada , Senescencia Celular , G-Cuádruplex , Humanos , Ligandos , Estructura Molecular , Telomerasa/metabolismo , Triazinas/química , Células Tumorales CultivadasRESUMEN
A series of platinum dichloroethylenediamine complexes [PtCl2(R-en)] bearing a side chain on one carbon atom of the ethylenediamine ligand, with or without a functional group on the side chain, have been prepared and investigated for antitumor activity against L1210 leukemia. They were tested both in vitro, with cisplatin-sensitive and resistant cell lines, and in vivo, with cisplatin-sensitive and resistant tumors grafted i.p. in B6D2F1 mice. The rationale for this study was to test how charge, polarity and shape of the R side chain influence antitumor activity. Complexes carrying one or more ammonium groups on the side chain were all inactive. Derivatives with a carbamate function attached by the nitrogen atom, via a methylene group, to the ethylenediamine moiety ('N-bound' carbamate) were highly active in vitro and in vivo. The best results were obtained with these carbamates bearing hydrophobic substituents of intermediate size. Replacement of N-bound by O-bound carbamate or by urea groups led to decreased in vivo activity. Sulfonamide derivatives were all inactive. Good to excellent activities were also recorded for complexes bearing bulky bicycloalkyl substituents, without any functional group, attached to one ethylenediamine carbon atom. Thus, it is the steric features of the side chain rather than its polarity that appear to favor the antitumor activity of the complex. Compared to cisplatin and oxaliplatin, the present complexes do not exhibit advantages in terms of experimental antitumor activities in solid tumor models.
Asunto(s)
Antineoplásicos/síntesis química , Compuestos Organoplatinos/síntesis química , Animales , Antineoplásicos/farmacología , Carbamatos/química , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Técnicas In Vitro , Leucemia L1210 , Masculino , Ratones , Ratones Endogámicos , Compuestos Organoplatinos/farmacología , Oxaliplatino , Compuestos de Amonio Cuaternario/química , Relación Estructura-Actividad , Sulfonamidas/química , Células Tumorales CultivadasRESUMEN
In order to obtain non-degradable and more potent protein-tyrosine kinase inhibitors, derived from the 5-(2,5-dihydroxybenzyl)-aminosalicylates already described, we have developed a new series of 5-(2,5-dihydroxybenzyl)phenylamines. The compounds, diversely substituted on the phenyl ring by alcohol, nitrile, ether, ketone, amide and thioamide groups, were tested for their ability to inhibit epidermal growth factor (EGF) receptor-associated tyrosine kinase activity in vitro. They inhibit the phosphorylation of the peptide substrate RR-Src by the EGF receptor purified from ER 22 cells, with IC50 values in the range 0.02-0.45 microns. Several of these compounds inhibit EGF-dependent DNA synthesis in ER 22 cells with IC50 values of around 1 micron and furthermore their inhibition has been found to be specific for various protein kinases.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Compuestos de Anilina/síntesis química , Compuestos de Anilina/farmacología , Animales , División Celular/efectos de los fármacos , Membrana Celular/enzimología , Cricetinae , ADN/biosíntesis , Inhibidores de Crecimiento/síntesis química , Inhibidores de Crecimiento/química , Humanos , Cinética , Relación Estructura-Actividad , TransfecciónRESUMEN
The interactions of the Escherichia coli endonuclease UvrAB proteins with the DNA mono- and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatin um(II) (complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with DNA than cis-DDP and gives monoadducts with a longer lifetime (8 h 20 min chelation t 1/2 compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [3H]DNA, the filter binding assay was associated with the analysis of the nucleoprotein complexes to characterize the UvrAB recognition of the platinum adducts and to demonstrate the occurrence of platinum-mediated DNA-protein cross-linking. First, it is shown that the UvrAB proteins recognize the complex 1 mono- and diadducts with a higher affinity than those of cis-DDP. Fifteen times more cis-DDP adducts per plasmid are required than complex 1 adducts, to lead to similar UvrAB binding. However, the UvrAB proteins recognize monoadducts and diadducts of each complex with a similar affinity. Second, it is shown that UvrB is the protein involved in the nucleo-protein complexes formed from mono- and diadducts of complex 1 and cis-DDP. This protein is also partly cross-linked to DNA with a similar efficiency by monoadducts derived from complex 1 and cis-DDP. However, as UvrB has a greater affinity for the DNA adducts of complex 1 than for those of cis-DDP, more UvrB-platinum-DNA cross-links are formed with complex 1 than with cis-DDP. This study, using a bacterial repair system as a model, points to a possible strategy for making new cytotoxic platinum complexes for mammalian cells.