|dc.description.abstract||Abnormal protein aggregation has been linked to many neurodegenerative diseases, including Parkinson’s disease (PD). The main pathological hallmark of PD is the formation of Lewy bodies and Lewy neurites, both containing the pre-synaptic protein α-synuclein (α-syn). Native α-syn, under normal conditions, exists in a soluble unfolded state but undergoes misfolding and aggregation into toxic aggregates under pathological conditions. Toxic α-syn species can cause oxidative stress, membrane penetration, synaptic and mitochondrial dysfunction, leading to neuronal death and eventually neurodegeneration. Currently, early diagnosis and treatments targeting PD pathogenesis are urgently needed. Given its critical role in PD, α-syn is an attractive target for the development of both diagnostic tools and effective therapeutics.
This thesis consists of a series of published and unpublished papers. In Chapter 1, which was published as a review, the progress towards discovering imaging probes and aggregation inhibitors for α-syn was summarized. Since a key property of such required therapeutic agents is specific binding to the target protein, relevant strategies and techniques in the discovery of α-syn-targeted drugs are discussed. As my PhD project aimed to screen small molecules capable of binding to α-syn specifically and then discover new α-syn aggregation inhibitors from the screened structures, relevant techniques were discussed at the end of Chapter 1. Mass spectrometry was chosen to discover specific α-syn binding molecules as this technique allows rapid detection of direct interactions between molecules and proteins. The materials and methods that were used in the included publications, were summarized in detail in Chapter 2.
To provide sufficient protein for our study, the in-house α-syn having equally good quality as the commercial protein, was successfully generated in Chapter 3. Also, high yield of pure protein can be acquired from medium scale of bacteria culture, saving plenty of time and money for preparing proteins for large-scale screening. The protein expression and purification was a part of the supplementary data in the publication included in Chapter 4, where an automated screening system based on the connection of a mass spectrometer and the auto-sampler from a high performance liquid chromatograph was successfully established. This system allows computer-controlled sample loading and data acquisition with high stability and reproducibility. We first discovered a new inhibitor by screening over 4,300 pure molecules. The new compound, 3-[(3-methoxyphenyl)carbamoyl]-7-[(E)-2-phenylethenyl]-4,7- dihydropyrazolo [1,5-a]pyrimidine-5-carboxylic acid, not only significantly inhibited the misfolding and aggregation of α-syn, protected neuroblastoma cells from α-syn toxicity, but also has a more specific binding site compared with positive controls. The capability of the MS-based screening was further extended to the discovery of active components from natural products (manuscript in submission). A total of 29 marine fractions from our collaborators, were tested by MS and a new cholesterol derivative with significant inhibition of α-syn aggregation, was discovered and isolated from the active fraction. This MS-guided isolation of active components from natural products can also be applied to investigating traditional Chinese medicines with known therapeutic effects.
Post-translational modifications (PTMs) of α-syn, especially enzymatic glycosylation with N-acetylglucosamine (GlcNAc) onto the proteins hydroxylated amino acid residues, have been reported to affect the pathogenic self-assembly of α-syn. As such, manipulation of the proteins’ O-GlcNAcylation statuses has been proposed to offer a therapeutic route toward addressing PD. In Chapter 5, small peptides with different sequences and modification sites were synthesized by our collaborators. In the thioflavin-T assay, which is a golden standard for measuring α-syn aggregation, two peptides with O-GlcNAcylation at the serine site exhibited significant inhibition. Therefore, small glycopeptides that couple the protective effects of O-GlcNAc with the selectivity of recognition sequences may prove useful tools to modulate α-syn aggregation (manuscript under review). Other sources of compounds including new analogs of anle138b, which is a well-studied α-syn aggregation modulator, were evaluated. Two derivatives of anle138b exerted promising effects on the aggregation of α-syn. Interestingly, these synthesized compounds and peptides did not form protein-ligand complexes in the mass spectra, indicating that these molecules, unlike the compounds we discovered in Chapter 4, may interact with α-syn aggregates instead of α-syn monomers. In the last chapter, general conclusions of the thesis were made and future directions were also discussed.||