Binding Sites of a Positron Emission Tomography Imaging Agent in Alzheimer’s β-Amyloid Fibrils Studied Using ¹⁹F Solid-State NMR

Abstract

Amyloid imaging by positron emission tomography (PET) is an important method for diagnosing neurodegenerative disorders such as Alzheimer’s disease. Many 11C- and 18F-labeled PET tracers show varying binding capacities, specificities, and affinities for their target proteins. The structural basis of these variations is poorly understood. Here we employ 19F and 13C solid-state NMR to investigate the binding sites of a PET ligand, flutemetamol, to the 40-residue Alzheimer’s β-amyloid peptide (Aβ40). Analytical high-performance liquid chromatography and 19F NMR spectra show that flutemetamol binds the current Aβ40 fibril polymorph with a stoichiometry of one ligand per four to five peptides. Half of the ligands are tightly bound while the other half are loosely bound. 13C and 15N chemical shifts indicate that this Aβ40 polymorph has an immobilized N-terminus, a non-β-sheet His14, and a non-β-sheet C-terminus. We measured the proximity of the ligand fluorine to peptide residues using 19F–13C and 19F–1H rotational-echo double-resonance (REDOR) experiments. The spectra show that three segments in the peptide, 12VHH14, 18VFF20, and 39VV40, lie the closest to the ligand. REDOR-constrained docking simulations indicate that these three segments form multiple binding sites, and the ligand orientations and positions at these sites are similar across different Aβ polymorphs. Comparison of the flutemetamol-interacting residues in Aβ40 with the small-molecule binding sites in other amyloid proteins suggest that conjugated aromatic compounds preferentially bind β-sheet surface grooves lined by aromatic, polar, and charged residues. These motifs may explain the specificity of different PET tracers to different amyloid proteins.

The clinical drug candidate anle138b binds in a cavity of lipidic α-synuclein fibrils

“…This level of commonality across different fibrils could have implications for positron emission tomography radiotracer development. So far, tracer targets are usually identified in exterior binding sites similar to those reported for fibrils of HET-s 28 , amyloid-β (Aβ) 29 , and tau 30 , 31 . While theoretical studies have predicted the existence of binding sites in the core of fibrillar aggregates 32 , experimental data did not allow for a high-resolution structure 33 .…”

Probing Molecular Packing of Drug Substances in Nanometer Domains in Pharmaceutical Formulations Using 19F Magic Angle Spinning NMR

“…During the 19 F CODEX experiment, polarization transfers among fluorine spin with the identical isotropic chemical shift but distinct anisotropic chemical shifts, which encodes information about molecular reorientations on a time scale of ≥1 ms and intermolecular distances. , When slow molecular motions are frozen, interatomic 19 F– 19 F spin diffusion becomes the dominant mechanism of spin exchange and can be utilized to quantify oligomerization, for instance, quantify protein aggregation and intermolecular distances up to 15 Å. ,− Results from a 19 F CODEX experiment proposed a trimer structure and mapped out a triangular geometry of a cell-penetrating peptide, penetratin, in trehalose-protected DMPC/DMPG bilayers, by utilizing 4- 19 F-phenylalanine labeling . Considering the relatively large gyromagnetic ratio and natural abundance, 19 F NMR has increasingly been implemented in the pharmaceutical analysis of natural abundance drugs. ,− ,,,− In recent years, 19 F spin-exchange NMR has also been applied to study fluorinated drugs. For example, Roos et al have studied sitagliptin phosphate, a medicine for treating type 2 diabetes, and comprehensively investigated interatomic 19 F distances up to 9.6 Å …”

Vanillin Benzothiazole Derivative Reduces Cellular Reactive Oxygen Species and Detects Amyloid Fibrillar Aggregates in Alzheimer’s Disease Brain

“…It is well known that ThT binds strongly with Aβ 42 fibril. The benzothiazole moiety of ThT is responsible for binding with Aβ 42 fibrillar aggregates. − Inspired from previously described reports and lacuna in this area of research domain, herein, we showcased the design and development of a new vanillin benzothiazole derivative (VBD), which can specifically reduce the intracellular H 2 O 2 (ROS) upon reaction with it and generates new in situ molecule VBD-1, which specifically binds with the Aβ 42 fibrillar aggregates of AD brain. In our probe design strategy, we have considered benzothiazole in one site, and in the other site, we have introduced a boronate ester group attaching benzaldehyde.…”