Dissecting the Mechanisms of Direct Activation for Proapoptotic BAK and BAX / Chemistry and Chemical Biology

Programmed cell death by apoptosis is required for normal development and tissue homeostasis. Perturbations of the critical signaling pathways that regulate apoptosis drive a number of pathologic diseases; therefore, a deep understanding of the apoptotic regulatory networks and methods for therapeutically modulating them is highly warranted. As constituents of the intrinsic pathway of apoptosis, pro-apoptotic BCL-2 family proteins respond to internal signals of cell stress to activate cell death through permeabilization of the outer mitochondrial membrane to release cytochrome c and other apoptogenic factors. Anti-apoptotic BCL-2 proteins block apoptosis by forming stable heterodimers with pro-apoptotic BAX and BAK. Specifically, the C-terminal binding groove of BCL-2 sequesters the BCL-2 homology 3 (BH3) death domain of BAX to prevent BAX oligomerization. The N-terminal BH4 domain of BCL-2 is also believed to confer anti-apoptotic activity but the mechanism by which this occurs remains unknown. A direct, inhibitory interaction between the BCL-2 BH4 domain and BAX was established through application of a peptide stapling technology to preserve the α-helical character of the BH4 domain outside the context of full-length BCL-2. Photoaffinity labeling identified a new mode of BH4 domain interaction at the C-terminal face of BAX, revealing an additional layer of apoptotic regulation. Examination of the conformational activation of full-length BAX in a lipid membrane by hydrogen-deuterium exchange mass spectrometry revealed that the BCL-2 BH4 helix blocks the BAX conformational changes triggered by an activating BH3 peptide to preserve the inactive BAX fold. Thus, the mechanistic paradigm for BCL-2 inhibition of BAX has been expanded to allow future opportunities for modulation of apoptosis by mimicking or inhibiting the BH4 motif.

Le but de cette thèse a concerné le développement de deux types de structures oligomériques contraintes, basées soit sur des dérivés non naturels d’acides aminés cycliques, soit sur des dimères cycliques de Peptide Nucleic Acids (PNA), en vue d’analyser leur tendance à adopter des conformations pré-organisées, imitant les conformations actives des protéines ou des acides nucléiques. La première partie de cette thèse a trait à l’élaboration de synthons clefs dérivant d’acides α-aminés cycliques non naturels (acides 4-oxopipécoliques 6-substitués), à leur utilisation potentielle dans la synthèse de peptidomimétiques contraints, homogènes et hétérogènes, ainsi qu’à leurs limites dans cette utilisation. Dans le cadre de ce travail, une nouvelle voie de synthèse permettant d’accéder à des résidus N-protégés d'acides 4-oxo-pipécoliques 6substitués a été mise au point. La deuxième partie de cette thèse concerne l’élaboration de dimères d’α-PNA (di-α-PNA) cycliques, dans lesquels les chaînes latérales de deux monomères d’α-PNA consécutifs sont «agrafées» via un pont lactame. Une stratégie de synthèse a tout d'abord été développée en phase liquide, puis appliquée à la synthèse en phase solide de di-α-PNA «agrafés» modèles, incorporant des bases nucléiques thymine. / The purpose of this thesis concerned the development of two kinds of constrained oligomeric structures, based either on unnatural cyclic α-amino acids derivatives or on cyclic Peptide Nucleic Acid (PNA) dimers, in view of analyzing their propensity to adopt pre-organized conformations mimicking the active conformations of proteins or nucleic acids. The first part of this thesis reports on the elaboration of new cyclic αamino-acids (6-substituted 4-oxopipecolic acids) building blocks, their potential use in the synthesis of constrained homogenous and heterogeneous peptidomimetics as well as their limitations in this use. As part of this work, a new methodology has been developed for the synthesis of N-protected 6-substituted 4-oxo-pipecolic acids residues. The second part of this thesis reports on the elaboration of constrained α-PNA dimers (di-α-PNA), in which the side-chains of two consecutive α-PNA monomers are “stapled” via a lactam bridge. A synthetic orthogonal strategy has been first developed in liquid-phase then applied to the solid-phase synthesis of models “stapled” di-α PNA incorporating thymine nucleobases.