Addressing retromer dysfunction via PPI-orchestrating small molecules as a therapeutic strategy for neurodegeneration

Alzheimer's disease (AD) and Parkinson's disease (PD) are the most frequent neurodegenerative conditions worldwide, and the urgency to discover disease-modifying therapies represents a strong unmet medical need. Both proteinopathies involve pathological protein-protein interactions.

To unveil proteome-wide different protein interactions in cellular models of neurodegeneration, we set up a structural proteomics method combining Limited Proteolysis-coupled Mass Spectrometry (LiP-MS) with Terminal Amine Isotopic Labeling of Substrates (TAILS) in the SH-SY5Y cellular model of altered dopamine homeostasis, an early step in PD pathogenesis. Among the proteins that changed their surface exposure, dopamine showed to affect retromer-mediated trafficking, and in particular the cargo-recognition core (composed by the vacuolar sorting protein VPS35-VPS26-VPS29 heterotrimer). Validation by indirect immunofluorescence of VPS35 allowed us to hypothesize cytoplasmic accumulation of dysfunctional vesicles. Our observations are in line with increasing evidence highlighting dysfunctional endolysosomal trafficking as a potential pharmacological target in neurodegenerative diseases.

Here, we propose the rational design of therapeutic molecular glues, i.e., small molecules to enhance the stability and restore the functionality of the retromer complex, by stabilizing the VPS35-VPS29 and the VPS35-VPS26 protein-protein interfaces.

Two distinct small molecule libraries were considered: a laboratory library, and one generated and refined through a computational pipeline based on physicochemical properties favoring oral bioavailability (Lipinski rules), cell permeability and blood brain barrier crossing, and, when available, considering cytotoxicity, binding profiles, and additional early Absorption, Distribution, Metabolism and Excretion (ADME) data or further pharmacokinetics information in the case of more advanced compounds.

After a computationally-enabled determination of the binding pockets of interest on the VPS35-VPS29 and the VPS35-VPS26 complexes, molecular-docking based in silico screening was performed with flexibility conditions for the amino acid side chains of the most relevant residues in the protein sites identified, and complete flexibility for the ligand candidates. After collecting SAR insights and performing hit optimization, the mechanism of action of the resulting lead compounds will be verified in vitro.

In conclusion, this approach offers a new therapeutic strategy for treating neurodegenerative diseases by leveraging molecular glues to stabilize intracellular protein-protein complexes.