Development of a sensitive in vitro system for anti-alpha-synuclein aggregation drug screening.

Parkinson's disease is a neurodegenerative disease, without cure, which affects one person in every 500. The current treatment for this disease is only symptomatic, partially relieving the characteristic motor symptoms. Unfortunately, these treatments do not stop the disease progression and therefore with time the therapy in Parkinson's patients starts to fail and they become increasingly disabled and affected by the drugs side effects. Parkinson's patients are therefore in the need of new therapies that impede further degeneration once the disease has been diagnosed.
At the brain level Parkinson's disease is characterized by reduced dopamine levels due to the death of a specific type of neurons in the substantia nigra. Without dopamine patients suffer tremors, muscle stiffness and slowness of movement. The cause of the neurons death is still unknown, however it has been associated with the presence of specific inclusions called Lewy bodies. These PD characteristic inclusions contain a misfolded version of an otherwise normal protein called alpha synuclein.
A current hypothesis, well supported by scientific data, to explain the progression of the PD pathology throughout the brain involves the aggregation of alpha synuclein and its spread from cell to cell. In view of this we focused on this protein and on trying to develop a system for drug screening that would stop the aggregation associated with the PD pathology.
Alpha synuclein shares several biochemical characteristics with the prion protein, which also misfolds and aggregate in the patients' brain. An in vitro technique, the PMCA (Protein misfolding cyclic amplification) reproduces the aggregation of prions in a test tube by subjecting recombinant protein to cycles of incubation and sonication. We postulated that such a technique could be also used to form recombinant alpha synuclein fibrils and subsequently become a new tool to study how to stop its conversion into the pathological conformation.
Our preliminary results indeed show that we can generate alpha synuclein fibrils by PMCA. These fibrils, which formation reaches a maximum after just 12 hours of reaction, show the typical characteristics of Parkinson's disease synuclein fibrils, such as a high content in beta-sheet structure (indicative of amyloid formation) and partial resistance to enzymatic digestion.
In addition we tested the effect of a panel of 10 drugs with diverse chemical structures and uses in the PMCA system. The reaction was reproducibly inhibited only by the two already reported anti amyloid compounds among the 10 drugs. This suggests that the alpha synuclein PMCA can be used to specifically select drugs that would inhibit the aggregation of recombinant alpha synuclein. Using this system replaces animal use until hits with real potential have been shortlisted.
In the view of our results, we propose to test drug libraries containing compounds already approved for its use in other disease areas, and therefore had all the "animal research" already done, with our alpha synuclein PMCA. Using these drug-approved libraries instead of new drug development has an economical advantage, but more importantly, it drastically shortens the research animal models needed to effectively bring the drug to the patient.
Our alpha synuclein PMCA system can be used as a high throughput assay, giving the advantage of testing 96 samples per experiment and therefore providing potential drug hits in just 24 hours. After selecting the drugs with inhibitory effect for alpha synuclein aggregation we propose to validate their efficacy and biological relevance using existing PD cellular and mice models.
Undertaking this project would allow us to gain knowledge about the mechanisms of alpha synuclein aggregation and accumulation. More important, it would produce results that could translate into slowing down or even stopping the progression or Parkinson's disease with minimal animal use.

Parkinson's disease is a brain disorder characterized by aggregated alpha synuclein (a-syn) inside intraneuronal inclusions known as Lewy bodies.
We studied if protein misfolding cyclic amplification (PMCA), used to replicate prion protein aggregates could be used to grow a-syn aggregates. Briefly, proteins are subjected to cycles of sonication and incubation and fibril formation is monitored by increased Thioflavin T fluorescence.
Our electron microscopy, circular dichroism and native gels results indicate that stable a-syn fibrils were formed. We also tested the a-syn anti-aggregation activity of several drugs and the results suggest that the a-syn PMCA is robust system for anti-aggregating drug screening. So we propose to use previously approved drugs, with animal testing already done, to repurpose into the treatment for Parkinson disease using the a-syn PMCA. The proposal has two aims:
1. Drug screening by high throughput a-syn PMCA: We propose to screen a library from previously FDA approved drugs with high chemical and pharmacological diversity and other with compounds marketed in Europe and/or Asia but not yet in the USA. A cut off of 50% inhibition in the HTS a-syn PMCA will be set up to prioritize the study of the drugs with higher potential. Other prioritization will be done based on the EC50, route of administration and side effects to reduce suffering in mice models.
2. Study of the hits biological relevance in cell and mice models of Parkinson's disease We plan to use PC12 cells overexpressing human alpha synuclein to study the presence or absence of insoluble alpha synuclein in the cells after transducing them with fibrils and in the presence of the hit drugs. We also want to use the A53T mice model that has widespread a-syn aggregation in the brain. The drug effect will be tested, before and simultaneously to the clinical symptoms appearance. With this time points we hope to stop the progression of the disease and reduce animal suffering.

The first 3Rs impact (replacing) of this grant application derives from the development of a high throughput in vitro assay that would allow us to screen for anti-amyloid drugs that could prevent the aggregation of alpha-synuclein without the need of using animal models. Our aim is to replace animal models during the whole initial process of hit selection by using a fast and reliable method of drug screening.

The second impact (refining) impact is due to our interest in drug repurposing. The generation of new drugs from the beginning of the process until they go into the market and are made available for the patients in need of them is from 10 to 15 years. Also, hundreds of animals are used to test those drugs, many of which will never be approved due to their side effects or biological irrelevance once they are used in animal models. Repurposing has two main advantages, the first related to time and money, as the drugs have been already approved for other use they already meet criteria such as "drug likeliness", stability, membrane permeability, solubility and a long etc. The second advantage relates to the animal use. As those drugs have been already approved the entire animal related work to test toxicity, therapeutic doses, etc has been done. This allows us to reduce the number of animals that we need to use and also to refine the experiments in a way that would be the least harmful for the experimental animals. Also, part of the proposal is to establish a cell model assay. This assay will allow us to further shortlist the hits from the high throughput assay and study the biological relevance of those hits in a still animal-free experimental design.

Finally we have a third impact of the 3Rs (reduction). At the last stage of the project we cannot escape from using animal models, transgenic mouse expressing a pathological form of the alpha synuclein, however we expect to have reduced the hundreds of initial drugs to less than 1% thru the initial screen assay and the cellular assay, meaning a great reduction of the numbers than one would have use otherwise. At this point priority will be given to those drugs whose side effects are lower to ensure minimizing animal distress.