Desarrollo y validación de nuevas metodologías para la caracterización de la interacción de ligandos con ADN. Estudio de la unión de cromóforos catiónicos heteroaromáticos con potencialidad intercalante

espanolEl diseno de compuestos con capacidad de union a secuencias especificas de ADN tiene gran importancia en farmacologia dado que existen numerosos farmacos que tienen el ADN como diana y cuya actividad terapeutica se basa en la afinidad y selectividad de su interaccion con el mismo. Por otro lado, estos ligandos de bajo peso molecular con capacidad de union a secuencias predeterminadas de ADN pueden ser herramientas de gran utilidad en biologia molecular. Para comprender en toda su complejidad la interaccion entre el ADN y estos ligandos se debe combinar la caracterizacion del mecanismo de union a resolucion practicamente atomica, con datos termodinamicos de las energias de union a oligonucleotidos. Existen diversos metodos para estudiar esta union pero generalmente presentan desventajas como el alto coste por ensayo (en tiempo, reactivos o fungible), falta de sensibilidad o imposibilidad de automatizacion. De ahi la utilidad del objetivo principal de esta Tesis: desarrollar un ensayo rapido y eficaz, capaz de identificar el sitio preferente de interaccion entre el ADN y compuestos de diferente naturaleza. A lo largo de esta Tesis se expondra el desarrollo, validacion y aplicaciones de un metodo novedoso, basado en el analisis de curvas de fusion de ADN y que puede ser considerado como metodo de alto rendimiento para cribado de un gran numero de compuestos que se unan al ADN. Este metodo aporta mejoras significativas en aspectos como consumo de reactivos y de tiempo por ensayo, que tradicionalmente han limitado la determinacion experimental de la selectividad de secuencia ligando-ADN. Asi, finalmente, ha podido aplicarse esta metodologia al estudio de la interaccion con ADN de una quimioteca formada por decenas de compuestos heteroaromaticos cationicos con N cuaternario en posicion cabeza de puente. EnglishThe goal of promoting or repressing gene expression by low molecular weight ligands is closely related with advances in our understanding of the DNA recognition reaction. Besides, there are notable applications in molecular biology of ligands with sequence selective DNA binding. The central objective of this dissertation is to deepen in the knowledge of DNA recognition of several ligands, mainly by the development of a new miniaturised assay. Available analytical assays for the determination of the potential selectivity interaction of a particular compound to DNA suffer the drawback that none are applicable to the highthroughput screening required to explore libraries of compounds in a systematic search for new and more selective DNA binders. In the introduction to this dissertation there is a brief review of concepts, data and methodologies related with DNA binding ligands. Special focus is made with intercalative agents. In addition, we describe the development and characteristics of a method that fulfil the expected purposes and its application to almost a hundred of ligands of diverse DNA binding modes. For this screening method we use synthetic oligonucleotides containing a fluorophore attached to deoxyribose at the 5’ position in one strand and a quencher in the 3’ position of the complementary. In double strand DNA fluorescence is quenched as fluorophore and quencher are in close proximity. When the strands melt, both are separated and there is a large increase in fluorescence. Ligand presence causes an increment in the temperature at which half DNA molecules are dissociated, therefore their strands are separated (melting temperature, Tm). These experiments are carried out in the real time thermocycler Fast-7500 Sequence Detection System from ABI-PRISM. This equipment provides us with a spectrofluorimeter where constant temperature variations can be established. It requires small amounts of material (typically 20 µl reaction mix total volume) and can perform 96 melting profiles in parallel. This makes feasible that the determination of the preferred sequence be fast, sensitive and quantitative and also allows carrying out the analysis of a high amount of specific double stranded DNA sequences. The procedure has relevant advantages comparing to DNA denaturalization detection by other techniques, like UV absorbance. Method’s utility has been validated with compounds binding DNA by different mechanisms. Thus, we have analysed minor groove DNA binding ligands (DAPI and Hoescht 33258), the monointercalative agents etidium bromide and doxorubicin, several bisintercalators (TOTO-I, thiocoraline-A and echinomycin), compounds that bind covalently to DNA (ecteinascidins and derivatives) and ligands with a complex way of DNA binding, such as the γ-carboline bis-salt AMP-10.15 or S223906-1, which binds to single-stranded DNA. For model compounds, results are consistent with those obtained by traditional procedures such as footprinting. This assay has also been applied to a library of cationic heteroaromatic compounds based on benzimidazole, benzothiazole, carboline and azolodiazines heterocycles, with potential intercalative properties, that were synthesized by our group. For these ligands, gel intercalation assays where performed as well before the determination of their general DNA preference (AT versus GC).