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Generation of clickable Pittsburgh Compound B for the detection and capture of #-amyloid in Alzheimer’s Disease brain Ian Samuel Diner, Jeromy Dooyema, Marla Gearing, Lary Walker, and Nicholas T. Seyfried Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00500 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 3, 2017
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Bioconjugate Chemistry
Generation of clickable Pittsburgh Compound B for the detection and capture of β-amyloid in Alzheimer’s Disease brain Ian Diner†, Jeromy Dooyema‡, Marla Gearing , Lary C. Walker‡§*, Nicholas T. Seyfried†,§*
†
Department of Biochemistry, ‡ Yerkes National Primate Research Center, Department of Pathology and Laboratory Medicine, § Department of Neurology, Emory University, Atlanta, GA 30322, USA
*Corresponding Authors: Nicholas T. Seyfried, Departments of Biochemistry and Neurology,
[email protected] and Lary C. Walker,
[email protected], Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 30322, USA
Key words: Pittsburgh Compound B, PiB, 6-OH-BTA-1, benzothiazole-aniline, amyloid imaging, fluorescence, click chemistry, PEGylation, Alzheimer’s disease, PET imaging, amyloid, affinity capture, histofluorescence
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Bioconjugate Chemistry
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Abstract: The benzothiazole-aniline derivative Pittsburgh Compound B (PiB) is the prototypical amyloid affinity probe developed for the in vivo positron emission tomography (PET) detection of amyloid beta (Aβ) deposits in Alzheimer’s disease (AD). Specific high-affinity binding sites for PiB have been found to vary among AD cases with comparable Aβ load, and they are virtually absent on human-sequence Aβ deposits in animal models, none of which develop the full phenotype of AD. PiB thus could be an informative probe for studying the pathobiology of Aβ, but little is known about the localization of PiB binding at the molecular or structural level. By functionalizing the 6-hydroxy position of PiB with a PEG3 spacer and a terminal alkyne (propargyl) moiety, we have developed a clickable PiB compound that was derivatized with commercially available azide-labeled fluorophores or affinity-tags using copper-catalyzed azidealkyne cycloaddition reactions, commonly referred to as “click” chemistry. We have determined that both the clickable PiB derivative and its fluorescently-labeled conjugate have low nanomolar binding affinities for synthetic Aβ aggregates. Furthermore, the fluorescent-PiB conjugate can effectively bind Aβ aggregates in human AD brain homogenates and tissue sections. By covalently coupling PiB to magnetic beads, Aβ aggregates were also affinity-captured from AD brain extracts. Thus, the clickable PiB derivative described herein can be used to generate a wide variety of covalent conjugates, with applications including the fluorescence detection of Aβ, the ultrastructural localization of PiB binding, and the affinity capture and structural characterization of Aβ and other co-factors from AD brains. Word Count: 249
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Bioconjugate Chemistry
Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder pathologically characterized by the accumulation of proteinaceous β-amyloid (Aβ) plaques and neurofibrillary (tau) tangles in the brain.
1
The plaques are primarily composed of the peptides Aβ(1-40) and
Aβ(1-42), which are cleaved from the Aβ-precursor protein (APP) by β- (BACE) and γsecretases.
1, 2
Of particular importance, the accumulation of Aβ is an initiating event that
precedes the development of cognitive decline by 10 or more years.3-6 For this reason, sensitive diagnostic biomarkers have been sought to detect the disease process at an early stage. Use of radiolabeled amyloid-binding ligands in conjunction with positron emission tomography (PET) has enabled real-time in vivo imaging and quantification of Aβ deposition for the diagnosis of AD. As this diagnostic methodology is based on Aβ deposition rather than cognitive deficits, it can be used to diagnose the early-stage or pre-symptomatic forms of AD.7-10 Such amyloid-binding radioligands can also be used to monitor disease progression in real-time and quantify the effects of novel drug therapies or treatment regimens.11 One of the earliest and most studied amyloid tracers is Pittsburgh Compound B (PiB),12-15 also referred to by its chemical abbreviation, 6-OH-BTA-1.15 Originally conceived in the search for high-affinity amyloid ligands, PiB was developed from the amyloid-binding dye Thioflavin T. PiB exhibits a high binding affinity (
