Small-molecule PET Tracers for Imaging Proteinopathies

Abstract

In this chapter, we provide a review of the challenges and advances in developing successful PET imaging agents for 3 major types of aggregated amyloid proteins: amyloid-beta (Aβ), tau, and alpha-synuclein (α-syn). These 3 amyloids are involved in the pathogenesis of a variety of neurodegenerative diseases, referred to as proteinopathies or proteopathies, that include Alzheimer disease, Lewy body dementias, multiple system atrophy, and frontotemporal dementias, among others. In the Introduction section, we briefly discuss the history of amyloid in neurodegenerative diseases and describe why progress in developing effective imaging agents has been hampered by the failure of crystallography to provide definitive ligand-protein interactions for rational radioligand design efforts. Instead, the field has relied on largely serendipitous, trial-and-error methods to achieve useful and specific PET amyloid imaging tracers for Aβ, tau, and α-syn deposits. Because many of the proteopathies involve more than 1 amyloid protein, it is important to develop selective PET tracers for the different amyloids to help assess the relative contribution of each to total amyloid burden. We use Pittsburgh compound B to illustrate some of the critical steps in developing a potent and selective Aβ PET imaging agent. Other selective Aβ and tau PET imaging compounds have followed similar pathways in their developmental processes. Success for selective α-syn PET imaging agents has not been realized yet, but work is ongoing in multiple laboratories throughout the world. In the tau sections, we provide background regarding 3-repeat (3R) and 4-repeat (4R) tau proteins and how they can affect the binding of tau radioligands in different tauopathies. We review the ongoing efforts to assess the properties of tau ligands, which are useful in 3R, 4R, or combined 3R-4R tauopathies. Finally, we describe in the α-syn sections recent attempts to develop selective tracers to image α-synucleinopathies.

Figure 1

Venn diagram of the three aggregated amyloid proteins discussed in the chapter and their associated neurodegenerative diseases. Abbreviations: cerebral amyloid angiopathy (CAA); Alzheimer’s disease (AD); AD Parkinson’s disease (PD); dementia with Lewy bodies (DLB); Parkinson’s disease with dementia (PDD); multiple system atrophy (MSA); Lewy body variant Alzheimers’s disease (LBVAD); chronic traumatic encephalopathy (CTE); frontal temporal dementia with parkinsonism-17 (FTDP-17); corticobasal degeneration (CBD); and progressive supranuclear palsy (PSP).

Figure 2

Secondary structures of proteins showing alpha-helix (A) and beta-pleated sheet (B) configurations. Hydrogen bonding between an amide nitrogen (blue) and an amide oxygen (red) in beta-pleated sheet structures takes place at the interfaces between two protein strands (edge-on-edge interactions), which is very different than the intramolecular hydrogen bonding of alpha-helix proteins. Note that the R groups on the α-carbons are either above the beta-sheet or below the beta-sheet. This presents sites for potentially different binding interactions with ligands on the two sides of the beta-sheet. Figure 4 displays the R group orientations from a different perspective.

Figure 3

Fluorescent images of consecutive paraffin sections from the frontal cortex of an Alzheimer’s disease brain stained with: (A) the pan-amyloid dye X-34 (100 μM), which binds to both Aβ-containing plaques and cerebral amyloid angiopathy (CAA) and tau-containing neurofibrillary tangles (NFTs); and (B) the Aβ-selective dye 6-CN-PiB (10 μM), which is a highly fluorescent, close structural analogue of PiB with identical binding properties. Note that X-34 stains Aβ plaques and CAA as well as NFTs while 6-CN-PiB stains only Aβ-containing plaques and CAA. Panels C and D are high magnification images of the area boxed in A and B, respectively. Bar = 50 μm in C.

Figure 4

Hypothetical binding orientation of thioflavin-T (A) to a beta-sheet structure (B) resulting from interactions with R groups oriented above the plane of the fibril’s long axis. PanelC shows multiple thiofavin-T molecules binding to the fibril. (adapted from (14))

Figure 5

Structure of cationic thioflavin-T and three neutral thioflavin-T analogues resulting from the removal of the quaternary methyl group on the benzothiazole nitrogen. The resulting compounds were termed benzothiazole anilines or BTAs and possessed different degrees of N-methylation (denoted by the trailing number).

Figure 6

Relationship of normal mouse whole brain uptake and logP_C18 (logarithm of the octanol-water partition coefficient estimated by relative HPLC retention on a lipophilic C₁₈ column) of different ¹¹C-labeled-BTA compounds at 2 min following intravenous tail-vein injection. Note that the brain uptake SUV of most BTAs exceeded 1.0. (adapted from (56)).

Figure 7

Competition binding plot of three neutral BTA analogues shown in Fig. 5 and thioflavin-T (Th-T) for Aβ_1–40 fibrils with [³H]BTA-1. All three BTA analogues demonstrated much higher affinity for Aβ_1–40 fibrils than thioflavin-T.

Figure 8

Pittsburgh Compound B (PiB) standardized uptake value ratio (SUVR) images relative to cerebellar grey matter in an Aβ-negative (Aβ−) cognitively normal elderly subject (top row) and an Aβ-positive (Aβ+) Alzheimer’s disease (AD) subject (bottom row) determined 40–60 min post injection. Retention of PiB in the cognitively normal subject is primarily a result of non-specific binding in white matter while the AD subject shows extensive tracer retention in frontal cortex, posterior cingulate, and parietal regions.

Figure 9

Structures of three ¹⁸F-labeled Aβ imaging agents approved for clinical scanning in the US, Europe, and Asia. Vizamyl is an ¹⁸F-labeled derivative of PiB, and Amyvid and Neuraceq differ in structure by one atom in the central ring (N or C).

Figure 10

Diagrammatic representation of the six isoforms of tau varying in length from 352 to 441 amino acids. Three isoforms contain 3-repeats (3R) and three isoforms contain 4-repeats (4R) in the critical microtubule binding region that is prone to aggregation and subsequent beta-sheet formation.

Figure 11

Structures of tau PET imaging tracers reported in human research studies in the scientific literature. ¹⁸F-FDDNP is a pan-amyloid imaging agent, while the other compounds are relatively selective for tau over other amyloids. THK-5351 has been shown recently to bind with high affinity to MAO-B. The relative binding affinities of all tau PET imaging tracers for 3R and 4R tauopathies remain to be clearly defined.

Figure 12

[¹⁸F]AV-1451 standardized uptake value ratio (SUVR) images in a tau-negative (tau-) cognitively normal elderly subject (top row) and a tau-positive (tau+) Alzheimer’s disease subject (bottom row) determined 80–100 min post injection. These are the same subjects shown in Fig. 8. Retention of [¹⁸F]AV-1451 in the cognitively normal subject shows elevated retention in basal ganglia (indicated by arrow) that is representative of off-target binding in elderly subjects. The AD subject also exhibits [¹⁸F]AV-1451 off-target binding in basal ganglia, but also high levels of radiotracer retention in the lateral temporal lobes.

Figure 13

Diagrammatic representation of the 140 amino acid α-synuclein molecule. The amphipathic repeat region contains both hydrophilic and hydrophobic sub-regions, and the NAC (non-amyloid-β component) is involved in beta-sheet formation. Some of the important post-translational modification amino acid sites are shown.

Figure 14

Co-localization of α-synuclein immunoreactivity and X-34 fluorescence in the amygdala from a case of dementia with Lewy bodies. A 10 μm thin paraffin section was first processed using anti-α-synuclein antibody LB509 immunohistochemistry with hematoxylin counterstain to visualize cell bodies (panel A). The section was cleared of chromogen using potassium permanganate, overstained with the pan-amyloid dye X-34 (100 μM), and re-imaged (panel B). Arrows point to Lewy bodies double-labeled with LB509 antibody and X-34.