Exploiting A Cryptic Hydrophobic Pocket In ApoE4 To Suppress Neurotoxic Aggregation

Author Block: Aliaksandra Kursit, Madhumalar Subramanian, Saltuk Mustafa Eyrilmez, Sergio Manuel Marques, Jiri Damborsky, Zbynek Prokop, David Bednar, Martin Marek.
Experimental Biology and RECETOX – Faculty of Science, Masaryk University, Brno, Czech Republic.

Abstract:
Background: Apolipoprotein E (ApoE) is a glycoprotein that plays a key role in lipid and cholesterol transport in the brain. The ApoE4 isoform is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). ApoE4 differs from the non-pathological ApoE3 by a single C112R mutation, which triggers small-to-large structural changes. These rearrangements confer the neurotoxicity of ApoE4 through higher aggregation propensity. The rotation of tryptophan 34 (W34) exposes a hydrophobic pocket, which could be a target for drugs aimed at inhibiting ApoE4 aggregation. Additionally, when ApoE4 binds to heparan sulfate through its heparin-binding site, the W34 residue also rotates. In this study, we developed a pipeline to explore small molecules that target the hydrophobic pocket and heparin-binding site of the ApoE4 protein, starting with a blind docking approach, followed by experimental evaluation of the binding affinity and anti-aggregation effects.
Methods: We conducted a virtual screening of FDA-approved drugs against the ApoE4 protein. The compounds’ prioritisation was based on their predicted binding affinity, commercial availability and their predicted ability to cross the blood-brain barrier. We selected the compounds with the best predicted binding affinities for further experimental evaluation. We have established a platform for determining binding affinity by monitoring tryptophan fluorescence and neurotoxic aggregation propensity using static light scattering measurements.
Results: We identified sixteen small molecules exhibiting binding affinities towards the ApoE4 isoform in micromolar ranges. Complementary screening of the compounds using light scattering revealed a total of ten compounds that effectively inhibited ApoE4 aggregation. Upon sorting, two compounds showed both higher binding affinity and aggregation inhibition potential. Additionally, seven compounds demonstrated either moderate binding affinity or suppression of neurotoxic aggregation.
Conclusion: In this work, we established a complex pipeline for the identification and validation of small molecules targeting the pathological properties of ApoE4. Our findings, along with further structural analysis, will enhance the understanding of the pathophysiological behaviour of ApoE4 and offer valuable chemical scaffolds for developing new candidate drugs for AD treatment.