Date of Degree

2-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor

Guillermo Gerona-Navarro

Committee Members

Maria Contel

Ryan P. Murelli

David R. Mootoo

Lissette Delgado-Gruzata

Subject Categories

Analytical Chemistry | Biochemistry | Biophysics | Medicinal-Pharmaceutical Chemistry | Organic Chemistry | Structural Biology

Keywords

peptides, stapled peptides, epigenetics, PRC2, Inhibitors

Abstract

Interactions between proteins play a key role in nearly all cellular process, and therefore, disruption of such interactions may lead to many different types of cellular dysfunctions. Hence, pathologic protein-protein interactions (PPIs) constitute highly attractive drug targets and hold great potential for developing novel therapeutic agents for the treatment of incurable human diseases. Unfortunately, the identification of PPI inhibitors is an extremely challenging task, since traditionally used small molecule ligands are mostly unable to cover and anchor on the extensive flat surfaces that define those binary protein complexes. In contrast, large biomolecules such as proteins or peptides are ideal fits for these so-called “undruggable” sites. However, their poor pharmacokinetic properties have limited their application as therapeutics. In this context, peptidomimetic molecules have emerged as an alternative and viable solution to this problem, since they conserve the architectural and structural features of peptides and also exhibit substantially improved pharmacokinetic profiles. Given the great promise of this class of compounds as therapeutics, new protocols granting easy access to them continue to be of great interest. This thesis describes the development of an efficient solid phase methodology for the chemoselective synthesis of bisthioether stapled peptides of multiple architectures and its application to discovering three families of potent allosteric inhibitors of the polycomb repressive complex 2 (PRC2) of proteins.

PRC2 is a multimeric complex consisting of four core proteins: EZH2, EED, SUZ12 and RBAP46/RBAP48, which is involved in the initiation of gene repression through its methyltransferase activity, specific for lysine 27 on Histone H3 (H3K27). The enzymatic activity of PRC2 is conferred by the catalytic SET domain of EZH2, but also to the other core components of the complex. Hence, a catalytically active PRC2 complex must contain EZH2 and at least EED and SUZ12, which underscores the role of the latter proteins as scaffolds for the proper assembly of PRC2 into its bioactive conformation. The biological relevance of PRC2 proteins is highlighted by their known role in the development and progression of different types of cancers, and thus targeting them has emerged as a high-priority strategy in the field of cancer epigenetics.

The thesis first describes the development of an innovative solid phase approach for the preparation of bisthioether stapled peptides of multiple architectures, including single-, double-turn and double stapled peptides. This methodology allows for ligation with all-hydrocarbon linkers of various lengths, avoiding the use of unnatural amino acids and expensive catalysts, and affords cyclopeptides with improved bioactive conformation and remarkable resistance to proteolytic degradation. Next, we describe the rational design, synthesis and biological evaluation of three new families of allosteric inhibitors of PRC2 function, targeting for the first time three protein interfaces in PRC2 that are crucial for its proper assembly and function: the intramolecular SANT1L/SBD interaction of EZH2, the SUZ12-VEFS/EZH2-SANT2 binary complex and the interaction between SUZ12-NBE domains. Remarkably, these inhibitors have demonstrated cell permeability, potent activity in vitro and in physiological conditions, as well as strong antiproliferative effects on Caki-1 renal cancer cells, which highlights their potential as novel therapeutics for the treatment of PRC2-dependent human cancers.

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