Funded Studies
Objective/Rationale:
We want to develop a series of secondary assays that will allow us to shortlist likely drug candidates for therapy against Parkinson’s disease. These assays will bridge the gap between a primary and a tertiary assay. The primary assay is a high throughput screen (developed in collaboration with Wyeth Research Ltd.) which is expected to yield several thousands of potential drugs. The tertiary assay is the final selection screen based on cellular assays, which will involve around a hundred compounds.
Project Description:
Aggregation of the protein α-synuclein is a key event in the development of PD. Our high throughput assay selects compounds for their ability to prevent this aggregation under one specific set of conditions. In our secondary assays, we will test these compounds for their anti-aggregative properties under a more diverse set of conditions to allow us to make a more qualified assessment of their therapeutic potential. In practice we will test their performance under more challenging conditions (conditions that make it easier for α-synuclein to aggregate) as well as their ability to block the toxicity of already formed aggregates. We will also analyze in more detail how the best of these candidates bind to α-synuclein at different stages of aggregation.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The secondary assay is an important step in the selection of compounds that feed into a drug development programme at Wyeth Research Ltd. We anticipate that the best of these compounds will ultimately keep aggregation of α-synuclein at bay and therefore act as a deterrent to the development of PD. Our assumption is that by blocking the aggregation of α-synuclein in the brain, we can effectively prevent the series of biochemical events leading to PD.
Anticipated Outcome:
By studying a large and diverse group of compounds which presumably interact with our target protein in many different ways, we expect to learn a great deal about how we can control the unwanted aggregation of a brain protein and how proteins respond to different attempts to "rein them in". When we combine this with our subsequent cellular assays, we will also learn how easily we can transfer these "test tube" data to living cells.
Final Outcome
The primary high throughput screen to identify inhibitors of alpha-synuclein aggregation has now been completed, and 136 different compounds have been identified as potential candidates for further investigation. A few of these were identified as having nanomolar potency and numerous candidates had mid-micromolar effects, which are good starting points for subsequent drug development. The best 63 of these compounds have been evaluated to rank their anti-aggregation potency, identifying several compounds that perform very well in two orthogonal aggregation assays. Further characterization of their interactions with alpha-synuclein are expected to help establish a general understanding of the mechanism of interaction between alpha-synuclein and aggregation-inhibiting compounds and provide the basis for an actual drug development program.
Work associated with our development of high-throughput screening against Parkinson’s Disease (based on funding from the Danish Ministry of Science, Technology and Innovation, on which the MJFF award builds) has resulted in the following articles:
L. Giehm, C.L.P. Oliveira, G. Christiansen, J.S. Pedersen, D.E. Otzen, SDS-induced fibrillation of alpha-synuclein: An alternative fibrillation pathway, J. Mol. Biol. 401, 115-133 (2010).
L. Giehm, D.E. Otzen, Strategies to increase the reproducibility of alpha-synuclein fibrillation in plate reader assays, Anal. Biochem. 400, 270-281 (2010).
The following is submitted:
L. Giehm, D.I. Svergun, D.E. Otzen, B. Vestergaard, Low resolution structure of an alpha-synuclein oligomer that accumulates during fibrillation, Submitted (200X).
An article describing the development of the screening assay is underway in collaboration with Pfizer, once permission to publish has been granted.