NMR in drug discovery. From screening to structure-based design of antitumoral agents

Nuclear Magnetic Resonance has experienced an increasing interest in the drug discovery field that has led to its wide use on nearly every stage of drug development. For this reason, during the present thesis we propose to use some of the tools offered by NMR to target various systems related with cancer. Initially we intended to get acquainted with the NMR most outstanding methodologies for the detection and characterization of binding events; and for this goal various proteins involved in cellular apoptosis (XIAP and Bcl-XL) were used to set up both ligand and receptor based NMR experiments. First we developed two novel labeling methodologies for the selective NMR observation of Trp, which coupled to the use of receptor-based experiments provided insight in the characterization of XIAP-ligand complexes, as well as significant advantages for HTS. Later, ligand based experiments such as STD, WaterLOGSY and iLOE were applied to the screening of weak binders for Bcl-XL, and the resulting information applied to the reconstruction of dual binders in a fragment-based drug discovery approach. In a second stage the same NMR tools were applied to the development of protein-protein interaction inhibitors, in particular for the pro-angiogenic protein VEGF; and to this end two different strategies were proposed. First, a small diverse library of D-oligopeptides was screened for weak ligands using the former fragment-based strategy. A second approach consisted on the identification of active compounds in complex plant extract mixtures using a combination of methionine selective labeling schemes and receptor-based experiments. In the latter case various flavonoids were identified as VEGF binders and the information harvested from NMR experiments allowed us to propose a binding mode for this type of molecules. Lastly, in a much more classic context, we decided to attempt the characterization of a medium sized oligopeptide by NMR. In particular we focused on Kahalalide F a marine origin depsipeptide with very promising anticancer activity; and although a SAR profile would be very useful from a medicinal chemistry perspective such molecules still pose a challenge to their structural characterization particularly stemming from its intrinsic flexibility. Several media were explored including H2O, DMSO, and membrane mimics (SDS micelles); in the latter the peptide seems to adopt a preferred structure allowing us to propose a model for the monomeric insertion of Kahalalide F within membrane-like environments. Such a model supports that part of the peptides cytotoxic activity occurs at a membrane level and that this activity is linked to the formation of a turn within its N-terminus section. " RESUMEN: La resonancia magnetica nuclear se utiliza en la actualidad en practicamente todas las etapas del proceso de descubrimiento y caracterizacion de farmacos. Durante la presente tesis nos propusimos explorar estas facetas de la RMN tomando como modelo diferentes sistemas relacionados con cancer. En primer lugar utilizamos proteinas involucradas en apoptosis (XIAP y Bcl-XL) para implementar varias metodologias de RMN para la caracterizacion de fenomenos de interaccion intermolecular. Se pusieron a punto esquemas de marcaje selectivo de triptofano y se evaluo su aplicabilidad en procesos de cribado de compuestos. Otros experimentos de cribado masivo (STD, WaterLOGSY) se emplearon para identificar nuevos ligandos de Bcl-XL; posteriormente, estrategias de reconstruccion de fragmentos nos permitieron disenar ligandos duales. Las mismas herramientas se utilizaron para el diseno y desarrollo de moleculas antiangiogenicas, inhibidoras de la interaccion entre VEGF y sus receptores. Para este fin, se plantearon dos estrategias diferentes en la identificacion de inhibidores: en primer lugar se pretendia disenar un ligando peptidico de afinidad elevada partiendo de dipeptidos de menor afinidad y utilizando estrategias de reconstruccion de fragmentos. Una segunda estrategia consistia en la identificacion de compuestos activos presentes en extractos de plantas utilizando esquemas de marcaje selectivo para VEGF, en concreto marcaje selectivo de metioninas. En este ultimo caso se consiguieron identificar varios compuestos flavonoides capaces de interaccionar con VEGF y su modo de union se caracterizo utilizando informacion derivada de experimentos de RMN. Por ultimo se llevo a cabo la caracterizacion estructural de Kahalalido F mediante RMN. Este depsipeptido de origen marino se encuentra en la actualidad en fases clinicas de desarrollo contra varios tipos de cancer, y su caracterizacion estructural presenta enorme interes en la comprension de su modo de accion. Se exploraron las preferencias conformacionales del peptido en varios medios incluyendo: H2O, DMSO, y medios mimeticos de membrana (micelas SDS). El peptido adopta una estructura preferente en presencia de micelas de SDS, lo que nos permiten proponer un modelo estructural para la insercion monomerica de Kahalalido F en entornos de membrana lipidica; esto sugiere que parte de su actividad citotoxica ocurre a nivel de membrana para lo cual la formacion de un giro ? en la seccion N-terminal del peptido tambien parece relevante. "