Mechanistic insights into ACSL4 inhibition by mutation and domain interaction studies

Details

Supervisors

Frédérick, Raphaël

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enThe development of ferroptosis inhibitors represents a significant progression in the medicinal chemistry research, as these compounds not only serve as valuable tools to investigate the role of ferroptosis in various pathologies but also provide promising starting points for the development of therapeutic strategies. Among the different ferroptosis inhibitors, those specifically targeting Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) stand out due to their selective action on the ferroptotic pathway. Currently, the most potent ACSL4 inhibitor identified is rosiglitazone. The lack of selectivity of this molecule, as it not only inhibits ACSL4 but also impacts lipid metabolism through PPARγ activation, justifies further research of inhibitors of ACSL4. In this master thesis, two different ACSL4 inhibition approaches are considered. The first involves LIBX-A401, a compound developed through a structure-activity relationship (SAR) study based on rosiglitazone, but specifically engineered to lack PPARγ agonist activity. The aim is to confirm that its binding occurs in the fatty acid tunnel of ACSL4. To validate this binding mode, various orthogonal methods, including enzymatic activity assays and microscale thermophoresis (MST), are performed, further demonstrating that LIBX-A401 effectively inhibits ACSL4 by occupying this tunnel and interfering with substrate recognition. Moreover, since ACSL4’s catalytic mechanism is thought to rely on conformational changes, a second inhibition strategy was explored using DM01, a peptide-based inhibitor targeting these structural dynamics. DM01 was designed to disrupt the interactions between the C-terminal and N-terminal domains of the enzyme, which are characteristic of these conformational changes. The development of DM01 was guided by AlphaFold predictions, mimicking a sequence from the N-terminal domain that is predicted to interact with the C-terminal domain. In order to assess the potential of this inhibition approach, the MST conditions allowing the visualization of the interaction between the domains of ACSL4 are established. These conditions allowed the evaluation of the impact of DM01 on the conformational dynamics of ACSL4, although the results indicates a non-specific mode of action for this inhibitor. However, this inhibition approach remains relevant and requires further studies to assess its potential to strongly and selectively inhibit ACSL4. In conclusion, the results obtained in this master thesis contribute to paves the way for future research on targeted inhibition strategies.