Studies on Proteolysis Targeting Chimeras: platform technology for targeted protein degradation in drug discovery

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.