Exploring novel target combinations for treating neurodegenerative diseases

[eng] Neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), affect millions of people worldwide and share common multifactorial characteristics, including neuroinflammation, oxidative stress, and protein aggregation. Due to the complexity of these diseases, this thesis focuses on exploring novel therapeutic strategies that have been poorly exploited to address AD and PD. Firstly, for AD, a bibliometric study on the development of multitarget drugs has revealed a significant increase in publications since 2011, with a predominant interest in linked hybrids, which mostly hit acetylcholinesterase (AChE), oxidative stress, and β-amyloid aggregation, while targets such as tau aggregation or GSK-3β remain relatively underexplored. Secondly, efforts have been made during this thesis to deepen into the understanding of protein aggregation by performing, in the frame of several collaborations, the evaluation in genetically modified Escherichia coli cells of the anti- aggregation activity of two structural series synthesized in this thesis and four different synthesized by others. It was observed that active anti-aggregating compounds display similar potencies against the aggregation of β-amyloid and tau proteins. Based on this information, new hybrid compounds have been designed to combine the inhibition of soluble epoxide hydrolase (sEH) and AChE with anti-aggregation properties. These compounds have demonstrated nanomolar potency against both targets, while effectively inhibiting the aggregation of β-amyloid, tau, and TDP-43 proteins. Moreover, they have shown to be non-neurotoxic, capable of crossing the blood-brain barrier, and have exhibited moderate aqueous solubility, making them good candidates for future in vivo evaluation. Regarding PD, this thesis has focused on the design of sEH inhibitors with specific anti-aggregation activity against α-synuclein. The new compounds have demonstrated high sEH inhibitory potency, significant α-synuclein anti-aggregation activity, and good DMPK and safety profile, thereby emerging as promising candidates for future preclinical studies in murine models. In addition to conventional multitarget strategies, targeted protein degradation (TPD) was explored as an alternative modality to tackle CNS-related proteins. One of the main needs in the field is to increase the toolbox of E3 ligases. In this context, novel ligands for KEAP1, an E3 ligase that degrades Nrf2—a key transcription factor with antioxidant effects—have been investigated. The aim is to develop a dual-action PROTACs, which could simultaneously increase Nrf2 levels to enhance antioxidant activity while degrading a protein of interest via KEAP1’s E3 ligase activity. Using MDMix calculations and virtual screening, 20 potential ligands were identified, five of which were biophysically validated. Among them three ligands showed micromolar affinity (Kd) towards the Kelch domain of KEAP1 and hints of upregulation of Nrf2-regulated genes. These results provided the foundation for the design and evaluation of new KEAP1-recruiting PROTACs incorporating the discovered ligands.