Studies on Proteolysis Targeting Chimeras: platform technology for targeted protein degradation in drug discovery
Creators: name: ZOPPI, VITTORIA
Publisher: name: Universit� degli studi di Genova
Year: 2019

description: Proteolysis Targeting Chimeras (PROTACs) represent an innovative approach to chemical
intervention into biology, with attractive therapeutic potential. In contrast to protein inhibition,
PROTACs trigger targeted protein degradation inside cells via hijacking the ubiquitinproteasome
machinery. PROTACs are bifunctional compounds composed by two small
molecules connected by a linker; one molecule binds to a protein of interest and the other one
binds to an E3 ubiquitin ligase. The ligases most commonly recruited are the von Hippel-
Lindau (VHL) protein complex CRL2VHL and the cereblon (CRBN) complex CRL4CRBN. To date,
different classes of target proteins have been successfully degraded, including epigenetic
targets such as bromodomain-containing proteins BRD2, BRD3, and BRD4, BRD9, TRIM24,
SIRT2, PCAF/GNC5, protein kinases, nuclear receptors and E3 ubiquitin ligases to selfdegrade.
The work described in this thesis is divided into two parts: the first part is about the research
activity carried out at the University of Dundee, under the supervision of Professor Alessio
Ciulli, while a second part involves the work done at the Department of Chemical Core
Technologies, within Nerviano Medical Sciences (Milan), under the supervision of Doctor
Eduard Felder.
The aim of the project carried out in Ciulli Lab was to investigate PROTAC-mediated
degradation of BRD7 and BRD9 proteins, subunits of the human SWI/SNF chromatin
remodelling complexes. These subunits have gained interest as therapeutic target especially in
hematopoietic cancers, for example supporting growth of acute myeloid leukemia (AML) cells.
Despite inhibitors able to disrupt the interaction of the BRD7/9 bromodomains are known, their
cellular activity has remained limited as other non-bromodomain functions of the target remain unaffected. PROTAC molecules able to induce BRD9 degradation would more profoundly
impact on BRD7 and BRD9 function and therefore could be used as chemical tools to better
understand its role in BAF complex and in oncogenesis.
Following analysis of the available co-crystal structures of the individual target and ligase
proteins with their respective ligands, a library of degraders has been designed and synthesised,
involving convergent synthetic strategies to conjugate a diverse range of scaffolds and linkers.
All compounds were tested in vitro against different cancer cell lines and immunoblotting was
carried out to assess the target degradation profile. VZ185 was found as a highly selective,
potent and rapid dual BRD7/BRD9 degrader, with slight preference for BRD9 over BRD7.
Degradation was demonstrated to be dependent upon proteasomal activity, cullin neddylation
and VHL binding.
The second section of the thesis reports the design, synthesis and biological characterization of
PROTACs targeting protein kinases, carried out at NMS for one-year stage. With the future
prospect of extending the development of PROTAC to other targets, it was planned to study a
literature example, in order to confirm the proof of concept. Amongst those available, we chose
DAS-6-2-2-6-CRBN, based on a potent tyrosine kinase inhibitor and a CRBN ligand that
induce degradation of c-ABL and BCR-ABL. Furthermore, we decided to extend the
development of degraders focusing on how target ligand and linker composition affect efficacy
and selectivity. The biological evaluation of the synthetized compounds was performed by the
Department of Biology of NMS throughout in vitro treatments of cancer cell lines and
immunoblotting.