Development of multifunctional compounds for the modulation and detection of amyloid beta species

Huang, Yiran

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https://hdl.handle.net/2142/110773 Copy

Description

Title

Development of multifunctional compounds for the modulation and detection of amyloid beta species

Author(s)

Huang, Yiran

Issue Date

2021-02-25

Director of Research (if dissertation) or Advisor (if thesis)

Mirica, Liviu M

Doctoral Committee Chair(s)

Mirica, Liviu M

Committee Member(s)

• Han, Hee-Sun
• Olshansky, Lisa
• Lu, Yi

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Aβ inhibition, multifunctional compounds

Abstract

Alzheimer’s disease (AD) is the most common neurodegenerative disease, associated with loss of memory and cognitive decline. An estimated 5.8 million Americans of all ages are living with Alzheimer’s dementia currently. The presence of amyloid plaques and neurofibrillary tangles in the brain is the hallmark of AD. Amyloid β (Aβ) peptides, the main component of amyloid plaques, are formed from the cleavage of amyloid precursor protein (APP) by β- and γ-secretases. The main alloforms of Aβ are Aβ40 and Aβ42, containing 40 and 42 amino acids, respectively. Even though Aβ40 is present in the deposits in larger amounts, Aβ42 exhibits higher neurotoxicity and aggregates more easily. In the past two decades, soluble Aβ oligomers have been found to be the most toxic form among all Aβ species through their interactions with membrane and synaptic receptors that influence intracellular systems and affect neurotransmission, leading to neurodegeneration. In addition, the amyloid deposits contain uncommonly high concentrations of metal ions such as Fe2+, Cu2+ and Zn2+, and it has been found that these metal ions promote the formation of neurotoxic Aβ aggregates. Cu and Fe ions can also cause the formation of reactive oxygen species (ROS), which exacerbates Aβ toxicity. Previously, we have reported that Cu2+ ions can slow down Aβ fibrillization and stabilize Aβ oligomers, and thus small molecules that can inhibit the interaction between metal ions and Aβ peptides, can be used as potential therapeutic compounds for AD. Positron emission tomography (PET) is a functional imaging technique that can be used for the diagnosis of AD. Recently, several PET imaging agents have been approved by FDA and can be used to visualize amyloid plaques in AD patients. However, these radiolabeled agents are employing short-lived radionuclides, such as 11C and 18F (t1/2 = 20.4 min and 109.8 min), respectively), thus limiting their widespread use. Thus, the development of longer-lived radiolabeled compounds is essential for further expanding the use of PET imaging in healthcare, and diagnostic agents employing longer-lived radionuclides such as 64Cu (t1/2 = 12.7 h, β+ = 17%, β– = 39%, EC = 43%, Emax = 0.656 MeV) are viewed as optimal PET imaging agents. Firstly, we developed a series of benzothiazole-based multifunctional compounds (MFCs) with an appreciable affinity for amyloid aggregates that can be potentially used for both the modulation of Aβ aggregation and its toxicity, as well as positron emission tomography (PET) imaging of Aβ aggregates. Among the six compounds tested HYR-16 is shown to be capable to reroute the toxic Cu-mediated Aβ oligomerization into the formation of less toxic amyloid fibrils. In addition, HYR-16 can also alleviate the formation of reactive oxygen species (ROS) caused by Cu2+ ions through Fenton-like reactions. Secondly, these MFCs can be easily converted to PET imaging agents by pre-chelation with the 64Cu radioisotope, and the Cu complexes of HYR-4 and HYR-17 exhibit good fluorescent staining and radiolabeling of amyloid plaques both in vitro and ex vivo. Importantly, the 64Cu-labeled HYR-17 is shown to have a significant brain uptake of up to 0.99 ± 0.04 %ID/g. Overall, by evaluating the various properties of these MFCs valuable structure-activity relationships (SAR) were obtained that should aid the design of improved therapeutic and diagnostic agents for AD. Then, we found the key limitation of 64Cu PET agents is that they release free radioactive isotopes due to the low binding affinity of ligands, which further decrease the signal-to-noise ratio and accuracy of imaging. As a result, a series of 1,4,7-triazacyclononane (TACN) and 2,11-diaza[3.3]-(2,6)pyridinophane (N4)-based pyridine metal-chelating compounds were designed and synthesized by incorporating Aβ interacting fragments with metal binding ligands, which allows excellent Cu chelation without losing Aβ binding affinity. Crystal structures of corresponding Cu complexes confirmed the N atom in pyridine was involved in metal binding process. In the following radiolabeling studies, the developed compounds could efficiently chelate with 64Cu according to their radio-HPLC traces and show up to 6.3 times higher radio-signal of AD over WT brain sections in autoradiography. Moreover, transition metal complexes have emerged as a viable alternative to organic compounds with distinct biological properties and have been wildly utilized for the treatment of cancer. Recently, transition metal complexes have been developed as chemical reagents capable of altering Aβ aggregation. Consequently, a series of benzothiazole-based luminescent Ir(III) complexes HN-1-8 were reported with appreciable Aβ inhibition ability in vitro and in living cells. In addition, they are capable of inducing obvious fluorescence increase when they bind to Aβ fibrils and oligomers. More importantly, compared to previously reported cationic Ir complexes, some of these inert complexes have higher log D values, which potentially allows them to have a better blood-brain barrier (BBB) permeability and hold promise to be used in vivo. Lastly, in order to understand and probe the Cu-mediated Aβ aggregation process, we rationally designed and synthesized a series of Cu-based activable sensors to detect the Cu-Aβ species in vitro and ex vivo. By linking the picolinic ester moiety with the strong Aβ binding fragment, the copper ions can rapidly catalyze the hydrolysis reaction of the ester bond to generate the high fluorescent Aβ binding molecules in vitro. More interestingly, the Cu-responsive sensors can also promptly react with Cu-Aβ oligomers and fibrils, resulting in a significant fluorescence turn-on, indicating that the probes are also able to detect Cu-Aβ species in vitro. To confirm the Aβ binding specificity of the probes, 5xFAD brain sections were stained with the developed sensors. As expected, if the brain sections are only stained with the compounds, the fluorescence images show that the sensor has poor ability to detect amyloid plaque with low fluorescence intensity. However, to mimic the Cu-rich environment in the AD brain, with the addition of excess amounts of Cu(II) to the solution, the fluorescence images clearly indicate that the Cu-responsive sensors were activated by Cu(II) and release the high fluorescent amyloid binding fluorophores which can specifically label the amyloid plaques on the 5xFAD brain sections.

Graduation Semester

2021-05

Type of Resource

Thesis

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http://hdl.handle.net/2142/110773

Copyright and License Information

Copyright 2021 Yiran Huang

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