Objective/Rationale:
Using high-throughput screening, we have identified a small-molecule compound that rescues toxicity in a yeast model of Parkinson’s Disease and in mammalian neurons. This compound appears to bind to GAPDH, a highly conserved enzyme that is also associated with cell death due to different kinds of stress. We will use the power of yeast genetics to quickly obtain enough information about the way the compound interacts with GAPDH to allow us to validate this potential drug target in neuronal models of PD.
Project Description:
We will characterize the activity of our compound by measuring its effects on GAPDH’s enzymatic activity, its physical modifications in response to stress, its interactions with other proteins, and its cellular localization. We will validate GAPDH as a drug target first in our yeast model, and then in cultured neurons, by introducing appropriate genetic alterations to mammalian GAPDH and measuring the effects of these manipulations on cell death. Finally, genetic manipulations that suppress toxicity in the yeast and cell culture systems will be tested for rescue in whole animal rodent models of PD.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
This research will provide a validated cellular target for further screening aimed at isolating clinically useful drug candidates for PD. Our current compound was identified using a functional approach in yeast. Validation of its molecular target will allow the development of potential drugs with low nanomolar target-binding affinities and druglike pharmacokinetic and toxicity profiles in animal models and human clinical trials.
Anticipated Outcome:
We hope to validate a specific mechanism of compound interaction with a regulator of cell viability as a promising therapeutic strategy for Parkinson’s disease.
Final Outcome
Using brewer's yeast modified to produce too much of the alpha-synuclein protein in its cells, the team screened 115,000 small compounds to see which ones alleviate the Parkinson's-like traits. During a screen, a compound is added to a small amount of yeast. Researchers can then easily and efficiently detect if that compound changes the yeast's growth rate, compared to a control. The technique takes advantage of the yeast's normally fast growth, which allows researchers to quickly test thousands of compounds, a process that is not possible in other common Parkinson's disease models.
Four compounds were found to restore the alpha-synuclein yeast cells' growth to 50 percent of normal yeast cells. Yeast cells that were not treated with the compounds died. The four compounds have similar chemical structures, a finding that indicates they may be acting on the same target or targets. The researchers also identified two commercially available compounds with similar chemical structures and used those in further tests.
To determine if the six compounds would work in animal models of Parkinson's, the scientists tested the compounds in C.elegans (a roundworm) and rat neurons. In both of these disease models, cells overproduce alpha-synuclein resulting in the same deleterious effects as in the yeast model. During testing, the first four compounds were able to rescue the round worms, while in the rat neurons, three of the four original compounds and one of the commercial compounds improved the nerve cells' growth.
In all of the models, the compounds improved protein trafficking and decreased mitochondrial damage.
Results of this project were published in the journal Disease Models and Mechanisms.
Researchers
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Susan Lindquist, PhD