New Insights Toward α-Synuclein Aggregation Inhibitors: Hope For Parkinson's Disease
Parkinson’s disease is a progressive disorder of the nervous system associated with rigidity, tremors, and slow movements that also accounts for about 15% of all dementias. The pathological hallmark of Parkinson’s disease is the formation of protein clumps termed Lewy bodies. As the primary component of Lewy bodies and because it is genetically linked to Parkinson’s disease, α-syn, a presynaptic neuronal protein, has recently attracted great interest. Current research suggests α-syn contributes to Lewy bodies through intermediary soluble oligomeric conformations called protofibrils. Deposition of these protofibrils in neuronal inclusions results in cell death by affecting intracellular targets and synaptic function.
It has previously been shown that the 71-82 region of α-syn is responsible for aggregation of the entire 140mer protein. While this would suggest focusing on this region for designing inhibitors, Dr. Mason’s group noted that the known point mutations associated with early onset Parkinson’s disease are found in a different segment of the protein. After observing that the majority of known mutations are in or very close to the 46-53 region, they chose to examine a 10mer based on this peptide segment. Specifically, they created a 209,952-member library of the 45-54 sequence that included known mutations and a range of alternative residue options in the segment as shown in Figure 1. The library was then screened using a multiplexed intracellular protein-fragment complementation assay (PCA). Peptides that bound to wild-type α-syn demonstrated recombination of the essential enzyme, murine dihydrofolate reductase (mDHFR), which acted as a reporter protein and conferred cell growth for those library members that were able to concomitantly reduce the toxicity associated with α-syn aggregation. For the PCA, mDHFR was selectively inhibited using trimethoprim. This initial screen resulted in approximately 200 candidates from the library. Subsequently, a competition based PCA was undertaken with sequential selective growth conditions elucidating differences in growth rates. The competition PCA provided a most promising sequence from the ~ 200 α-syn binders initially found, which could be determined by DNA sequencing.
Figure 1. The 45-54 wild-type sequence of α-syn (top) was used to create a 209,952 member peptide library. Included were residue positions and option associated with early-onset Parkinson’s disease (underlined and bold). The sequence selected after competition selection PCA is shown.
The leading peptide candidate identified from the competitive PCA round was capable of binding to the disease-relevant wild α-syn and reduce amyloid formation more than 90%. Dr. Mason then utilized solid phase peptide synthesis (SPPS) to synthesize the wild-type 45-54 α-syn peptide (as a control) and the PCA derived peptide candidate for studying its effects on binding to the 140mer wild-type α-syn. The peptide derived from the PCA study was able to prevent aggregation of wild-type α-syn at a 1:1 stoichiometry, which together with atomic force microscopy (Figure 2) and ThT dye-binding assays, was confirmed by circular dichroism experiments that showed almost complete prevention of β-sheet formation from the random coil structure. As anticipated from the selection method the inhibitor also led to a large decrease in the toxicity associated with α-syn aggregation. Therefore, this study not only verified the methodology for creating anti-α-syn aggregation peptide inhibitors, but also provided a promising peptide sequence that could function as a scaffold for potential drug candidates.
Figure 2. Shown on the left are atomic force microscopy images of toxic amyloid fibrils formed by the α-syn protein. These are found in the brain of sufferers of Parkinson’s disease. On the right is the same protein that has been mixed with the newly derived peptide. The peptide binds to the sticky parts within the α-syn protein and almost completely prevents the fibril from forming.
Dr. Mason acquired CEM’s Liberty Blue™ Peptide Synthesizer in late 2013. The system has allowed him to rapidly synthesize peptides needed for his research. It has also allowed him to obtain substantial cost and time savings over purchasing peptides which has been valuable for his work. As an added benefit, Dr. Mason is no longer concerned about having enough peptide material for experiments issues as he can quickly and efficiently make more. Dr. Mason commented “With the Liberty Blue, we are willing to try more research and can take on more risky challenges. The Liberty Blue has been a great addition to our laboratory and I would highly recommend the system to other researchers”.
Dr. Jody Mason’s research is published:
Intracellular Screening of a Peptide Library to Derive a Potent Peptide Inhibitor of a-Synuclein Aggregation
Journal of Biological Chemistry, 2015, 290 (12), 7426–7435