Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson's disease

Abstract

The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.

Figure 1. Fragment probe mapping identifies potential small-molecule binding pockets in monomeric αSyn.

A. Structural properties of the 100 analyzed conformations from the NMR-derived ensemble structure of αSyn; correlation between the radius of gyration (Rg), solvent accessible surface area (SASA) and the number of non-bonded contacts. The gray circles represent the αSyn conformations, which were used for the fragment probe mapping calculations. B. 2D chemical structure of small molecules used as fragment probes in the fragment probe mapping calculations. C. 3D representation of the eight pockets identified within αSyn conformations filled with clusters of small molecules resulting from the fragment probe mapping calculations. Residues of αSyn are colored according to the amino acid sequence.

Figure 2. ELN484228 provides protection in cellular models of αSyn-mediated dysfunction.

A. ELN484228 alleviates αSyn-mediated impairment of vesicular dynamics. H4 neuroglioma cells over-expressing αSyn from a tetracycline inducible promoter were cultured for 24 hours in the absence or presence of 1 µg/ml tetracycline to induce αSyn and ELN484228 or control compound ELN484217 (compound number 38 in table S4 in Supporting Information text file S1). Open bars: without compound, black bars: with indicated amount of compound. Cells were assayed for phagocytic activity as a measure of αSyn-mediated impairment of vesicular function. 4 μ beads were added for 90 minutes and a phagocytic index was calculated by microscopic visualization. Each sample was run in triplicate and experiments were run three independent times. The phagocytic indices for each individual experiment were averaged and statistical analyses run on the final averages from the three experiments. T-test analysis of the combined averages of the three experiments revealed a significant difference in phagocytosis between Tet-induced samples with and without ELN484228 (n = 3+/− s.e.m *p≤0.001 versus no compound Tet-induced sample). B. Microglia isolated from postnatal day 1 to 3 pups from hSNCA^E46K transgenic (αSyn ) or non-transgenic littermates were incubated for 24 hours with 100 µM ELN484217 or ELN484228 followed by addition of 10 µm beads for 90 minutes. A phagocytic index was calculated by microscopic visualization (n = 3+/− s.e.m *p≤0.001). C. ELN484228 alleviates loss of dopaminergic neurons and neurite retraction induced by the A53T mutant of αSyn. Primary rat embryonic midbrain cultures were non-transduced (‘control’) or transduced with adenovirus encoding A53T αSyn, in the absence or presence of 10 µM ELN484228. The cells were then stained immunocytochemically for MAP2 and TH. Preferential dopaminergic cell death was assessed by evaluating the percentage of MAP2-positive cells that also stained positive for TH. The lengths of neurites staining positive for both MAP2 and TH were measured using the NIS-Elements software. Data are plotted as the mean ± s.e.m. n = 3 for neuron viability analysis; n = 160–206 for neurite length analysis. *p-value≤0.05, ***p-value≤0.001 versus aSyn A53T virus in the absence of compound; one-way ANOVA with Newman-Keuls post-test. D. ELN484228 reduces translocation of αSyn to the phagocytic cup. To assess αSyn translocation, H4 cells were treated with 100 µM ELN484228 and 1 µg/ml tetracycline for 24 hours; cells were then stimulated with 4 μ beads for 90 minutes. Samples were fixed and stained with 5C12 antibody to detect αSyn (red). Cells were counterstained with 488-phalloidin (green) and Hoechts (blue). A dotted circle indicates the position of the bead. E. ELN484228 reduces translocation of αSyn to synapses. Rat hippocampal neurons (∼21DIV) grown in serum-free conditions were treated for 24 hours with 1 µM ELN484228 or 0.01% DMSO vehicle. On the left side is a representative confocal microscopic image showing localization of αSyn (red) detected with 5C12 antibody, and localization of the presynaptic marker synaptophysin (green). Scale bar is 5 µm. Images were subjected to quantitative analysis and synaptic αSyn levels were determined as the amount of signal that colocalizes with the synaptic synaptophysin marker. Automated measurements were performed in Metamorph imaging analysis software to determine synaptic αSyn and synaptophysin levels by integrated intensity or pixel area, respectively. Values represent mean +/− SEM, n = 1000 terminals (αSyn) or 18 optical fields (synaptophysin) per condition, and derived from 2–3 independent cultures. Quantitation demonstrates that ELN484228 reduces the synaptic levels of αSyn in rat hippocampal neurons.