Toward Novel [18F]Fluorine-Labeled Radiotracers for the Imaging of α-Synuclein Fibrils

Abstract

The accumulation of α-synuclein aggregates (α-syn) in the human brain is an occurrence common to all α-synucleinopathies. Non-invasive detection of these aggregates in a living brain with a target-specific radiotracer is not yet possible. We have recently discovered that the inclusion of a methylenedioxy group in the structure of diarylbisthiazole (DABTA)-based tracers improves binding affinity and selectivity to α-syn. Subsequently, complementary in silico modeling and machine learning (ML) of tracer–protein interactions were employed to predict surface sites and structure–property relations for the binding of the ligands. Based on this observation, we developed a small focused library of DABTAs from which 4-(benzo[d][1,3]dioxol-5-yl)-4′-(3-[18F]fluoro-4-methoxyphenyl)-2,2′-bithiazole [18F]d2, 6-(4′-(3-[18F]fluoro-4-methoxyphenyl)-[2,2′-bithiazol]-4-yl)-[1,3]dioxolo[4,5-b]pyridine [18F]d4, 4-(benzo [d][1,3]dioxol-5-yl)-4′-(6-[18F]fluoropyridin-3-yl)-2,2′-bithiazole [18F]d6, and 6-(4′-(6-[18F]fluoropyridin-3-yl)-[2,2′-bithiazol]-4-yl)-[1,3]dioxolo[4,5-b]pyridine [18F]d8 were selected based on their high binding affinity to α-syn and were further evaluated. Binding assay experiments carried out with the non-radioactive versions of the above tracers d2, d4, d6, and d8 showed high binding affinity of the ligands to α-syn: 1.22, 0.66, 1.21, and 0.10 nM, respectively, as well as excellent selectivity over β-amyloid plaques (Aβ) and microtubular tau aggregates (>200-fold selectivity). To obtain the tracers, their precursors were radiolabeled either via an innovative ruthenium-mediated (SNAr) reaction ([18F]d2 and [18F]d4) or typical SNAr reaction ([18F]d6 and [18F]d8) with moderate-to-high radiochemical yields (13% – 40%), and high molar activity > 60 GBq/μmol. Biodistribution experiments carried out with the tracers in healthy mice revealed that [18F]d2 and [18F]d4 showed suboptimal brain pharmacokinetics: 1.58 and 4.63 %ID/g at 5 min post-injection (p.i.), and 1.93 and 3.86 %ID/g at 60 min p.i., respectively. However, [18F]d6 and [18F]d8 showed improved brain pharmacokinetics: 5.79 and 5.13 %ID/g at 5 min p.i.; 1.75 and 1.07 %ID/g at 60 min p.i.; and 1.04 and 0.58 %ID/g at 120 min p.i., respectively. The brain uptake kinetics of [18F]d6 and [18F]d8 were confirmed in a dynamic PET study. Both tracers also showed no brain radiometabolites at 20 min p.i. in initial in vivo stability experiments carried out in healthy mice. [18F]d8 seems very promising based on its binding properties and in vivo stability, thus encouraging further validation of its usefulness as a radiotracer for the in vivo visualization of α-syn in preclinical and clinical settings. Additionally, in silico and ML-predicted values correlated with the experimental binding affinity of the ligands.

Advancing Parkinson’s Disease Diagnostics: The Potential of Arylpyrazolethiazole Derivatives for Imaging α-Synuclein Aggregates

“…17 A 1,3-benzodioxole moiety is also recurrent in other known ligands with high affinity for αsyn aggregates, including anle138b. 15,17,19 Furthermore, in known classes of compounds, a thiazole ring is reported for some selective ligands for α-syn over Aβ. 17,20 In this study, we designed a class of APT compounds in which a thiazole ring replaces a benzene ring of the DPP analogs.…”

“…The crude product was then purified by flash chromatography (PE/EA, gradient 40−95% B) to afford the desired product as a white solid (18 mg, 56%). 1 [17] (100 mg, 0.29 mmol, 1 equiv) in dry THF (3 mL) was added to the solution of NaH in THF at 0 °C, and the resulting mixture was stirred at room temperature for 30 min. A solution of MeI (0.054 mL, 0.63 mmol, 1.5 equiv) in THF (1 mL) was then added and the mixture was allowed to stir overnight at room temperature.…”

Insight into the Binding of First- and Second-Generation PET Tracers to 4R and 3R/4R Tau Protofibrils

“…For PSP tau, 7P65 is selected because it is from the canonical tissue with Richardson’s syndrome . Considering the movement along the principal axis of fibrils and the consumption of computational resources, a nine-chain protofibril was constructed for each Cryo-EM structure of PSP, CBD, and AD using the method mentioned in our previous study. , The automated preparation protocol implemented in the Protein Preparation Workflow of the Schrödinger Suite (Version 2021-4) was used, , which assigns the atom types, bond orders, hydrogen atoms, and protonation states of ionizable residues at the pH of 7.4 and minimizes the system by the convergence of heavy atoms to 0.3 Å of root-mean-square deviations (RMSD) with the OPLS4 force field . The assigned protonation states of the ionizable residues for each prepared protofibrils were visually inspected …”

“…To thoroughly explore the binding and unbinding of the tracer on the surface sites, we further performed well-tempered metadynamics simulations for each tracer on PSP protofibrils with a randomly placed position . The simulation protocols were adopted as our previous studies , using PLUMED (version 2.8.0) patched with GROMACS (version 2021.4). − Briefly, the tracers were parameterized with the general Amber force field with the charges fitted by the restrained electrostatic potential procedure, in which the electrostatic charges were firstly calculated by Gaussian 16 (rev. C01) at the Hartree–Fock level using the 6–31G­(d) basis set .…”

Selecting the Best Animal Model of Parkinson’s Disease for Your Research Purpose: Insight from in vivo PET Imaging Studies

“…The development of a radioligand selectively targeting α-syn aggregates is a major unmet need in the PD field and would have utility for the early and differential diagnosis of PD and other synucleinopathies. Indeed, several research teams have undertaken this task and have labelled different α-syn binding ligands with radioactive labels [ 82 , 83 ], including phenothiazines, indolinonedienes and chalcone-like derivatives, benzoxazoles, diaryl pyrazoles, and bisquinolines [ 74 , 84 ]. These were prepared with the ambitious overarching aim of understanding potential correlations between α-syn load and distribution, clinical symptom severity and disease progression.…”