Abstract:
The accumulation of misfolded α-synuclein (αSYN) is a hallmark of synucleinopathies, including Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies. Positron emission tomography (PET) imaging of αSYN aggregates would be a game changer in facilitating the diagnosis and monitoring of synucleinopathies and the development of novel disease-modifying therapies. Despite the considerable endeavors leading to the identification of several potential αSYN PET tracers, none have been successfully translated to clinical applications thus far. This thesis work aimed to advance PET tracer development efforts towards αSYN PET imaging in synucleinopathies by focusing on the preclinical evaluation of potential radiotracers targeting αSYN aggregates.
The primary part of this work comprises a comprehensive in vitro and in vivo assessment of MODAG-005. Its binding characteristics were studied in vitro using fibril binding assays and brain tissue autoradiography, and subsequently in vivo in three animal models of αSYN. Exceptional binding of [3H]MODAG-005 to aggregated αSYN was demonstrated by its high binding affinities to four distinct αSYN fibril strains (Kd = 0.2–1.9 nM). Autoradiography experiments on brain tissues corroborated the findings, presenting a clear macroscopic and microscopic evidence of [3H]MODAG-005 binding to glial cytoplasmic inclusions in MSA brain tissues with an unprecedented high binding affinity (Kd = 0.25 nM), exceeding those of other reported αSYN tracer candidates. We confirmed the feasibility of using [11C]MODAG-005 for in vivo imaging by demonstrating its specific target binding and improved imaging contrast in the acute αSYN fibril injection rat model, as well as its increased retention in the pathology-rich brain regions in the A30P αSYN mouse model of PD. Additionally, we generated a rat model with accelerated αSYN pathology progression by performing fibril seeding in the BAC αSYN transgenic rats. However, the target binding of [11C]MODAG-005 in these fibril-seeded BAC αSYN rats could not be conclusively determined due to relatively low levels of αSYN pathology. Furthermore, the potential applicability of MODAG-005 in facilitating the clinical development of a therapeutic candidate was demonstrated, which confirmed the drug target engagement of anle138b, both in vitro and in vivo. Limitations of this work include the inconclusive binding specificity of the tracer to Lewy bodies and Lewy neurites in PD tissues due to concomitant pathology, as well as the presence of off-target binding in brain tissues of Alzheimer’s disease and progressive supranuclear palsy, highlighting the pervasive challenge and the need for improvement in future development. Overall, the preclinical evidence strongly supports [11C]MODAG-005 as a promising tracer for αSYN, thus warranting its progression to clinical studies for in vivo validation in human subjects with synucleinopathies.
In the second part of this work, we aimed to identify and evaluate additional ligands targeting αSYN. Three novel classes of αSYN ligands, designed based on different approaches, were screened using fibril binding assays. The first library, developed via the molecular hybridization of diphenylpyrazole and phenothiazine, showed decreased binding affinity to αSYN fibrils compared to their parent molecules. Screening of the second library, consisting of 2-styrylbenzothiazole derivatives designed by modification of a fluorescent probe, led to the discovery and selection of two promising ligands exhibiting selectivity towards αSYN fibrils for 18F-radiolabeling. Nevertheless, both [18F]PFSB and [18F]MFSB displayed high non-specific binding in human brain tissues through autoradiography, which was corroborated by a very slow brain clearance of [18F]MFSB in PET pharmacokinetic study. For the third library, screening of the isoxazole derivatives, which were generated based on a pharmacophore modelling approach, led to the selection of five ligands for radiolabeling and further evaluation. Notably, we encountered several challenges commonly discussed in the literature for αSYN PET tracer development, such as low affinity, insufficient selectivity, and unfavorable pharmacokinetics. Our findings emphasized the use of diversified development strategies to drive a range of possible outcomes and highlighted the increasing significance of computational approaches to enhance efficiency. To overcome the persistent challenges and accomplish the aspiration of αSYN PET imaging, the adoption of upcoming innovative techniques will be pivotal.
Alpha-synuclein