Mimicry of discontinuous Epitopes employing the Truazacyclophane (TAC) scaffold for modulation of protein-protein interactions

Abstract

All biological processes are regulated by means of protein-protein interactions. In practically each step of the often complicated molecular cascades, protein-protein interactions play a crucial role. Defects in the regulation of these interactions are believed to be involved in several diseases Because protein-protein interactions are involved in many diseases, drug design in order to modulate and/or interfere with protein-protein interactions has become very important. The aim of this thesis is the development of methods to mimic discontinuous epitopes employing the TAC scaffold. Several biological relevant target systems are chosen for the development of a method for the mimicry of discontinuous epitopes, resulting in the “pars pro toto” mimicry of their parent biomolecules. The mimicry of a discontinuous epitope of cystatin B is described in chapters two and three. Cystatin B interacts through three epitope sequences viz. the N-terminus (6MMCGA10), a β-hairpin loop (53QVVAGT58) and the C-terminus (122LTYF125) with papain. Based on the crystal structure of papain in complex with cystatin B synthetic inhibitors were designed, synthesized and kinetic assays were carried out. Next to the assembly of linear epitopes an attempt was made to synthesize a TAC scaffold decorated with a cyclic epitope sequence. In chapter four the methodology developed in chapters two and three was applied to design en synthesize inhibitors of Caspase-3 based on the discontinuous epitope of XIAP. Caspase-3 is the pivotal protein of the intrinsic and the extrinsic apoptotic pathways. In the normal situation is Caspase-3 blocked selectively by XIAP (X-linked IAP) to prevent apoptosis. The crystal structure of Caspase-3 in complex with XIAP indicates three binding sequences (discontinuous epitope) of XIAP that interact with Caspase-3 viz. the “zinc” 225PNCFF229, the “hook” 138DYLLRTGQV146 and the “sinker” 148DISDTIYPR156 epitope. This discontinuous epitope was assembled on the TAC scaffold to yield synthetic inhibitors of Caspase-3. Chapters five and six deal with the mimicry of pertactin (P.69 Prn 1). Pertactin is an outer membrane protein of the bacterium Bordetella pertussis. Importantly, P.69 Prn 1 is a principal component of most current pertussis subunit vaccines. Antibody levels against P.69 Prn1 have shown to correlate with clinical protection. P.69 Prn1, the focus of this study, belongs to a class of so-called autotransporter proteins that undergo autoproteolytic processing. The epitopes of this protein were previously mapped by Hijnen et al. Two different discontinuous epitopes were used i.e. 11GERQH15, 97GDTWDDD103 and 247GGFGP251, which are sequences recognized by mAbs and 70DGIRRFL76, 156SLQPEDL162 and 247GGFGP251, which are sequences recognized by human sera. These discontinuous epitopes were assembled on the TAC scaffold yielding a series of PEPTAC’s. After conjugation to Tetanus Toxoid, mouse experiments were carried out to test the protective properties of the PEPTAC’s.