Date of Award

2022

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

College of Natural Science and Mathematics, Chemistry and Biochemistry

First Advisor

Sunil Kumar

Second Advisor

Scott Horowitz

Third Advisor

Daniel Linesman

Fourth Advisor

Daniel Paredes

Keywords

Parkinson's disease, Therapy, Biochemistry, Biophysics

Abstract

Alpha-Synuclein (αS) – a neuronal, disordered, presynaptic protein – aggregates into amyloid fibrils and accumulates in the substantia nigra pars compacta of Parkinson's Disease (PD) patients. The aggregation and accumulation of αS amyloid fibrils leads to death of dopaminergic neurons; a hallmark of PD. Although it’s not clear why αS aggregates, prior studies have found that intrastriatal injection of fibril alone is sufficient to cause PD pathology in mouse and non-human primates models. These observations implicate αS as a therapeutic target against PD.

Unfortunately, there are three caveats when attempting to target αS. First, αS is a neuronal protein expressed in the midbrain. A therapeutic agent should be able to cross the Blood Brain Barrier (BBB) and the cell membrane to target αS. Second, αS is intrinsically disordered and dynamic, and most drug design platforms target static tertiary protein structures. Third, it’s not clear which sequence domain initiates the aggregation process. αS has 140 residues with the first 90 N-terminal residues typically found in the amyloid core. The specific N-terminus sequence that nucleates aggregation isn’t characterized, thus there isn’t a known αS region to target.

To address these problem, we prepared and screened a library of small molecule alpha-helical mimetics called ‘Foldamers’ against αS aggregation. Foldamers fold to a helical conformation and present their functional groups at similar spatial and angular locations to peptide alpha helix, making them great tools for targeting protein-protein interactions. Our screen identified SK129, an oligoquinoline foldamer that is a potent inhibitor of in vitro and in vivo αS aggregation as demonstrated in human cells (HEK293 Cells) and C. elegans (NL5901, PD model). Then, we tested the hypothesis that SK129 could be binding αS in the sequence that nucleates aggregation. We performed Heteronuclear Single Quantum Coherence NMR experiments and identified sequences 6- 12, 15-23, 36-45 and 47-53 as potential binding sites for SK129. We made αS mutants with deletions in the identified sequences – Δ6-12, Δ15-23, Δ36-45 and Δ47-53 – and performed aggregation experiments. Mutants Δ36-45 and Δ47-53 couldn’t form amyloid fibrils suggesting these sequences are critical for aggregation.

Additionally, we developed an Oligopyridylamide (OP) based 2-Dimensional Fragment-Assisted Structure-based Technique (2D-FAST) and utilized it to develop a potent inhibitor of αS aggregation – NS163. This molecule was able to prevent cellular αS aggregation in body wall muscle cells (NL5901, C. elegans model) and dopaminergic neurons (UA196). NS163 was also able to halt disease progression in post-disease onset PD models.

We also explored the effect polyamines have on αS aggregation. Since one of hallmarks of PD is degradation resistant αS aggregates, we tested if aggregating αS in the presence of different polyamines confers degradation resistance to resulting aggregates. We found αS aggregated with spermine is resistant to enzymatic degradation. Spermine also perturbs the aggregation equilibrium to favor the formation of higher amounts of insoluble protein.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Jemil Ahmed

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

201 pgs

Discipline

Biophysics

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