Tricyclic Antidepressants as Prospective α-Synucleinopathy Fighters: Evidence of Protein Binding and Antiaggregation...

Intrinsically disordered proteins (IDPs) like α-synuclein are pivotal in neurodegenerative diseases but present formidable challenges for drug discovery due to their conformational heterogeneity and lack of defined binding pockets. Here, we report the finding that clinically approved tricyclic antidepressants imipramine, amitriptyline, and doxepin bind directly to monomeric α-synuclein and inhibit its fibrillation, a key process in Parkinson's pathology. Using a combination of NMR spectroscopy (WaterLOGSY and chemical shift perturbation, CSP) and molecular dynamics simulation methods, we elucidate a nonspecific, multiligand binding mechanism of tricyclic antidepressants predominantly targeting the C-terminal domain of the protein. The experimental CSP magnitudes correlate with the inhibitory potency of ligands, with imipramine emerging as the most potent inhibitor, and agree with the contact probabilities with different residues from simulations. The protein-ligand binding is driven by a dynamic combination of electrostatic attraction of positively charged ligands to anionic side chains, π-stacking with aromatic residues, and hydrophobic contacts. Moreover, simulations show that a single ligand molecule frequently engages in simultaneous salt-bridge and π-stacking interactions, unlike some previously studied α-synuclein binders. Our findings position tricyclic antidepressants as promising scaffolds for targeting α-synuclein and demonstrate the efficiency of molecular dynamics approaches for the description of interactions between small drug molecules and IDPs.