Heterobifunctional molecules: PROTACs and beyond

Abstract

[eng] Our body is an intricate, well-organized structure formed by trillions of cells. Each one of these cells is a highly complex entity composed of thousands of biomolecules that enable it to carry out its functions. Among them, proteins stand out as the machinery that orchestrates most cellular processes. Alterations in normal protein activity will lead to cellular malfunction and the appearance of diseases. For this reason, protein modification by small molecules has stood as the main pharmacological approach. However, the traditional occupancy-based mechanism of action of most drugs falls short at targeting the wide range of the human proteome. 80% of the disease associated proteins remain undruggable, mainly due to the lack of well-defined pockets in the protein. Recently, new technologies are being developed to overcome the limitations of this mechanism. One of the most promising is the use of Proteolysis Targeting Chimeras (PROTACs). PROTACs work by hijacking the endogenous protein homeostasis system to induce the degradation of the target protein. Their event-based mechanism of action allows PROTACs to bypass the need for high-binding affinities with the target protein, whilst maintaining high selectivity. Together with other advantages like a catalytic mechanism, more permanent effect, lower dosing and less appearance of resistance, puts PROTACs as the most promising alternative to traditional drugs. This doctoral thesis explores PROTAC’s design and synthesis process, as well as the following challenges that the field is facing. In Chapter 2, we explored the use of PROTACs in Castrate Resistant Prostate Cancer (CRPC). This disease comprises 10-20% of Prostate Cancer patients, and most of these cases arise from the appearance of an Androgen Receptor (AR) alternative splice variant that lacks the ligand binding domain (AR-V7). AR-V7 cannot be drugged efficiently due to the intrinsically disordered nature of its structure, leading to high mortality rates. We envisioned the use of PROTACs as a promising alternative to target this challenging protein. Its design is based on the CAM family of ligands, previously reported in the group, to recruit AR-V7. In Chapter 3, we focused on the resistance mechanisms to degraders. Previous work done by Winter’s group in 2019 highlighted that resistance arises away from the target protein. Namely, UBE2Mmut cells become pan-resistant to all degraders. Using this cellular model, we started a high throughput screening campaign followed by an SAR to identify its synthetic vulnerabilities. This study led to the disclosure of RBS-10; a small-molecule capable of differentially kill UBE2Mmut cells. Further work on its mechanism of action led to uncovering that RBS-10 is a prodrug bioactivated by NQO1, which generates metabolites that induce ROS and DNA damage. Chapter 4 showcases the ability of proximity-inducing pharmacology to modulate protein activity. We envisioned the use of heterobifunctional molecules to target the ETS2 Repressor Factor (ERF), an attractive oncogenic target that has traditionally remained undruggable. Through a high- throughput screening of an electrophilic library we identified a first-in-class covalent binder of ERF. After characterizing its binding, we designed and synthesized different bifunctional molecules to modulate ERF’s activity. On one hand, we prepared PROTACs to induce its degradation, inducing cell proliferation and differentiation. On the other hand, we worked on Phosphatase Recruiting Chimeras (PhoRCs), able to induce its dephosphorylation by proximity with a phosphatase. These molecules over-activate ERF, hijacking the cell in a non-proliferative state and acting as a tumor suppressor.

Finally, we also worked on the development of Relocalization Targeting Chimeras (ReloTACs), which induce the nuclear localization of ERF through the formation of a ternary complex with a nuclear protein, p300. In summary, the work in this doctoral thesis highlights the advantages that PROTACs and proximity-inducing approaches have over traditional pharmacology. It emphasizes both their ability to target previously considered undruggable proteins, as well as their design and synthesis process. Furthermore, it displays their vast potential to modulate protein activity and develop novel therapies.

[cat] Dins els milers de biomolècules que formen les nostres cèl·lules, les proteïnes ressalten com els directors d’orquestra de tots els processos cel·lulars. Alteracions en la seva activitat normal desemboquen en diferents malalties. Per aquest motiu, la modificació de l’activitat d’aquestes proteïnes a partir de compostos químics ha estat sempre el dogma principal de la farmacologia. Tanmateix, els mètodes tradicionals basats en ocupació tan sols són efectius en un 20% de les proteïnes associades a malalties. Recentment, noves tecnologies prometen sobrepassar aquestes limitacions. Una de les més prometedores és l’ús de Proteolysis Targeting Chimeras (PROTACs). Aquestes molècules són capaces d’induir la degradació de la proteïna diana segrestant el sistema d’homeòstasi endogen de la cèl·lula. Dividida en tres capítols, aquesta tesi doctoral explora el procés de disseny i síntesi de PROTACs, la seva capacitat de tractar proteïnes abans intractables, i els futurs reptes que aquest camp es planteja. En el Capítol 2, hem sintetitzat potencials degradadors per la proteïna AR-V7, un dels principals causants del Càncer de Pròstata Resistent a la Castració. Aquesta proteïna ha estat tradicionalment considerada intractable, motiu pel qual hi ha una taxa de mortalitat molt alta i és necessari estudiar nous tractaments. En el Capítol 3, hem estudiat les vulnerabilitats sintètiques de cèl·lules resistents a degradadors. A través d’un assaig de relació Estructura-Activitat hem identificat RBS-10 com al millor compost capaç d’acabar selectivament amb aquestes cèl·lules. A partir d’un estudi exhaustiu del seu mecanisme d’acció, hem observat que actua com a prodroga bioactivada per l’enzim NQO1. En el Capítol 4, hem estudiat ERF, una proteïna molt atractiva des del punt de vista oncogènic que fins ara ha escapat el seu tractament. Al llarg d’aquest capítol hem dissenyat i sintetitzat PROTACs per induir la seva degradació, PhoRCs per activar-la mitjançant desfosforilació i ReloTACs per mantenir la seva localització dins el nucli cel·lular. En resum, aquesta tesi doctoral ressalta els avantatges que els PROTACs i altres tecnologies basades en la inducció de proximitat tenen davant la farmacologia tradicional, el seu procés de disseny i síntesi, i les seves perspectives de futur.