Most synthetic small molecules function as inhibitors to modulate biological processes without affecting target level inside cells. Recent studies have demonstrated that inhibitors targeting more than a single protein i.e. pan-selective, can be turned into more selective and more effective chemical probes for individual target proteins, by using PROTACs (proteolysis targeting chimera). Using this approach, the ubiquitin proteasome pathway can be hijacked to induce degradation of the target protein as opposed to its simple blockade. PROTACs are usually hetero-bifunctional compounds containing a ligand for an E3 Ubiquitin Ligase, and a ligand for the target protein, tethered by a linker. By bringing the two proteins into proximity, PROTACs can induce ubiquitination and subsequent degradation of the target protein through the proteasome. One E3 ligase successfully targeted by our laboratory is the von Hippel-Lindau protein (VHL), which as part of Cullin2 complex naturally functions in the oxygen signalling pathway. The work described in this thesis aims to target two of the major players involved in this pathway – VHL itself, and the prolyl hydroxylase enzymes (PHDs) that catalyze the key hydroxylation step that triggers the series of event that regulate the sensing of oxygen. The crystal structures of PHD2 and VHL proteins with their respective inhibitor bound provided the start point for the PROTAC design, allowing to identify suitable positions for chemical derivatization. Two series of PROTACs against PHD (Hetero-PROTACs) and VHL (Homo-PROTACs) recruiting both VHL and a related E3 ligase (cereblon) were designed accordingly and synthetized. The biological evaluation of the compounds ability to degrade their target proteins was performed by in vitro treatments of different cancer cells lines and immunoblotting. Homo-PROTAC CM11 induced rapid and isoform specific degradation of VHL in the double-digit nanomolar concentrations. Degradation was shown to be dependent upon proteasomal activity, cullin neddylation and VHL binding. Formation of ternary complex was assessed by biophysical studies. Together, this study provide first proof-of-concept for developing the PROTAC approach to trigger an E3 ligase to induce its own degradation, with wide implications for chemical biology and potentially drug discovery.

Design, synthesis and biological evaluation of new bifunctional chemical degrader molecules (PROTACs) targeting hypoxia signalling pathway

MANIACI, CHIARA
2017-02-15

Abstract

Most synthetic small molecules function as inhibitors to modulate biological processes without affecting target level inside cells. Recent studies have demonstrated that inhibitors targeting more than a single protein i.e. pan-selective, can be turned into more selective and more effective chemical probes for individual target proteins, by using PROTACs (proteolysis targeting chimera). Using this approach, the ubiquitin proteasome pathway can be hijacked to induce degradation of the target protein as opposed to its simple blockade. PROTACs are usually hetero-bifunctional compounds containing a ligand for an E3 Ubiquitin Ligase, and a ligand for the target protein, tethered by a linker. By bringing the two proteins into proximity, PROTACs can induce ubiquitination and subsequent degradation of the target protein through the proteasome. One E3 ligase successfully targeted by our laboratory is the von Hippel-Lindau protein (VHL), which as part of Cullin2 complex naturally functions in the oxygen signalling pathway. The work described in this thesis aims to target two of the major players involved in this pathway – VHL itself, and the prolyl hydroxylase enzymes (PHDs) that catalyze the key hydroxylation step that triggers the series of event that regulate the sensing of oxygen. The crystal structures of PHD2 and VHL proteins with their respective inhibitor bound provided the start point for the PROTAC design, allowing to identify suitable positions for chemical derivatization. Two series of PROTACs against PHD (Hetero-PROTACs) and VHL (Homo-PROTACs) recruiting both VHL and a related E3 ligase (cereblon) were designed accordingly and synthetized. The biological evaluation of the compounds ability to degrade their target proteins was performed by in vitro treatments of different cancer cells lines and immunoblotting. Homo-PROTAC CM11 induced rapid and isoform specific degradation of VHL in the double-digit nanomolar concentrations. Degradation was shown to be dependent upon proteasomal activity, cullin neddylation and VHL binding. Formation of ternary complex was assessed by biophysical studies. Together, this study provide first proof-of-concept for developing the PROTAC approach to trigger an E3 ligase to induce its own degradation, with wide implications for chemical biology and potentially drug discovery.
bivalent molecules; PROTACs; E3 ligases; von Hippel-Lindau; hypoxia
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11570/3105207
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