Design, synthesis and biological evaluation of novel molecules with the capacity of inducing the degradation of different proteins by direct signalling to the proteasome

Abstract

In 2001, the initial hypothesis of targeted protein degradation (TPD) was born to announce the launching of a new era of therapeutics development not by inhibiting a component of ubiquitin-proteasome system with small molecule inhibitors, but by recruiting the whole system to induce the degradation of specific proteins. TPD techniques can efficiently target long-lived proteins since they are not constrained by protein turnover, thus overcoming some of the drawbacks of UPS inhibitors. The majority of these technologies also share the advantageous pharmacokinetic characteristics of small compounds resembling drugs. Importantly, due to their various modes of action, they may expand the conventional druggable space. Many technologies with the therapeutic potential of targeting protein for degradation have been under massive study and development including PROteolysis TArgeting Chimeras (PROTACs). PROTACs utilize the UPS to target a specific protein and induce its degradation by employing hetero-bifunctional molecules consisting of a ligand to bind the protein of interest (POI), another ligand to recruit an E3 Ub ligase and a linker to bind the two ligands. These molecules interact simultaneously and hijack the enzymatic machinery by forming a ternary complex (POI:PROTAC:E3 ligase) that facilitates the transfer of Ub moieties to form polyubiquitin chain(s) on the target protein. The polyubiquitinated protein will be recognized and promoted to degradation by the proteasome. Multiple PROTACs that degrade proteins relevant in several diseases have been developed, and the number is quickly increasing, indicating their therapeutic projection. Given some limitations of E3-based PROTACs such as the diversity and complexity of E3 ligases, alternative strategies in target protein degradation are pursued. The main objective of this thesis was to develop a novel type of chimeras with the capacity to induce protein degradation by direct signaling to the 26S proteasome by interacting with USP14, a 26S-associated deubiquitinating enzyme involved in substrate processing and allosteric regulation of 26S activity. The overall results obtained in the biological studies provide proof of concept for this 26S-directed PROTAC, which should expand the potential of target protein degradation. Although this novel approach lacks the limitations associated to ubiquitination in the classical E3-protacs (i. e. different tissue or cell types' expression patterns of targeted E3, inadequate levels of Ub and/or Ub signaling factors under stressful circumstances), the formation of ineffective ternary complexes threatening the effectiveness of classical E3-based PROTACs is not circumvented by USP14-directed PROTACS. The linker is one of the factors that may lead to weak ternary complexes. Until very recently, linker design and selection was a "trial and error" endeavor. However, future avenues for rational linker design, including advances in computational methods, will accelerate the identification of optimized PROTACs.