RESUMEN
The mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy, with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses, resistance to chemotherapy, and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9, which is associated with a poor prognosis, increased chemoresistance, and higher relapse rates. However, direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA, leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors, namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models, DB1055 and DB818 induced in vitro cell growth reduction, death, differentiation, and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore, DB1055 and DB818 exhibited potent antileukemic activities in a human THP-1 AML in vivo model, leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients, with DB1055 successfully reducing leukemia burden in patient-derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9.
RESUMEN
Anti-nuclear antibodies are the hallmark of autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma. However, the molecular mechanisms of B cell tolerance breakdown in these pathological contexts are poorly known. The study of rare familial forms of autoimmune diseases could therefore help to better describe common biological mechanisms leading to B cell tolerance breakdown. By Whole-Exome Sequencing, we identified a new heterozygous mutation (p.R594C) in ERN1 gene, encoding IRE1α (Inositol-Requiring Enzyme 1α), in a multiplex family with several members presenting autoantibody-mediated autoimmunity. Using human cell lines and a knock-in (KI) transgenic mouse model, we showed that this mutation led to a profound defect of IRE1α ribonuclease activity on X-Box Binding Protein 1 (XBP1) splicing. The KI mice developed a broad panel of autoantibodies, however in a subclinical manner. These results suggest that a decrease of spliced form of XBP1 (XBP1s) production could contribute to B cell tolerance breakdown and give new insights into the function of IRE1α which are important to consider for the development of IRE1α targeting strategies.
Asunto(s)
Enfermedades Autoinmunes , Proteínas Serina-Treonina Quinasas , Humanos , Ratones , Animales , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Transducción de Señal , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismo , Ratones TransgénicosRESUMEN
HOXA9 (Homeobox A9) is a homeotic transcription factor known for more than two decades to be associated with leukemia. The expression of HOXA9 homeoprotein is associated with anterior-posterior patterning during embryonic development, and its expression is then abolished in most adult cells, with the exception of hematopoietic progenitor cells. The oncogenic function of HOXA9 was first assessed in human acute myeloid leukemia (AML), particularly in the mixed-phenotype associated lineage leukemia (MPAL) subtype. HOXA9 expression in AML is associated with aggressiveness and a poor prognosis. Since then, HOXA9 has been involved in other hematopoietic malignancies and an increasing number of solid tumors. Despite this, HOXA9 was for a long time not targeted to treat cancer, mainly since, as a transcription factor, it belongs to a class of protein long considered to be an "undruggable" target; however, things have now evolved. The aim of the present review is to focus on the different aspects of HOXA9 targeting that could be achieved through multiple ways: (1) indirectly, through the inhibition of its expression, a strategy acting principally at the epigenetic level; or (2) directly, through the inhibition of its transcription factor function by acting at either the protein/protein interaction or the protein/DNA interaction interfaces.
RESUMEN
Most transcription factors were for a long time considered as undruggable targets because of the absence of binding pockets for direct targeting. HOXA9, implicated in acute myeloid leukemia, is one of them. To date, only indirect targeting of HOXA9 expression or multitarget HOX/PBX protein/protein interaction inhibitors has been developed. As an attractive alternative by inhibiting the DNA binding, we selected a series of heterocyclic diamidines as efficient competitors for the HOXA9/DNA interaction through binding as minor groove DNA ligands on the HOXA9 cognate sequence. Selected DB818 and DB1055 compounds altered HOXA9-mediated transcription in luciferase assays, cell survival, and cell cycle, but increased cell death and granulocyte/monocyte differentiation, two main HOXA9 functions also highlighted using transcriptomic analysis of DB818-treated murine Hoxa9-transformed hematopoietic cells. Altogether, these data demonstrate for the first time the propensity of sequence-selective DNA ligands to inhibit HOXA9/DNA binding both in vitro and in a murine Hoxa9-dependent leukemic cell model.
Asunto(s)
ADN/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Proteínas de Homeodominio/antagonistas & inhibidores , Leucemia/patología , Modelos Biológicos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN/química , Diseño de Fármacos , Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos/química , Leucemia/genética , LigandosRESUMEN
Transcription factors are involved in a large number of human diseases such as cancers for which they account for about 20% of all oncogenes identified so far. For long time, with the exception of ligand-inducible nuclear receptors, transcription factors were considered as "undruggable" targets. Advances knowledge of these transcription factors, in terms of structure, function (expression, degradation, interaction with co-factors and other proteins) and the dynamics of their mode of binding to DNA has changed this postulate and paved the way for new therapies targeted against transcription factors. Here, we discuss various ways to target transcription factors in cancer models: by modulating their expression or degradation, by blocking protein/protein interactions, by targeting the transcription factor itself to prevent its DNA binding either through a binding pocket or at the DNA-interacting site, some of these inhibitors being currently used or evaluated for cancer treatment. Such different targeting of transcription factors by small molecules is facilitated by modern chemistry developing a wide variety of original molecules designed to specifically abort transcription factor and by an increased knowledge of their pathological implication through the use of new technologies in order to make it possible to improve therapeutic control of transcription factor oncogenic functions.
Asunto(s)
Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Factores de Transcripción/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Animales , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Factores de Transcripción/metabolismoRESUMEN
In this paper novel isoindolines substituted with cyano and amidino benzimidazoles and benzothiazoles were synthesized as new potential anti-cancer agents. The new structures were evaluated for antiproliferative activity, cell cycle changes, cell death, as well as DNA binding and topoisomerase inhibition properties on selected compounds. Results showed that all tested compounds exerted antitumor activity, especially amidinobenzothiazole and amidinobenzimidazole substituted isoindolin-1-ones and benzimidazole substituted 1-iminoisoindoline that showed antiproliferative effect in the submicromolar range. Moreover, the DNA-binding properties of selected compounds were evaluated by biophysical and biochemical approaches including thermal denaturation studies, circular dichroism spectra analyses and topoisomerase I/II inhibition assays and results identified some of them as strong DNA ligands, harboring or not additional topoisomerase II inhibition and able to locate in the nucleus as determined by fluorescence microscopy. In conclusion, we evidenced novel cyano- and amidino-substituted isoindolines coupled with benzimidazoles and benzothiazoles as topoisomerase inhibitors and/or DNA binding compounds with potent antitumor activities.
Asunto(s)
Antineoplásicos/síntesis química , Bencimidazoles/química , Benzotiazoles/química , ADN/metabolismo , Isoindoles/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dicroismo Circular , ADN/química , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isoindoles/metabolismo , Isoindoles/farmacología , Células MCF-7 , Microscopía Fluorescente , Relación Estructura-ActividadRESUMEN
Within this manuscript design, synthesis of novel 2-imidazolinyl substituted benzo[b]thieno-2-carboxamides bearing either benzimidazole or benzothiazole subunit and biological activity are presented and described. The antiproliferative activities were assessed in vitro on a panel of human cancer cell lines. Tested compounds showed moderate activity while cytotoxicity on normal fibroblasts was lower in comparison with 5-fluorouracile. The variations of 2-imidazolinyl substituent at heteroaromatic subunits in different positions led to different cytotoxic properties. The strongest selective activity against HeLa cells was observed for the benzothiazole derivative 4d with 2-imidazolinyl group at the benzo[b]thiophene subunit with a corresponding IC50 = 1.16 µM. Additionally, several biological experiments were performed to explain the mode of biological action. Fluorescence microscopy evidenced nuclear subcellular localization of compounds 3a, 4a and 4c. Additionally, detailed DNA binding studies confirmed a strong DNA groove binding for derivatives 4a and 4c while DNase I footprinting experiments evidenced sequence-selective binding of compound 4c in the A-T rich side. Furthermore, topoisomerase suppressive effect was for compounds 4a-4c.
Asunto(s)
Amidinas/farmacología , Antineoplásicos/farmacología , Bencimidazoles/farmacología , Benzotiazoles/farmacología , ADN de Neoplasias/efectos de los fármacos , Tiofenos/farmacología , Amidinas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Benzotiazoles/síntesis química , Benzotiazoles/química , Sitios de Unión/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN de Neoplasias/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/química , Células Tumorales CultivadasRESUMEN
DNA alkylating drugs have been used in clinics for more than seventy years. The diversity of their mechanism of action (major/minor groove; mono-/bis-alkylation; intra-/inter-strand crosslinks; DNA stabilization/destabilization, etc.) has undoubtedly major consequences on the cellular response to treatment. The aim of this review is to highlight the variety of established protein recognition of DNA adducts to then particularly focus on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) function in DNA adduct interaction with illustration using original experiments performed with S23906-1/DNA adduct. The introduction of this review is a state of the art of protein/DNA adducts recognition, depending on the major or minor groove orientation of the DNA bonding as well as on the molecular consequences in terms of double-stranded DNA maintenance. It reviews the implication of proteins from both DNA repair, transcription, replication and chromatin maintenance in selective DNA adduct recognition. The main section of the manuscript is focusing on the implication of the moonlighting protein GAPDH in DNA adduct recognition with the model of the peculiar DNA minor groove alkylating and destabilizing drug S23906-1. The mechanism of action of S23906-1 alkylating drug and the large variety of GAPDH cellular functions are presented prior to focus on GAPDH direct binding to S23906-1 adducts.
Asunto(s)
Alquilantes/farmacología , Aductos de ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Alquilación , Núcleo Celular , Citoplasma , Daño del ADN , Replicación del ADN , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
DNA minor-groove-binding compounds have limited biological applications, in part due to problems with sequence specificity that cause off-target effects. A model to enhance specificity has been developed with the goal of preparing compounds that bind to two AT sites separated by G·C base pairs. Compounds of interest were probed using thermal melting, circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling methods. A new minor groove binder that can strongly and specifically recognize a single G·C base pair with flanking AT sequences has been prepared. This multi-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.
Asunto(s)
Emparejamiento Base , Derivados del Benceno/química , ADN/química , Secuencia de Bases , Simulación del Acoplamiento Molecular , Estructura MolecularRESUMEN
A series of 6-methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (4), 13-aza derivatives of benzo[b]acronycine, the isomeric 5-methoxy-2,2,13-trimethyl-2,13-dihydro-6H-benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-one (5), and related cis-diols mono- and diesters were designed and synthesized. Their in vitro and in vivo biological activities were evaluated. As previously observed in the acronycine series, esters were the most potent derivatives exhibiting submicromolar activities; among them monoesters are particularly active. Racemic diacetate 21 showed a strong activity against KB-3-1 cell lines and was selected for in vivo evaluation and proved to be active, inhibiting tumor growth by more than 80%. After separation of the two enantiomers, compounds 21a and 21b were also evaluated against C38 colon adenocarcinoma; their activities were found to be significantly different.
Asunto(s)
Acronina/química , Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Naftiridinas/síntesis química , Naftiridinas/farmacología , Adenocarcinoma/patología , Animales , Carcinoma de Células Escamosas/patología , Neoplasias del Colon/patología , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Ensayo de Cambio de Movilidad Electroforética , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Novel phenyl-substituted (3a-3d, 4a, 5, 8a, 8b and 9a) and pyridyl-substituted (3e-3i, 4b, 8c-8e, 9b and 9c) isoindolines were prepared in the reaction of o-phthalaldehyde and corresponding substituted aromatic and heteroaromatic amines by modification of reaction conditions from low to high temperature and from neutral to acidic environment. The antiproliferative activity of chosen substituted isoindolines was assessed on a panel of tumour cell lines and normal human fibroblasts. The majority of tested compounds was active at the highest tested concentrations phenyl-substituted isoindolines 3a and 3b and pyridyl-substituted isoindoline 3g showed a selective effect at micromolar concentrations on HepG2 cell line in comparison with other tested tumour cell lines and normal human fibroblasts. The strongest yet non-selective effect was observed for the pyridyl-substituted isoindoline 8c. These isoindoline derivatives showed diverse mechanism of action on tumour cell death induction as compounds 3a and 8c probably induced mitotic catastrophe while compound 3b induced apoptosis. Indeed, DNA binding properties evidenced that compounds 8a, 8c and 8d bind to DNA as highly potent DNA intercalators. By contrast, compounds 3b, 3e, 3i, 4a and 5 did not target the DNA. At last, the phenyl-substituted compound 8b proved to be a strong DNA binding compound with sequence selective binding and without DNA intercalation profile.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , ADN/metabolismo , Indoles/química , Indoles/farmacología , Piridinas/química , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Humanos , Indoles/síntesis químicaRESUMEN
DB1255 is a symmetrical diamidinophenyl-dithiophene that exhibits cellular activity by binding to DNA and inhibiting binding of ERG, an ETS family transcription factor that is commonly overexpressed or translocated in leukemia and prostate cancer [Nhili, R., Peixoto, P., Depauw, S., Flajollet, S., Dezitter, X., Munde, M. M., Ismail, M. A., Kumar, A., Farahat, A. A., Stephens, C. E., Duterque-Coquillaud, M., Wilson, W. D., Boykin, D. W., and David-Cordonnier, M. H. (2013) Nucleic Acids Res. 41, 125-138]. Because transcription factor inhibition is complex but is an attractive area for anticancer and antiparasitic drug development, we have evaluated the DNA interactions of additional derivatives of DB1255 to gain an improved understanding of the biophysical chemistry of complex function and inhibition. DNase I footprinting, biosensor surface plasmon resonance, and circular dichroism experiments show that DB1255 has an unusual and strong monomer binding mode in minor groove sites that contain a single GC base pair flanked by AT base pairs, for example, 5'-ATGAT-3'. Closely related derivatives, such as compounds with the thiophene replaced with furan or selenophane, bind very weakly to GC-containing sequences and do not have biological activity. DB1255 is selective for the ATGAT site; however, a similar sequence, 5'-ATGAC-3', binds DB1255 more weakly and does not produce a footprint. Molecular docking studies show that the two thiophene sulfur atoms form strong, bifurcated hydrogen bond-type interactions with the G-N-H sequence that extends into the minor groove while the amidines form hydrogen bonds to the flanking AT base pairs. The central dithiophene unit of DB1255 thus forms an excellent, but unexpected, single-GC base pair recognition module in a monomer minor groove complex.
Asunto(s)
Amidinas/química , ADN/química , Tiofenos/química , Amidinas/síntesis química , Amidinas/metabolismo , Secuencia de Aminoácidos , Técnicas Biosensibles , ADN/metabolismo , Huella de ADN , Desoxirribonucleasa I/metabolismo , Guanina/química , Guanina/metabolismo , Modelos Moleculares , Resonancia por Plasmón de Superficie , Tiofenos/síntesis química , Tiofenos/metabolismo , Temperatura de TransiciónRESUMEN
A series of new anilides (2a-c, 4-7, 17a-c, 18) and quinolones (3a-b, 8a-b, 9a-b, 10-15, 19) with nitrogen-bearing substituents from benzo[b]thiophene and thieno[2,3-c]thiophene series are prepared. Benzo[b]thieno[2,3-c]- and thieno[3',2':4,5]thieno[2,3-c]quinolones (3a-b, 8a-b) are synthesized by the reaction of photochemical dehydrohalogenation from corresponding anilides. Anilides and quinolones were tested for the antiproliferative activity. Fused quinolones bearing protonated aminium group, quaternary ammonium group, N-methylated and protonated aminium group, amino and protonated amino group (8a, 9b, 10-12) showed very prominent anticancer activity, whereby the hydrochloride salt of N',N'-dimethylaminopropyl-substituted quinolone (14) was the most active one, having the IC50 concentration at submicromolar range in accordance with previous QSAR predictions. On the other hand, flexible anilides were among the less active. Chemometric analysis of investigated compounds was performed. 3D-derived QSAR analysis identified solubility, metabolitic stability and the possibility of the compound to be ionized at pH 4-8 as molecular properties that are positively correlated with anticancer activity of investigated compounds, while molecular flexibility, polarizability and sum of hydrophobic surface areas were found to be negatively correlated. Anilides 2a-b, 4-7 and quinolones 3a-b, 8a-b, 9b and 10-14 were evaluated for DNA binding propensities and topoisomerases I/II inhibition as part of their mechanism of action. Among the anilides, only compound 7 presented some DNA binding propensity whereas the quinolones 8b, 9b and 10-14 intercalate in the DNA base pairs, compounds 8b, 9b and 14 being the most efficient ones. The strongest DNA intercalators, compounds 8b, 9b and 14, were clearly distinguished from the other compounds according to their molecular descriptors by the PCA and PLS analysis.
Asunto(s)
Anilidas/química , Anilidas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Quinolonas/química , Quinolonas/farmacología , Línea Celular Tumoral , Citostáticos/química , Citostáticos/farmacología , ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias/tratamiento farmacológico , Nitrógeno/química , Relación Estructura-Actividad Cuantitativa , Tiofenos/química , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Heterocyclic diamidines are strong DNA minor-groove binders and have excellent antiparasitic activity. To extend the biological activity of these compounds, a series of arylimidamides (AIAs) analogues, which have better uptake properties in Leishmania and Trypanosoma cruizi than diamidines, was prepared. The binding of the AIAs to DNA was investigated by Tm , fluorescence displacement titration, circular dichroism, DNase I footprinting, biosensor surface plasmon resonance, X-ray crystallography and molecular modeling. These compounds form 1:1 complexes with AT sequences in the DNA minor groove, and the binding strength varies with substituent size, charge and polarity. These substituent-dependent structure and properties provide a SAR that can be used to estimate K values for binding to DNA in this series. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for AIAs.
Asunto(s)
Amidas/metabolismo , ADN/metabolismo , Amidas/química , Secuencia de Bases , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , ADN/química , Desoxirribonucleasa I/metabolismo , Leishmania/metabolismo , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Temperatura de Transición , Trypanosoma cruzi/metabolismoRESUMEN
Novel amidino-derivatives of phenylene-bisbenzothiazoles were synthesized and tested for their antiproliferative activity against several human cancer cell lines, as well as DNA-binding properties. The synthetic approach used for preparation of isomeric amidino substituted-phenylene-bis-benzothyazoles 3a-3f was achieved by condensation reaction of isophthaloyl dichloride 1a and terephthaloyl dichloride 1b or with phthalic acid 1c with 5-amidinium-2-aminobenzothiolate 2a and 5-(imidazolinium-2-yl)-2-aminobenzothiolate 2b in good yields. The targeted compounds were converted in the desired water soluble dihydrochloride salts by reaction of appropriate free base with concd HCl in ethanol or acetic acid. All tested compounds (3a-3f) showed antiproliferative effects on tumour cells in a concentration-dependant manner. The strongest activity and cytotoxicity was observed for diimidazolinyl substituted phenylene-bisbenzothiazole compound 3b. These effects were shown to be related to DNA-binding properties, topoisomerase I and II poisoning effects and apoptosis induction. The highest tested selectivity towards tumour cells was observed for the imidazolyl substituted phenylene-benzothiazole 3d that showed no cytotoxic effects on normal fibroblasts making it an excellent candidate for further chemical optimization and preclinical evaluation.
Asunto(s)
Antineoplásicos/farmacología , Benzotiazoles/farmacología , Proliferación Celular/efectos de los fármacos , ADN/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Benzotiazoles/síntesis química , Benzotiazoles/química , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Células MCF-7 , Modelos Químicos , Estructura Molecular , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Espectrofotometría , Relación Estructura-ActividadRESUMEN
In a view to develop new DNA alkylating antitumour drugs, evaluating the precise mechanism of action and the molecular/cellular consequences of the alkylation is a point of major interest. The benzo-b-acronycine derivative S23906-1 alkylates guanine nucleobases in the minor groove of the DNA helix and presents an original ability to locally open the double helix of DNA, which appears to be associated with its cytotoxic activity. However, the molecular mechanism linking adduct formation to cellular consequences is not precisely known. The objective of the present study was to identify proteins involved in the recognition and mechanism of action of S23906-DNA adducts. We found that GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a protein that binds to S23906-alkylated single-stranded, double-stranded and telomeric sequences in a drug-dependent and DNA sequence/structure-dependent manner. We used the CASTing (cyclic amplification of sequence targeting) method to identify GAPDH DNA-binding selectivity and then evaluated its binding to such selected S23906-alkylated sequences. At the cellular level, alkylation of S23906-1 results in an increase in the binding of GAPDH and its protein partner HMG (high-mobility group) B1 to the chromatin. Regarding the multiple roles of GAPDH in apoptosis and DNA repair, the cytotoxic and apoptotic activities of GAPDH were evaluated and present opposite effects in two different cellular models.
Asunto(s)
Acronina/análogos & derivados , Aductos de ADN/química , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Proteínas Nucleares/química , Acronina/química , Alquilación , Aductos de ADN/genética , Aductos de ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Células HT29 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica/genéticaRESUMEN
Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.
Asunto(s)
Amidinas/farmacología , Antineoplásicos/farmacología , Tiofenos/farmacología , Transactivadores/antagonistas & inhibidores , Activación Transcripcional/efectos de los fármacos , Amidinas/química , Amidinas/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión , Línea Celular Tumoral , ADN/química , ADN/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Tiofenos/química , Tiofenos/metabolismo , Transactivadores/metabolismo , Regulador Transcripcional ERGRESUMEN
A series of new N,N-dimethylaminopropyl- and 2-imidazolinyl-substituted derivatives of benzo[b]thienyl- and thieno[2,3-b]thienylcarboxanilides and benzo[b]thieno[2,3-c]- and thieno[3',2':4,5]thieno[2,3-c]quinolones were prepared. Quinolones were prepared by the reaction of photochemical dehydrohalogenation of corresponding anilides. Carboxanilides and quinolones were tested for the antiproliferative activity. 2-Imidazolinyl-substituted derivatives showed very prominent activity. By use of the experimentally obtained antitumor measurements, 3D-derived QSAR analysis was performed for the set of compounds. Highly predictive 3D-derived QSAR models were obtained, and molecular properties that have the highest impact on antitumor activity were identified. Carboxanilides 6a-c and quinolones 9a-c and 11a were evaluated for DNA binding propensities and topoisomerases I and II inhibition as part of their mechanism of action assessment. The evaluated differences in the mode of action nicely correlate with the results of the 3D-QSAR analysis. Taken together, the results indicate which modifications of the compounds from the series should further improve their anticancer properties.
Asunto(s)
Anilidas/síntesis química , Antineoplásicos/síntesis química , ADN/química , Relación Estructura-Actividad Cuantitativa , Quinolonas/síntesis química , Tiofenos/síntesis química , Anilidas/química , Anilidas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Desnaturalización de Ácido Nucleico , Procesos Fotoquímicos , Análisis de Componente Principal , Quinolonas/química , Quinolonas/farmacología , Tiofenos/química , Tiofenos/farmacología , Inhibidores de Topoisomerasa I/síntesis química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Small molecule complexes with DNA that incorporate linking water molecules are rare, and the DB921-DNA complex has provided a unique and well-defined system for analysis of water-mediated binding in the context of a DNA complex. DB921 has a benzimidazole-biphenyl system with terminal amidines that results in a linear conformation that does not possess the appropriate radius of curvature to match the minor groove shape and represents a new paradigm that does not fit the classical model of minor groove interactions. To better understand the role of the bound water molecule observed in the X-ray crystal structure of the DB921 complex, synthetic modifications have been made in the DB921 structure, and the interactions of the new compounds with DNA AT sites have been evaluated with an array of methods, including DNase I footprinting, biosensor-surface plasmon resonance, isothermal titration microcalorimetry, and circular dichroism. The interaction of a key compound, which has the amidine at the phenyl shifted from the para position in DB921 to the meta position, has also been examined by X-ray crystallography. The detailed structural, thermodynamic, and kinetic results provide valuable new information for incorporation of water molecules in the design of new lead scaffolds for targeting DNA in chemical biology and therapeutic applications.
Asunto(s)
ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Agua/química , Amidinas/química , Amidinas/metabolismo , Secuencia de Bases , Bencimidazoles/química , Bencimidazoles/metabolismo , Sitios de Unión , ADN/genética , Desoxirribonucleasa I/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Peso Molecular , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
The in vitro anticancer activity and toxicity of phyllostictine A, a novel oxazatricycloalkenone recently isolated from a plant-pathogenic fungus (Phyllosticta cirsii) was characterized in six normal and five cancer cell lines. Phyllostictine A displays in vitro growth-inhibitory activity both in normal and cancer cells without actual bioselectivity, while proliferating cells appear significantly more sensitive to phyllostictine A than non-proliferating ones. The main mechanism of action by which phyllostictine displays cytotoxic effects in cancer cells does not seem to relate to a direct activation of apoptosis. In the same manner, phyllostictine A seems not to bind or bond with DNA as part of its mechanism of action. In contrast, phyllostictine A strongly reacts with GSH, which is a bionucleophile. The experimental data from the present study are in favor of a bonding process between GSH and phyllostictine A to form a complex though Michael attack at C=C bond at the acrylamide-like system. Considering the data obtained, two new hemisynthesized phyllostictine A derivatives together with three other natural phyllostictines (B, C and D) were also tested in vitro in five cancer cell lines. Compared to phyllostictine A, the two derivatives displayed a higher, phyllostictines B and D a lower, and phyllostictine C an almost equal, growth-inhibitory activity, respectively. These results led us to propose preliminary conclusions in terms of the structure-activity relationship (SAR) analyses for the anticancer activity of phyllostictine A and its related compounds, at least in vitro.