Neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, and ALS affect more than 50 million people today and are projected to cost over $9 trillion by the year 2050. On top of that, there are currently no cures for any of these diseases. The overarching theme of these diseases is the formation of protein aggregates and insoluble fibrils which result in neuron death. The fibrils then provide a template to further the aggregation of monomeric protein in a prion-like fashion.
The complete biological role of α-Synuclein is not fully understood; however, it is known that this protein is vital to vesicle recycling and neurotransmitter release in the dopaminergic neurons of the brain. α-Synuclein aggregate formation has a toxic effect on these neurons leading to a drastic decrease in dopamine in the brain and resulting in the well-known symptoms of Parkinson’s disease. These symptoms include a tremor, slowness of movement, a loss of balance and coordination, and even a loss of smell.
The development of small molecules that can prevent abnormal protein interactions related to these diseases is one of the strategies used by scientists in the development of therapeutics. An example of a small molecule scaffold that has been tested in several protein aggregation-related diseases is the oligopyridylamide (OP). The OP is ideal for inhibiting protein aggregation because it mimics the structure of a peptide by presenting its functional groups in the exact spacing to interact with proteins in their native fold.
Our Novel 2D-FAST Approach
Previous synthetic methods for OPs were far more laborious, requiring 14 synthesis reactions and 11 chromatography purification steps to generate one tripyridyl. Following this methodology, an individual monopyridyl must be synthesized for each functional group which limits the diversity of the library. There is no systematic optimization involved.
In this study, we developed a new method that pairs a more simplified synthesis of oligopyridylamides with stepwise Thioflavin T (ThT) screenings to increase the affinity for the target protein. Our method is called the 2-dimensional Fragment-Assisted Structure-based Technique (2D-FAST), because it forms a library of molecules with an increasing variety and number of functional groups that are presented on the OP scaffold.
By performing a substitution reaction with an aromatic chlorine, we can generate a diverse library of di- and tripyridyls from one scaffold and virtually any amine or thiol containing molecule. Stepwise screening of the mono- and dipyridyl libraries allows us to identify which fragment should be expanded to identify the molecule that is most effective at inhibiting the aggregation of the target protein.
We utilized the 2D-FAST method to find the best molecule to inhibit the aggregation of α-Synuclein, which is the hallmark of Parkinson’s disease. By screening a library of monopyridyls in a ThT dye-based aggregation assay, we determined that the carboxylic acid functional group (NS41) was the most active antagonist of α-Synuclein aggregation. After synthesizing and screening a library of di-pyridyls with a carboxylic acid in the first position, we determined that the molecule with a cyclohexyl group (NS55) was the most active. In the final stage of synthesizing and screening a library of trimers with the established carboxylic acid and cyclohexyl functional groups, we found that the indole in the third position (NS132) was the best antagonist of α-Synuclein aggregation.
As anticipated, we found that our molecule had limited cell permeability which we attributed to the carboxylic acid functional group. In order to perform cellular and in vivo biophysical experiments, we synthetically converted the carboxylic acid to a hydroxamic acid analog (NS163) as commonly performed in medicinal chemistry. This modification increased the cell permeability of the molecule while maintaining its antagonistic activity towards α-Synuclein.
Efficacy of OPs in various Parkinson’s disease models
We used a HEK-293 cell line, which expresses a known Parkinson’s disease mutant of α-Synuclein (A53T) tagged with a yellow fluorescent protein to assess our molecule’s ability to inhibit the disease-relevant fibril catalyzed aggregation of α-Synuclein. By treating the cells with preformed fibrils of α-Synuclein followed by our molecule, we observed a substantial decrease in aggregate puncta formation with confocal microscopy. This marked decrease was observed in cells treated with NS163, as well as the less cell permeable analog NS132, compared to the control.
To test for the ability of our molecules to rescue Parkinson’s disease phenotypes and prevent the loss dopaminergic neurons, we used 2 different strains of microscopic worms known as C. elegans as models for the disease. The NL5901 strain of C. elegans expresses human α-Synuclein protein with a yellow fluorescent protein tag in their body wall cells. The UA196 strain expresses human α-Synuclein and a green fluorescent protein in their 6 dopaminergic neurons. As the neurons degenerate, the observed fluorescence dissipates making this organism ideal for testing neuroprotective effects. Both of these strains exhibit the symptoms of Parkinson’s disease as they age, including reduced motility and an inability to sense their food.
In the NL5901 strain, we observed a significant decrease in protein aggregate formation and a significant increase in motility after treatment with both ligands, NS132 and NS163, compared to the untreated diseased worms. We observed the same motility rescue as well as neuroprotection of the dopaminergic neurons in the UA196 C. elegans when treated with our molecules. Using a chemotaxis assay, we showed that NS132 and NS163 are also able to rescue the food sensing behavior of both diseased strains of the worms.
Since diseases like Parkinson’s are most often diagnosed after the onset of symptoms, we wanted to test the activity of our ligands in a post-disease onset model. After treating the worms on day 5 (following ~28% neuron degeneration), we observed a rescue of the dopaminergic neurons by both NS163 and NS132, albeit less so than NS163.
Altogether, our data suggests that NS163 and NS132 are potent inhibitors of α-Synuclein aggregation both in vitro and in vivo. Most notably, our OPs can effectively rescue Parkinson’s disease phenotypes in a post-disease onset model of the disease. We are optimistic that our technique will develop lead therapeutics for Parkinson’s disease and other neurodegenerative diseases.