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Article

Development and In Vitro Characterization of [3H]GMC-058 as Radioligand for Imaging Parkinsonian-Related Proteinopathies

by
Andrea Varrone
1,*,
Vasco C. Sousa
2,
Manolo Mugnaini
3,
Sandra Biesinger
4,
Gunnar Nordvall
5,
Lee Kingston
6,
Ileana Guzzetti
7,
Charles S. Elmore
6,
Dan Sunnemark
8,9,
Dinahlee Saturnino Guarino
10,
Sjoerd J. Finnema
11 and
Magnus Schou
12
1
Department of Clinical Neuroscience, Centre for Psychiatry Research, Karolinska Institutet and Stockholm Health Services, BioClinicum, J4-14, Akademiska Stråket 1, 17176 Solna, Sweden
2
Department of Clinical Neuroscience, Division of Imaging Core Facilities, Karolinska Institutet, Stockholm, Sweden, BioClinicum, J5, Akademiska Stråket 1, 17176 Solna, Sweden
3
Translational Sciences, Neuroscience & Ophthalmology Discovery Research, AbbVie Deutschland GmbH & Co. KG, Knollstraße 50, 67061 Ludwigshafen, Germany
4
Advanced Cell Technologies & Screening, Neuroscience & Ophthalmology Discovery Research, AbbVie Deutschland GmbH & Co. KG, Knollstraße 50, 67061 Ludwigshafen, Germany
5
AlzeCure Pharma AB, Hälsovägen 7, 14157 Huddinge, Sweden
6
Isotope Chemistry, Early Chemical Development, Pharmaceutical Sciences, R&D, AstraZeneca, Pepparedsleden 1, 43183 Mölndal, Sweden
7
Medicinal Chemistry, RIA, Biosciences R&D, AstraZeneca, Pepparedsleden 1, 43183 Mölndal, Sweden
8
Offspring Biosciences, Forskargatan 20 J, 15136 Södertälje, Sweden
9
Department of Clinical Neuroscience, Karolinska Institutet, Center for Molecular Medicine, Karolinska University Hospital, 17176 Stockholm, Sweden
10
Department of Radiology, Perelman School of Medicine, University of Pennsylvania, 1012, 231 S. 34th Street, Philadelphia, PA 19104, USA
11
Translational Sciences, Neuroscience & Ophthalmology Discovery Research, AbbVie, 1 North Waukegan Road, North Chicago, IL 60064, USA
12
Precision Medicine Diagnostic Development and HBS Science, AstraZeneca R&D Oncology, AstraZeneca J4:14, BioClinicum, Akademiska Stråket 1, 17176 Solna, Sweden
 
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*
Author to whom correspondence should be addressed.
Cells 2025, 14(12), 869; https://doi.org/10.3390/cells14120869
Submission received: 4 April 2025 / Revised: 18 May 2025 / Accepted: 3 June 2025 / Published: 9 June 2025
(This article belongs to the Special Issue Development of PET Radiotracers for Imaging Alpha-Synuclein)
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Abstract

The molecular imaging of α-synuclein (α-syn) pathology in Parkinson’s disease (PD) and related movement disorders is a clinically unmet need. The aim of this study was to discover and characterize in vitro a radioligand for imaging α-syn pathology. A library of 78 small molecules was developed and screened using recombinant α-syn fibrils and brain homogenates from Alzheimer’s disease (AD) donors. The selection criteria were as follows: Kiα-syn < 30 nM, Kitau and KiA-β > 200 nM. Three compounds, GMC-073 (Kiα-syn: 8 nM), GMC-098 (Kiα-syn: 9.7 nM), and GMC-058 (Kiα-syn: 22.5 nM), fulfilled the criteria and were radiolabeled with 3H. [3H]GMC-058 was the only compound with negligible binding in controls, and was further evaluated using tissue microarrays, autoradiography on fresh-frozen brain slices, and in vitro saturation binding assay on brain homogenates. [3H]GMC-058 binding co-localized with α-syn inclusions in Parkinson’s disease (PD) and multiple-system atrophy (MSA), with dense A-β plaques in cerebral amyloid angiopathy and AD and with p-tau inclusions in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Specific binding was highest in PSP and CBD. In vitro KD was highest in AD (5.4 nM), followed by PSP (41 nM) and CBD (75 nM). The KD in MSA, PD, and controls was >100 nM. [3H]GMC-058 is a novel radioligand displaying a low affinity for aggregated α-syn in tissue, with an in vitro profile also suitable for detecting tau pathology in 4R tauopathies.

1. Introduction

The accumulation of intracellular aggregates of misfolded α-synuclein (α-syn) is a pathological hallmark of neurodegenerative disorders called synucleinopathies [1]. Such disorders include Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), characterized by the accumulation of pathological α-syn in neurons, in the form of Lewy bodies and Lewy neurites [2], and multiple-system atrophy (MSA), characterized by the accumulation of pathological α-syn in glial cells, in the form of glial cytoplasmic inclusions [3].
In the last decade, extensive research and efforts have been devoted to the search and discovery of imaging agents able to detect in vivo pathological α-syn in patients with synucleinopathies using positron emission tomography (PET). Recent clinical studies with newly developed PET tracers [18F]ACI-12589 [4], [18F]SPAL-T-06 [5], and [18F]C05-05 [6] have shown promising results in patients with MSA ([18F]ACI-12589 and [18F]SPAL-T-06) and in patients with Lewy-body disease ([18F]C05-05). However, these PET radioligands present some shortcomings (Table 1).
[18F]ACI-12589 displays in vitro selectivity for α-syn vs. tau and Aβ, and a KD in PD and MSA tissue ~30 nM. [18F]ACI-12589 is able to successfully image α-syn pathology only in MSA, likely because the Bmax/KD of the radioligand is not adequate enough to detect a lower level of pathology in PD. In the case of [18F]SPAL-T-06 and [18F]C05-05, an IC50 = 2–3 nM in brain homogenates from DLB or MSA cases has been reported using homologous competition binding studies. However, both radioligands have been developed from the same scaffold as PBB3 and also display an affinity to Aβ and tau. Given that concomitant Aβ and tau pathology is frequent in Lewy-body disease, particularly DLB and PD with dementia, selectivity for α-syn over Aβ and tau is a requirement for a fit-for-purpose α-syn PET radioligand. All clinical PET radioligands developed so far lack the properties of combined high Bmax/KD and selectivity for α-syn, required to image α-syn pathology in Lewy-body disease with high specificity.
Therefore, a selective PET radioligand able to image α-syn in all synucleinopathies is still a clinically unmet need. The aim of this study was to discover small molecules displaying suitable in vitro profiles (affinity in nM range and >10-fold selectivity for α-syn vs. Aβ and tau) for development as α-syn PET radioligands. The work included in vitro screening using recombinant α-syn fibrils and brain homogenates from Alzheimer’s disease (AD) donors. Compounds with suitable in vitro affinity profiles were radiolabeled with 3H. Potential candidates were evaluated with in vitro autoradiography (ARG). Brain tissue from control donors and from patients with different proteinopathies was used for ARG experiments using fresh-frozen tissue sections and paraffine-embedded tissue microarrays (TMAs). High-resolution ARG and immunohistochemistry on consecutive TMA sections were performed to study the co-localization of an ARG signal with α-syn, p-tau and Aβ pathology.

2. Materials and Methods

2.1. In Vitro Binding Assays

2.1.1. Radioligand Binding to α-Syn Fibrils

Compound affinity for α-syn fibrils was measured by means of radioligand binding displacement experiments of an AbbVie tool radioligand with high affinity for α-syn fibrils ([3H]-α-syn-tool). The aggregation of α-syn monomers into fibrils was obtained as described by Bousset et al. [7]. In brief, monomers (5 mg/mL, prepared in AbbVie (Worcester, MA, USA) were incubated in 150 mM of KCl and 50 mM of Tris-HCl (pH 7.5) for 7 days at 37 °C, under constant shaking conditions (650 rpm, Eppendorf Thermomixer C, Eppendorf, Hamburg, Germany). After incubation, the fibrils were harvested via ultra-centrifugation (40,100× g, 30 min, 20 °C), resuspended in buffer, sonicated (40% amplitude, 10 s pulse on, 5 s pulse off) for 4.5 min at 20 °C, divided into aliquots (5.5 mg/mL), and stored at −80 °C until the day of the experiment. The characterization of the fibrils included protein content determination (BCA method) and an analysis of Proteinase K degradation pattern [7].
On the day of the experiment, α-syn fibrils were thawed for a few minutes at 37 °C, diluted in Dulbecco’s Phosphate Buffered Saline (PBS) without calcium and magnesium, pH = 7.4 (DPBS) plus 0.1% bovine serum albumin (BSA), and incubated (at the final fibrils concentration of 2.5 µg/mL) for 60 min, at room temperature (RT), with 5 nM [3H]-α-syn-tool, in the absence or presence of different concentrations of non-radiolabeled compounds. Bound radioligand was separated from free radioligand via rapid filtration and washings with cold DPBS, using a Unifilter-96 GF/B filter plate (PerkinElmer, Rodgau, Germany) presoaked in 0.3% polyethylenimmine (PEI) and a Unifilter Cell Harvester (PerkinElmer, Rodgau, Germany). Plates were air-dried, and the scintillation liquid (Betaplate Scint, PerkinElmer) was added and counted using a MicroBeta2 2450 (Perkinelmer, Rodgau, Germany). Non-specific binding (NSB) was defined as the binding in the presence of 10 µM unlabeled α-syn tool. Data fitting and the constant of inhibition (Ki) calculation was performed using the software platform from Dotmatics (Boston, MA, USA).

2.1.2. Radioligand Binding to Native Tau and Aβ Fibrils

Compounds’ affinity for tau and Aβ fibrils was measured by means of radioligand binding displacement experiments on AD brain homogenates, using [3H]NFT-355 [8] (AbbVie, North Chicago, IL, USA) and [3H]Pittsburg Compound-B (PiB; Novandi Chemistry AB, Södertälje, Sweden [9]) to label tau and Aβ aggregates, respectively. Flash-frozen tissue from the frontal cerebral cortex of an AD (Braak stage V) brain was purchased from Analytical Biological Services Inc. (ABS; Wilmington, DE, USA), homogenized in DPBS, divided into aliquots (10 mg/mL), and stored at −80 °C until the day of the experiment.
On the day of the experiment, the brain homogenate was brought to RT, diluted in DPBS plus 0.1% BSA and incubated for 90 min, at RT, with 0.5 nM [3H]NFT-355 or 1 nM [3H]PiB, in the absence or presence of different concentrations of non-radiolabeled compounds. Bound radioligand was separated from free radioligand via rapid filtration and washings with cold DPBS, using a Unifilter-96 GF/B filter plate (presoaked in 0.3% PEI) and a Unifilter Cell Harvester (PerkinElmer). Plates were air-dried, and the scintillation liquid (Betaplate Scint, PerkinElmer) was added and counted using a MicroBeta2 2450. NSB was defined as the binding in the presence of 10 µM T808 [10] (AbbVie Germany) and 10 µM PiB for [3H]NFT-355 and [3H]PiB, respectively. Data fitting was performed using the software platform from Dotmatics (Boston, MA, USA).

2.2. Radiosynthesis of [3H]GMC-058, [3H]GMC-073, and [3H]GMC-098

Chemicals and solvents were obtained from commercial sources and were used without further purification. NMR spectra were recorded on a Bruker 500 MHz AVANCE III system using standard Bruker pulse sequences. Experiments were run in D6-DMSO at 25 °C. 1H NMR chemical shifts were referenced relative to the residual solvent peak at 2.50 ppm, and 13C NMR chemical shifts were referenced to 39.5 for D6-DMSO. Flash column chromatography was carried out using prepacked silica gel columns supplied by Biotage and using a Biotage automated flash system with UV detection. Preparative HPLC was performed using a Waters Xbridge C18 5 µ OBD 19X150 mm, 25 mL/min, 30% for 2 min and then was ramped to 95% over 18 min and held for 3 min MeCN-0.2% aq. NH4OH was used unless otherwise indicated. Reactions with tritium gas were performed on an RC Tritec tritium manifold. Analytical HPLC was carried out using an Agilent 1100 series HPLC using a Waters 4.6 × 100 mm Xbridge C18 3.5 μ, with the following elution profile: 5% for 3 min and then ramped to 95% over 22 min and hold 95% for 5 min MeCN/water 10 mM NH4HCO3 pH 10 with radioactive detection using a LabLogic Beta-Ram 4 detector. LCMS analysis was carried out using a Waters 1100 HPLC system with a Waters 3100 mass detector on an XSelect CSH C-18 4.6/150 mm, 3.5 μm with a gradient of 5–100% MeCN–0.1% formic acid (adjusted to pH 3) over 10 min followed by a 2 min wash with 100% MeCN or on a Waters1200 series UPLC with a Waters 4.6 × 100 mm Xselect CSH C18 3.5 μ, 5 to 95% MeCN/water 0.2% formic acid pH 3 for 1.85 min then isocratic elution for 0.1 min with mass detection using a QDA mass detector. The molar activities of the products were determined via LC/MS using Isopat2 to deconvolute the MS signals [11]. Liquid scintillation counting was performed with a Beckman LS 6500 scintillation counter.

2.3. Autopsy Material

Frozen human brain tissues from patients with Parkinson’s disease (PD), Lewy-body disease (LBD), multiple-system atrophy (MSA), corticobasal degeneration (CBD), and non-dementia controls were obtained from the Netherlands brain bank (Table 2) and frozen human tissues from patients with MSA, PSP, and one control were obtained from the UK brain bank (Table 3). Formalin-fixed paraffine-embedded tissue blocks from patients with different synucleinopathies were obtained from the Netherland Brain Bank (Table A1). Approval for the use of the autopsy material for the project was obtained from the Swedish Ethical Research Authority. Brain homogenates were obtained from tissue of AD, CBD, PSP, MSA-P patients, and healthy controls, obtained from the Alzheimer’s Disease Research Center of the University of Pittsburgh (Table A2), from the University of California San Francisco (Table A3), and from the Banner Sun Health Research Institute (Table A4).

2.4. Preparation of Human Brain Tissue for In Vitro Binding Studies

As described in Bagchi et al. [12], to prepare insoluble fractions from PD and MSA-P patients, fresh-frozen tissue blocks were sequentially homogenized in four buffers (3 mL/g wet weight of tissue) with glass Dounce tissue grinders (Kimble, Vineland, NJ, USA): (1) high salt (HS) buffer: 50 mM Tris-HCl pH 7.5, 750 mM NaCl, 5mM EDTA; (2) HS buffer with 1%Triton X-100; (3) HS buffer with 1%Triton X-100 and 1M sucrose; and (4) phosphate-buffered saline (PBS). Homogenates were centrifuged at 100,000× g after each homogenization step, and the pellet was resuspended and homogenized in the next buffer in the sequence. For AD, CBD, PSP, and CT cases, the frozen tissue blocks were prepared as described by Stehouwer et al. [13]. In brief, tissue blocks were thawed and homogenized in ice-cold pH 7.0 phosphate-buffered saline (PBS) at 300 mg/mL on ice using a glass homogenizer, diluted 30-fold with PBS to 10 mg/mL, and homogenized a second time with a Brinkmann Polytron homogenizer before storage at −80 °C.

2.5. Saturation Binding Assays

Saturation binding assays were performed in AD, CBD, PSP, MSA-P, PD, and healthy patients brain homogenates (0.1 mg/mL tissue) using a [3H]GMC-058 concentration range of 0.9 nM to 80 nM and incubation for 90 min at room temperature (RT) with PBS + 20% EtOH. Non-specific binding was determined using 10 μM of unlabeled GMC-044. After incubation, samples were filtered under vacuum on equilibrated GF/B UniFilter plates (PerkinElmer) using the FilterMate 196 (PerkinElmer). Afterwards, filters were washed three times with 250 μL chilled buffer (PBS + 20% EtOH). [3H]GMC-058 binding was measured with a beta scintillation counter (PerkinElmer). The saturation data was fitted and analyzed using the non-linear regression function of GraphPad Prism 10 software to calculate the dissociation constant (KD) and maximum number of binding sites (Bmax). Scatchard plots were prepared with GraphPad Prism 10 software to display the saturation binding data.

2.6. Autoradiography Experiments on Fresh-Frozen Tissue Sections

Selections of frozen human sections (described in Table 2 and Table 3) were used for binding autoradiography. Sections were first pre-incubated for 20 min in binding buffer [50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 0.1% BSA] and then incubated with [3H]GMC-058 (specific activity 22 Ci/mmol); [3H]GMC-073 (specific activity 23.4 Ci/mmol); or [3H]GMC-073 (specific activity 26.4 Ci/mmol) in binding buffer for 180 min at RT. In the pilot autoradiography experiment (Figure A1). [3H]GMC-058 was tested at 25 and 50 nM, while [3H]GMC-073 and [3H]GMC-098 were each tested at 10 and 20 nM. For all other autoradiography in fresh-frozen tissue sections, 25 nM [3H]GMC-058 was used. To determine NSB, adjacent brain sections were incubated with [3H]-tracer mixed with 5 μM of unlabeled GMC-044. The binding reaction was stopped with two 10 min washes in washing buffer [50 mM Tris-HCl (pH 7.4) at 4 °C] and a brief dip in cold distilled water. Slides were allowed to air-dry before being placed under storage phosphor screens (Fujifilm Plate BAS-TR2025, Fujifilm, Tokyo, Japan) in imaging cassettes for 90 h together with ART0123C and ART0123B tritium standards on glass slides (American Radiolabeled Chemicals Inc., St. Louis, MO, USA). Storage phosphor screens were scanned using an Amersham Typhoon FLA-9500 phosphor imaging scanner (Cytiva, Marlborough, MA, USA), and the resulting images were analyzed using Multi Gauge 3.2 phosphor imager software (Fujifilm, Tokyo, Japan) for ROI delimitation, density calibration, and quantification. Quantitative binding data from duplicates of the total, and NSB were plotted and analyzed using graphPad Prism v10 (GraphPad Software, Boston, MA, USA). Specific binding was determined by subtracting the non-specific signal from the total signal. The toluidine blue staining of adjacent sections was used to obtain anatomical references for white- and gray-matter ROI selection. Care was taken not to include artifact signals in the ROIs analyzed. These artifacts were identified as randomly placed punctate signals appearing in both total and NSB images, and they often coincided with small folds or tears in the tissue sections.

2.7. Autoradiography and Emulsion Autoradiography on Tissue Microarrays

Tissue micro-arrays (TMAs), prepared from paraffin-embedded tissue from human post-mortem tissue as described in [14], were first de-paraffinized with two 1 h and one overnight incubation in Neo-Clear Xylene Substitute (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and then dehydrated in three short incubations at RT with decreasing concentrations of ethanol before autoradiography with [3H]GMC-058 was performed as described above. For the saturation binding autoradiography, duplicate slides of each TMA set were incubated with 0.3 nM to 200 nM [3H]GMC-058. Adjacent sections were co-incubated with 5 μM of unlabeled GMC-044 to calculate NSB. The quantification of total and non-specific binding was performed via delineation of the ROIs on the whole core, except for MSA, where the ROI followed the areas in which the binding of [3H]GMC-058 corresponded to areas with α-syn inclusions. Those cores that had imperfections or were incomplete were not included in the analysis, or only the area of remaining tissue that adhered to the glass slide was delineated. For the cores from the substantia nigra of PD patient cases, the cores selected for analysis were those containing α-syn inclusion, but no A-β deposits of pTau tangles. The information was collected from IHC analysis of adjacent TMA slides.
For high-resolution emulsion autoradiography, an autoradiography experiment with TMAs using 25 nM [3H]GMC-058 was performed as described above. At the end of the 90 h exposure to the storage phosphor screen, slides were stored in a vacuum until dipping in a photoemulsion. In the dark, using a sodium lamp, Agar Scientific, AGP9284 type NTB emulsion was melted in a heated water bath and diluted (1:1) with ddH2O. The emulsion was poured into dipping chambers, and the slides were dipped and placed vertically to air-dry. When completely dry, the slides were placed in a light-tight slide box with desiccant and exposed at 4 C for 1–12 weeks. After exposure, the slides were developed in diluted Phenisol Developer for 2 min, rinsed in water, and fixed in hypam Fixer at 17 °C in a water bath. After rinsing in water, the slides were counterstained with Harris HTX, dehydrated in graded ethanol, cleared in Xylene, and mounted in Pertex. Images were taken using a 3D Histech P250 III scanner (3DHISTECH Kft. Hungary) at up to 15 focal layers with a distance of 0.2 μm.

2.8. Immunohistochemistry

Immunohistochemical chromogenic (IHC) staining was made essentially as in [15], and the reference antibodies for the respective proteinopathies used were Signet laboratories (Abeta 6E10, Abeta 4G8), Abcam (Anti-Alpha-synuclein antibody [LB 509]), and Thermo Fisher Scientific (Waltham, MA, USA) (AT-8 Phospho-PHF-Tau). Briefly, 5 μm slide-mounted tissue sections were performed according to a standardized protocol with modifications to optimize the specificity of the observed staining patterns. IHC staining was performed using the Discovery Ultra platform (Ventana, Roche Diagnostics International AG, Rotkreuz, Switzerland) automated immunostaining robot, using the OmniMap DAB chromogenic staining kit (Ventana Medical Systems) according to the manufacturer’s instructions. In brief, initial deparaffinization, followed by heat-activated antigen retrieval in a pH 8.0 buffer for 92 min (Ventana Medical Systems’ (Ventana) DISCOVERY CC1 (DISCOVERY Cell Conditioning Solution 1, 06414575001, 950–500), was performed to improve the detection of antigens in the FFPE tissue. Endogenous tissue peroxidases (Inhibitor CM, Ventana), which may interfere with the assays, were blocked with 0.3% hydrogen peroxide. The primary antibod forphosphorylated Tau detection (AT-8 pTau [In vitrogen, MN1020] at 2 µg/mL), and re for total Abeta (Mouse anti-Abeta 4G8 [Signet, Covance, 800709 (SIG-39200)], were mixed and co-incubated at a final concentration of 1 µg/mL) and applied on the tissue sections. For the IHC staining of α-synuclein inclusions, the anti-human α-synuclein antibody LB509 (Abcam, GR300971) 0,5 µg/mL was used. Prior to staining, sections were treated for 24 min with protease (Ventanas Protease 1, [760–2018]) to degrade soluble α-synuclein species. Antibodies were followed by incubation with the HRP-conjugated secondary antibody (the HPR labeled OmniMap goat anti-Mouse Ab, 05269652001 760–4310), and the visualization of the positively stained cells was performed through the addition of hydrogen peroxide and DAB (DISCOVERY ChromoMap DAB Kit [Ventana, 760–159) (single IHC)], resulting in an insoluble brown (DAB) staining precipitate at the site of antibody binding. Counterstaining for IHC was performed with hematoxylin [Hematoxylin II, Ventana, 760–2208 and Bluing Reagent, Ventana, 760–2037]. The stained slides were subsequently scanned at up to 10 focal layers in brightfield (20× -40 objective) using a digital whole slide scanner (Pannoramic 250 III Scanner, 3DHistech, Budapest, Hungary). Analysis was performed of the IHC stained tissue sections via manual evaluation using a digital image viewer software (CaseViewer_2.0_RTM_v2.0.2.61392). All analyses of the stained and labeled tissue sections with reference antibodies for Aβ, p-tau, and α-syn and/or ligands were compared, side by side, to each other. Adjacent fresh-frozen tissue sections or TMAs were used when comparing IHC immunoreactivity with an autoradiography binding signal.

3. Results

The work plan for this study included a first part that was devoted to the discovery of small molecules with an in vitro profile suitable for the development of a selective α-syn ligand and a second part that included the in vitro characterization of lead candidates.
The discovery part included compound design and in vitro competition experiments for the identification of lead candidates. The in vitro characterization included 3H-radiolabeling, in vitro saturation binding, and autoradiography experiments in tissues from donors with different proteinopathies.

3.1. Discovery and In Vitro Characterization of GMC-058

3.1.1. Discovery of GMC-058, GMC-073 and GMC-098

A library of 78 small molecules was generated based on available information from the public domain. Four optimization cycles were performed with radioligand binding assays to measure the affinity of compounds for in vitro-assembled α-syn fibrils and native tau and Aβ fibrils in AD homogenates.
Sixteen of these compounds showed affinity for α-syn fibrils < 30 nM and for Aβ and tau > 50 nM (Table A5). The criteria for selection were Ki for α-syn < 30 nM and Ki for tau and Aβ > 200 nM.
Three compounds, GMC-058, GMC-073, and GMC-098, were selected based on those criteria and on the feasibility of radiolabeling. The Ki of GMC-058, GMC-073, and GMC-098 using recombinant α-synuclein fibrils was 22.5 nM, 8 nM, and 9.7 nM, respectively.
All three compounds showed a lower affinity for Aβ (Ki = 1490 nM for GMC-058, 2630 nM for GMC-073, and 226 nM for GMC-098) and tau (Ki= 1320 nM for GMC-058, 248 nM for GMC-073, and 805 nM for GMC-098) in AD brain homogenates. GMC-058 (Figure 1), GMC-073, and GMC-098 were, therefore, selected for radiolabeling with 3H.

3.1.2. Autoradiography Experiments with [3H]GMC-058, [3H]GMC-073 and [3H]GMC-098

The three [3H]-labeled compounds were initially evaluated through autoradiography in tissue sections from the cingulate cortex of healthy controls.
[3H]GMC-073 and [3H]GMC-098 displayed clear binding in control cingulate cortex tissue, approximately 50% of which was displaced via 5 µM GMC-044, whereas [3H]GMC-058 displayed negligible binding (Figure A1 and Table A6). The nature of this off-target displaceable binding is not known. [3H]GMC-058 was selected for further evaluation in pathological tissue sections.

3.1.3. In Vitro Saturation Binding Assays with [3H]GMC-058 in Brain Homogenates

Saturation binding assays with [3H]GMC-058 were conducted in brain homogenates from cases with AD, PSP, CBD, MSA-P, PD, and Controls (Figure 2 and Figure A2).
The KD estimated in AD tissue was 5.4 nM, and in PSP tissue, it was 46 nM, while in CBD tissue, it was 71 nM. In brain homogenates from MSA-P and control cases, the KD was >100 nM. No evidence of saturation binding was observed in PD tissue.

3.1.4. Autoradiography Experiments with [3H]GMC-058

Autoradiography experiments using 25 nM [3H]GMC-058 in tissue sections from the cingulate cortex of controls and PD patients and from the cerebellum of MSA patients did not show clear evidence of specific binding higher than the displaceable (off-target) binding in controls (Figure 3 and Figure A3 and Table A7). Saturation [3H]GMC-058 binding autoradiography experiments in TMAs showed that specific binding did not reach saturation in any of the tissues (Figure A4 and Figure A5) and did not permit us to obtain reliable KD estimates.
However, the specific binding data obtained at the concentration of 25 nM [3H]GMC-058 showed clear differences in specific binding and were thus selected for group comparison.
In synucleinopathies (PD and MSA), cerebral amyloid angiopathy (CAA), AD, and 4R tauopathies (PSP and CBD), the specific binding of 25 nM [3H]GMC-058 was significantly higher than the displaceable binding in controls (p < 0.01, ANOVA with Dunnett’s multiple comparisons test; see Table 4 and Figure A6). High-resolution autoradiography showed that the binding of 25 nM [3H]GMC-058 co-localized with α-syn inclusions in PD and MSA, with dense Aβ plaques in CAA and AD, and with p-tau inclusions in PSP and CBD (Figure 4, Figure A7, Figure A8 and Figure A9).
We then conducted autoradiography experiments in fresh-frozen tissue for which α-syn, Aβ, and p-tau expression was measured via IHC. We selected tissue sections of cerebellum from two donors with MSA, of superior frontal gyrus from one donor with CBD, of globus pallidus (GP) from two donors with PSP, and of the cingulate cortex, caudate, GP, and putamen from two controls (Figure 5 and Figure A10 and Table A8).
In the two MSA cases, specific binding measured in cerebellar gray matter was two-fold higher than the average displaceable binding measured in the gray matter of the cingulate cortex, caudate, the globus pallidus, and the putamen of the controls (Figure 5A,D,E). In CBD and PSP cases, the specific binding in the gray matter of the superior frontal gyrus and the globus pallidus was, on average, three-fold and ten-fold higher, respectively, than the displaceable binding measured in controls (Figure 5B,C,E). No specific binding was observed in the white matter of control cases, whereas in MSA and CBD cases, the average specific binding was 35 to 80 fmol/mg (Figure 5A,B,E and Table A8).

4. Discussion

This study was designed to identify a small molecule with in vitro properties suitable for development as ligand binding to pathological α-syn. We identified three compounds, GMC-058, GMC-073, and GMC-098, that displayed selective binding to recombinant α-syn fibrils. The three compounds were radiolabeled with 3H, and among the three compounds, [3H]GMC-058 was selected since it showed the lowest non-displaceable binding in control tissue. However, when [3H]GMC-058 was further evaluated in human brain tissue homogenates and sections, it displayed low specific binding in synucleinopathy cases but clear specific binding in PSP and CBD cases. This observation was unexpected, considering that the Ki of GMC-058 measured in recombinant α-syn fibrils was 22.5 nM, and in tau-enriched AD tissue was >200 nM. It is possible that the affinity of GMC-058 measured using recombinant α-syn fibrils was different from the affinity of [3H]GMC-058 to native α-syn fibrils in tissue slices or brain homogenates, resulting in a discrepancy between the two different assay results. With regards to tau, since the competition studies were performed using the reference tau tracer [3H]NF-355, it is possible that GMC-058 recognizes a different binding site on tau fibrils. Several radioligands have been developed for imaging tau pathology [16]. Second-generation tau PET radioligands [18F]PI-2620 and [18F]PM-PPB3 (also known as [18F]APN-1607, or florzolotau) have been studied in patients with AD [17,18] and PSP [19,20]. Those studies showed that both radioligands can image 3R and 4R tau pathology in vivo. More recently, the discovery, in vitro characterization, and in vivo pre-clinical assessment of [18F]OXD-2314 has been reported [21], indicating that this radioligand also has the potential to image 3R and 4R tauopathies. Although [3H]GMC-058 has been designed and developed as a potential α-syn PET radioligand, the in vitro properties suggest that the Bmax/KD is not high enough to enable imaging of α-syn pathology in vivo. On the other hand, the evidence of higher specific binding of [3H]GMC-058 in PSP and CBD than in MSA, suggests that GMC-058 might have potential as a PET radioligand for imaging 4R tauopathies. A discrepancy was observed between the high KD (>50 nM) of [3H]GMC-058 in brain homogenates from PSP and CBD cases and the clear evidence of specific binding in fresh-frozen brain sections from CBD and PSP cases. A similar discrepancy between the results of in vitro saturation studies in brain homogenates and autoradiography studies on brain sections has been observed for another potential α-syn tracer, [3H] -4i, and is difficult to explain [22].

5. Conclusions

[3H]GMC-058 is a novel radioligand displaying a low in vitro affinity for aggregated α-syn, with an in vitro profile also suitable for imaging tau pathology in 4R tauopathies. Further evaluations, including radiolabeling with 11C and in vivo imaging in non-human primates, are warranted to further assess the potential of GMC-058 as a potential PET radioligand for 4R tau.

Author Contributions

Conceptualization, A.V., S.J.F. and M.S.; methodology, V.C.S., M.M., S.B., G.N., L.K., I.G., C.S.E., D.S. and D.S.G.; formal analysis, V.C.S., M.M., S.B., D.S. and D.S.G.; investigation, A.V., V.C.S., M.M., S.B., G.N., L.K., I.G., C.S.E., D.S., D.S.G., S.J.F. and M.S.; resources, A.V., V.C.S., M.M., S.B., G.N., C.S.E., D.S., D.S.G., S.J.F. and M.S.; data curation, A.V., V.C.S., M.M., S.B., G.N., C.S.E., D.S., D.S.G., S.J.F. and M.S.; writing—original draft preparation, A.V.; writing—review and editing, A.V., V.C.S., M.M., G.N., L.K., I.G., C.S.E., D.S., D.S.G., S.J.F. and M.S.; visualization, A.V., V.C.S., M.M., D.S., D.S.G. and M.S.; supervision, A.V.; project administration, A.V.; funding acquisition, A.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Michael J Fox Foundation for Parkinson Research, grant number 10908, by the Swedish Parkinson Foundation (Parkinsonfonden), grant number 1268/20, by AstraZeneca and AbbVie. Dinhalee Saturnino Guarino was supported by NIH grant U19-NS110456.

Institutional Review Board Statement

The study was approved by the Regional Ethics Committee of Stockholm, Sweden (protocol code 2015/4:6 and date of approval: 17 June 2015).

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Acknowledgments

We would like to acknowledge David Holzinger (Translational Sciences, Neuroscience & Ophthalmology Discovery Research, AbbVie Deutschland GmbH & Co., Knollstraße 50, 67061 Ludwigshafen, Germany) for α-syn fibrils generation and Åsa Södergren for technical assistance with autoradiography. All autoradiography experiments were performed at the autoradiography core facility of Karolinska Institutet.

Conflicts of Interest

Charles S. Elmore and Magnus Schou are employed at AstraZeneca. Manolo Mugnaini, Sandra Biesinger, and Sjoerd J. Finnema are employed at AbbVie. Gunnar Nordvall is employed at AlzeCure. Dan Sunnemark is employed at Offspring Biosciences.

Abbreviations

The following abbreviations are used in this manuscript:
Aβ amyloid beta
AD Alzheimer’s disease
ARG autoradiography
α-syn α-synuclein
CAA cerebral amyloid angiopathy
CBD corticobasal degeneration
DLB dementia with Lewy bodies
IHC immunohistochemistry
LBD Lewy-body disease
MSA multiple-system atrophy
PD Parkinson’s disease
PSP progressive supranuclear palsy
TMA tissue microarray

Appendix A

Synthesis of [3H]GMC-058.
1-(3-bromo-4-methoxyphenyl)-4-(4-nitrophenyl)piperazine.
A suspension of 1-(3-bromo-4-methoxyphenyl)piperazine as the HCl salt (198 mg, 0.64 mmol) and cesium carbonate (524 mg, 1.61 mmol) in DMF (5 mL) was stirred under N2 as 1-fluoro-4-nitrobenzene (0.075 mL, 0.71 mmol) was added, and the reaction was stirred at 50 °C for 4.5 h and at rt overnight. The reaction was poured into water (30 mL), and the aqueous portion was washed with isopropyl acetate (3 × 10 mL). The combined organics were dried by passing through a phase separator, and the solvent was removed under reduced pressure. The residue was washed with heptane, and the resultant solid was dried under a vacuum overnight to afford 1-(3-bromo-4-methoxyphenyl)-4-(4-nitrophenyl)piperazine (130 mg, 51.5%) as an orange solid. LCMS (M + H)+: 392.1 (100%), 394 (90%). 1H NMR (500 MHz, DMSO) δ 8.08 (m, 2H), 7.22 (d, J = 2.5 Hz, 1H), 7.09 (m, 2H), 7.01 (m, 2H), 3.77 (s, 3H), 3.60 (m 4H), 3.19 (m, 4H). 13C NMR (126 MHz, DMSO) δ 155.1, 149.7, 146.3, 137.5, 126.2, 121.3, 117.1, 113.9, 113.3, 111.6, 56.9, 49.2, 46.7.
4-(4-(3-bromo-4-methoxyphenyl)piperazin-1-yl)aniline.
A suspension of 1-(3-bromo-4-methoxyphenyl)-4-(4-nitrophenyl)piperazine (130 mg, 0.33 mmol) in ethanol (1 mL) and acetic acid (1 mL) was stirred at room temperature under a stream of nitrogen. Iron (130 mg, 2.32 mmol) was added, and the mixture stirred at 80 °C for 90 min and at rt overnight. The mixture was filtered, and the filtrate was diluted with sat. aq. sodium bicarbonate solution. The aqueous portion was washed with ethyl acetate (2 × 5 mL), and the combined organics were passed through a phase separator. The residue was purified via a reversed-phase HPLC (MeCN/0.2% w/w NH4OH) to afford an off-white solid (25 mg, 20%). The manipulation of the compound in solution resulted in rapid coloration, so the compound was used immediately in the next reaction. LCMS (M + H)+: 362.2 (95%), 364.2 (100%).
N-(4-(4-(3-bromo-4-methoxyphenyl)piperazin-1-yl)phenyl)pivalamide.
A solution of pivaloyl chloride (0.013 mL, 0.10 mmol) and NEt3 (0.029 mL, 0.21 mmol) in CH2Cl2 (1 mL) was stirred for 15 min at rt and then 4-(4-(3-bromo-4-methoxyphenyl)piperazin-1-yl)aniline (25 mg, 0.07 mmol) in CH2Cl2 (1 mL) added. The resulting mixture was stirred for 120 min.
Water (5 mL) was added to the reaction mixture and the layers separated. The aqueous layer was washed twice with CH2Cl2 (2 × 1 mL), and the combined organic layers passed through a phase separator. The organic layer was concentrated to dryness, and the residue was purified via reversed phase HPLC (35–95% gradient over 7 min). Fractions containing the pure product were combined and lyophilized to afford N-(4-(4-(3-bromo-4-methoxyphenyl)piperazin-1-yl)phenyl)pivalamide (22.00 mg, 71.4%) as an off-white solid. LCMS (M + H)+: 446.1 (90%), 448.1 (100%). 1H NMR (500 MHz, DMSO) δ 8.99 (s, 1H), 7.48 (d, J = 9.1 Hz, 2H), 7.20 (d, J = 2.4 Hz, 1H), 7.01 (m, 2H), 6.93 (d, J = 9.1 Hz, 1H), 3.77 (s, 3H), 3.18 (m, 8H), 1.20 (s, 9H). 13C NMR (126 MHz, DMSO): 175.9, 149.0, 147.0, 146.1, 131.7, 121.5, 120.7, 116.5, 115.8, 113.4, 111.1, 56.4, 49.2, 48.9, 27.3.
N-(4-(4-(4-methoxy-[3-3H]phenyl)piperazin-1-yl)phenyl)pivalamide ([3H]GMC-058)
A slurry of N-(4-(4-(3-bromo-4-methoxyphenyl)piperazin-1-yl)phenyl)pivalamide (2.5 mg, 5.60 µmol), 10% Pd-C (2.1 mg, 0.02 mmol) and NEt3 (20 µL, 0.14 mmol) in DMF (0.5 mL) was degassed using freeze-thaw methodology [22] and T2 (274 GBq, 129 µmol) was added. The reaction mixture was stirred at rt for 4 h. The unused tritium was recovered, and the volatiles were removed by blowing a stream of N2 over the slurry. MeOH (0.5 mL) was added and removed under a N2 stream twice. The residue was taken up in EtOH and the catalyst was removed via filtration. HPLC showed 86% radiochemical purity. The residue was purified via preparative HPLC, and product-containing fractions were combined and concentrated to dryness. The residue was taken up in 10 mL of EtOH to give 3554 MBq. LCMS (M − H)−: 368.2 (24%), 370.2 (100%). Molar activity: 854 GBq/mmol. 1H NMR: 9.00 (s, 1H), 7.49 (d, J = 8.5 Hz, 2H) 6.95 (t, J = 10.7 Hz, 4H), 6.86 (s, 1H), 3.71 (s, 3H). 3H NMR: 6.9 (s). LC Rad: 99.5%
Synthesis of [3H]GMC-073.
1-(3-bromo-2-methoxyphenyl)-3-(3-cyano-4-(4-(4-fluorophenyl)piperazin-1-yl)phenyl)urea.
A solution of 5-amino-2-(4-(4-fluorophenyl)piperazin-1-yl)benzonitrile (167 mg, 0.56 mmol), 3-bromo-2-methoxyaniline (114 mg, 0.56 mmol), and NEt3 (0.47 mL, 3.4 mmol) in THF (2 mL) under N2 was cooled in an ice-bath and was stirred as triphosgene (120 mg, 0.40 mmol) in THF (2 mL) was added. The mixture was stirred for 90 min in the ice-bath. Water (25 mL) was added to the mixture, and the mixture was stirred for 15 min. The sample was diluted with EtOAc, and the organic portion was separated. The aqueous layer was washed with EtOAc, and the combined organics were dried (Na2SO4). The solids were removed via filtration, and the solvent was concentrated to dryness. The residue was purified through silica gel chromatography to afford 1-(3-bromo-2-methoxyphenyl)-3-(3-cyano-4-(4-(4-fluorophenyl)piperazin-1-yl)phenyl)urea (34.8 mg, 11.78%) as a pale yellow solid. LCMS (M + 1)+: 524.4 (80%), 526.4 (100%) 1H NMR (500 MHz, DMSO) δ 9.52 (s, 1H), 8.53 (s, 1H), 8.20 (dd, J = 8.3, 1.5 Hz, 1H), 7.91 (d, J = 2.6 Hz, 1H), 7.57 (dd, J = 8.9, 2.7 Hz, 1H), 7.22 (m, 2H), 7.04 (m, 5H), 3.79 (s, 3H), 3.25 (m, 8H).
1-(3-cyano-4-(4-(4-fluorophenyl)piperazin-1-yl)phenyl)-3-(2-methoxy-[3-3H]-phenyl)urea ([3H]GMC-073).
A slurry of 1-(3-bromo-2-methoxyphenyl)-3-(3-cyano-4-(4-(4-fluorophenyl)piperazin-1-yl)phenyl)urea (2.4 mg, 4.58 µmol) and 10% Pd/C (2 mg, 18.79 µmol) in DMF (0.5 mL) and NEt3 (20 µL, 143.49 µmol) was degassed via two freeze/thaw cycles (1), and T2 (280 GBq, 131 µmol) was added. The reaction mixture was stirred at rt for 4 h. The unused tritium was recovered, and then volatiles were removed by blowing a stream of N2 over the slurry. MeOH (0.5 mL) was added and removed under a N2 stream twice. The sample was taken up in 0.5 mL of EtOH and was filtered to yield 4117 MBq. HPLC analysis showed the radiochemical purity to be 47%. The EtOH solution was concentrated to dryness and purified via preparative HPLC. Product-containing fractions were combined and concentrated under vacuum. The sample was dissolved in EtOH (30 mL) to yield 832.8 MBq of [3H]GMC-073. LCMS (M + 1): 446 (19.7%), 448 (100%). Molar activity: 865 GBq/mmol. 1H NMR (500 MHz, DMSO) δ 9.54 (s, 1H), 8.29 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.92 (s, 1H), 7.56 (s, 1H), 7.23 (d, J = 9.2 Hz, 1H), 7.10 (s, 2H), 7.05 (s, 2H), 6.91 (s, 1H), 3.89 (s, 3H). 3H NMR (533 MHz, DMSO): 7.08 (d, 4.3 Hz). LC Rad: 98.9%.
Synthesis of [3H]GMC-098.
N-(2-cyano-4′-methoxy-[1,1′-biphenyl]-4-yl)-4-(4-iodophenyl)piperazine-1-carboxamide.
A solution of N-(2-cyano-4′-methoxy-[1,1′-biphenyl]-4-yl)-4-phenylpiperazine-1-carboxamide (10 mg, 24 µmol) and N-iodosuccinimde (12.5 mg, 55.5 µmol) in MeCN (5 mL) was stirred at rt overnight. LCMS analysis showed an incomplete reaction; therefore, N-iodosuccinimide (5 mg, 20 µmol) was added. After 3.5 h, a third portion of N-iodosuccinimde (5 mg, 20 µmol) was added. After a further 90 min, the reaction was concentrated to dryness under a stream of nitrogen, and the residue was purified via preparative HPLC. Product-containing fractions were pooled and lyophilized to afford (1.8 mg, 14%) as a white solid. The location of the iodide was inferred from the tritiation product. LCMS (M + H)+: 539.1.
N-(2-cyano-4′-methoxy-[1,1′-biphenyl]-4-yl)-4-([4-3H]phenyl)piperazine-1-carboxamide ([3H]GMC-098).
A slurry of N-(2-cyano-4′-methoxy-[1,1′-biphenyl]-4-yl)-4-(4-iodophenyl)piperazine-1-carboxamide (0.75 mg, 1.4 µmol), 10% Pd-C (1 mg), and NEt3 (20 µL, 149 µmol) in DMF (0.5 mL) was degassed via two freeze/thaw cycles [23] and T2 (282 GBq, 132 µmol) was added. The reaction mixture was stirred at rt for 4 h. The unused tritium was recovered, and the volatiles were removed by blowing a stream of N2 over the slurry. MeOH (0.5 mL) was added and removed under a N2 stream twice. The sample was taken up in 0.5 mL of EtOH and was filtered. HPLC analysis showed the radiochemical purity to be 56%. The EtOH solution was concentrated to dryness, and the residue was purified via preparative HPLC. Product-containing fractions were combined and concentrated under vacuum. The sample was dissolved in EtOH (30 mL) to give 281 MBq of [3H]GMC-098. LCMS (M + H)+: 413.3 (8.8%), 415.3 (100%). Molar activity: 974 GBq/mmol. 1H NMR (500 MHz, DMSO) δ 9.02 (s, 1H), 8.55 (m, 2H), 8.04 (s, 1H), 7.83 (d, J = 8.9 Hz, 1H), 7.49 (m, 3H), 7.23 (m, 3H), 7.07 (d, J = 8.2 Hz, 2H), 7.00 (d, J = 8.2 Hz, 2H). 3H NMR (533 MHz, DMSO): 6.88. LC-RAD 98.5%.
Table A1. Demographic data of cases from the Netherlands Brain Bank selected for the construction of tissue microarrays.
Table A1. Demographic data of cases from the Netherlands Brain Bank selected for the construction of tissue microarrays.
Case n.DiagnosisAgeSexPMI (h)Brain RegionAβ/pTau/α-syn *
S11/010Parkinson’s disease82M6.1Cingulate gyrus−/+/+++
S10/47Lewy-body disease76M6.3Hippocampus+++/+++/+++
S14/013Lewy-body disease68F4.6Hippocampus+/+++/+++
S13/054Dementia with Lewy bodies91F4.7Inferior parietal gyrus+++/+/+++
Multiple-system atrophy66M4.9Cerebellum−/−/+++
S10/134Multiple-system atrophy65M4.75Cerebellum−/−/+++
S95/120Multiple-system atrophy69F4.5Cerebellum−/−/+++
S11/074Corticobasal degeneration63M6.5Superior frontal gyrus−/+++/−
S98/105Corticobasal degeneration72M5.3Thalamus and subthalamic nucleus−/+++/−
S11/085Corticobasal degeneration65F6Thalamus−/+++/−
S13/064Progressive supranuclear palsy70M6.8Cerebellum−/+++/−
S12/004Progressive supranuclear palsy65F5.8Cerebellum−/+++/−
S12/083Cerebral amyloid angiopathy79M9.75Hippocampus+++/+++/−
S98/071Cerebral amyloid angiopathy79M9.75Cerebellum+++/−/−
S98/071Cerebral amyloid angiopathy79M9.75Parietal cortex+++/+/+
S98/071Non-dementia control78F7.17Cingulate gyrus−/−/−
S14/029Non-dementia control51F7.7Cingulate gyrus−/−/−
S94/325Non-dementia control63F6.4Cingulate gyrus−/−/−
S95/258Non-dementia control56M5.4Cingulate gyrus−/−/−
S98/235Non-dementia control56M5.4Medial temporal gyrus−/−/−
S98/235Non-dementia control56M5.4Superior frontal gyrus−/−/−
S98/235Alzheimer’s disease85M4.6Inferior frontal gyrus+++/+++
M05/340Alzheimer’s disease67F5.8Hippocampus+++/+++
M05/332Alzheimer’s disease88F5.2Hippocampus+++/+++
M05/331Alzheimer’s disease84F5.9Hippocampus+++/+++
M05/327Parkinson’s disease98M5.7Substantia nigra−/+/+++
S09/252Parkinson’s disease78M5.8Substantia nigra−/+/+++
S13/059Parkinson’s disease86M4.2Substantia nigra+/−/+++
S13/077Parkinson’s disease57M6.6Substantia nigra−/−/+++
S17/055Parkinson’s disease87F7.9Substantia nigra−/−/+++
S17/056Parkinson’s disease90F5.3Substantia nigra−/+/+++
S17/101Parkinson’s disease85F5.2Substantia nigra+/+/+++
S17/162Parkinson’s disease82M6.1Cingulate gyrusAβ/pTau/α-syn *
Aβ/pTau/α-syn = ICH was performed using antibodies for Aβ (6E10/4G8), pTau (AT-8), and α-syn (LB509). * Qualitative score of pathology: − = negative to pathology; + = low abundance of aggregates/inclusions; +++ = high amount of aggregates/inclusions.
Table A2. Demographic data of cases from the Alzheimer’s Disease Research Center selected for brain homogenates.
Table A2. Demographic data of cases from the Alzheimer’s Disease Research Center selected for brain homogenates.
DiagnosisSex(M/F)Age (years)PMT
(h)		ApoEABCRegion Sampled
ADF88434333MFG
ADF67334333MFG
ADF81834333MFG
ADM61633333MFG
ADM62333333MFG
ADM84744333MFG
ControlM4414.2n/a000prefrontal, occipital,
temporal, parietal.
Table A3. Demographic data of cases from the University of California San Francisco selected for brain homogenates.
Table A3. Demographic data of cases from the University of California San Francisco selected for brain homogenates.
DiagnosisSex(M/F)Age (years)PMT
(h)		Abeta Pathology:Tau Pathology:
Specific Tau Lesions		Region Sampled
PSPM634.4n/aNeuronal and glial inclusions, tufted astrocytesL-SFG
PSPF707.2n/aNeuronal and glial inclusions, tufted astrocytesL-SFG
PSPM6810.1n/aNeuronal and glial inclusions, tufted astrocytesR-SFG
CBDM687.40Neuronal and glial inclusions, astrocytic plaquesR-M/IFG(A)
CBDF707.40Neuronal and glial inclusions, astrocytic plaquesR-M/IFG(A)
CBDF635.00Neuronal and glial inclusions, astrocytic plaquesR-M/IFG(A)
Table A4. Demographic data of cases from Banner Sun Health Research Institute selected for brain homogenates.
Table A4. Demographic data of cases from Banner Sun Health Research Institute selected for brain homogenates.
DiagnosisSex(M/F)Age (years)PMTLB Stage
PDM812.5lll. Brainstem/Limbic
PDDM701.83lll. Brainstem/Limbic
PDDM752.25lV. Neocortical
PDM752.33lV. Neocortical
MSA-PF5732n/a.
MSA-PM5812n/a
Table A5. Compounds displaying Ki values < 30 nM for α-syn and >50 nM for Aβ and tau. In bold are shown the compounds that were selected for 3H-radiolabeling.
Table A5. Compounds displaying Ki values < 30 nM for α-syn and >50 nM for Aβ and tau. In bold are shown the compounds that were selected for 3H-radiolabeling.
StructureNameMolecular WeightXLogPα-syn (Ki, nM)Aβ (Ki, nM)Tau-NFT (Ki, nM)
Cells 14 00869 i001GMC013_FR-1397.53.3725.563151
Cells 14 00869 i002GMC015413.53.2628.3294761
Cells 14 00869 i003GMC021442.32.6322.28892510
Cells 14 00869 i004GMC023427.52.8621.215144
Cells 14 00869 i005GMC024427.52.8625.38168
Cells 14 00869 i006GMC025427.52.8629.714150
Cells 14 00869 i007GMC044411.53.3225.23440692
Cells 14 00869 i008GMC045402.53.1524.7412239
Cells 14 00869 i009GMC058367.53.4922.514901320
Cells 14 00869 i010GMC070403.52.646.86490
Cells 14 00869 i011GMC071428.52.3614.2818086
Cells 14 00869 i012GMC073445.53.378.02630248
Cells 14 00869 i013GMC079420.53.2423.94042510
Cells 14 00869 i014GMC086444.53.8715.280926
Cells 14 00869 i015GMC095438.52.9814.526302510
Cells 14 00869 i016GMC096442.54.136.5512510
Cells 14 00869 i017GMC098442.54.139.7226805
Table A6. Average total, non-specific binding (NSB) and total minus NSB (in fmol/mg tissue equivalent) ± standard error of the mean (SEM), measured at two concentrations for each radioligand from the gray matter of the cingulate cortex of n = 1 control subject (C03). Average calculated from 4 technical replicates per condition.
Table A6. Average total, non-specific binding (NSB) and total minus NSB (in fmol/mg tissue equivalent) ± standard error of the mean (SEM), measured at two concentrations for each radioligand from the gray matter of the cingulate cortex of n = 1 control subject (C03). Average calculated from 4 technical replicates per condition.
[3H]GMC-058[3H]GMC-073[3H]GMC-098
25 nM50 nM10 nM20 nM10 nM20 nM
TotalNSBTotalNSBTotalNSBTotalNSBTotalNSBTotalNSB
Average (fmol/mg) ± SEM57.6 ± 1.837.6 ± 3.8114.1 ± 9.374.6 ± 4.1664.6 ± 24.3346.9 ± 14.51870 ± 100.21022 ± 452705 ± 62.21878 ± 74.15146 ± 386.22336 ± 48.5
Total minus NSB (fmol/mg)20403188488272810
Table A7. Binding (fmol/mg tissue equivalent) of 25nM [3H]GMC-058 in tissue sections from controls, PD and MSA patients. Each gray matter (GM) or white matter (WM) value represents an average from the ROIs of four replicates.
Table A7. Binding (fmol/mg tissue equivalent) of 25nM [3H]GMC-058 in tissue sections from controls, PD and MSA patients. Each gray matter (GM) or white matter (WM) value represents an average from the ROIs of four replicates.
Binding in fmol/mg
ConditionCasesROITotalNSB
(5 µM GMC044)		Total Minus NSB
Non-dementia control
(cingulate gyrus)		C01GM56.860.3−3.5
WM56.959.8−2.9
C02GM44.324.619.8
WM45.331.813.6
C03GM45.328.616.7
WM48.739.09.7
MSA
(cerebellum)		MSA1GM46.927.419.5
WM50.133.316.8
MSA2GM30.319.910.3
WM52.527.724.8
PD
(cingulate gyrus)		PD1GM55.642.313.3
WM52.550.61.9
PD2GM16.217.1−0.9
WM55.453.71.6
PD3GM59.342.017.2
WM55.447.18.3
Table A8. Specific 25 nM [3H]GMC058 binding (fmol/mg tissue equivalent) from autoradiography of brain tissue from different proteinopathies. Binding was quantified from gray (GM) and white matter (WM) regions of interest (ROI) in all cases. Bars represent the average ± s.e.m. of two technical replicates quantified from each case. MSA = multiple-system atrophy; CBD = cortical-basal degeneration; PSP = progressive supranuclear palsy; NDE = non-dementia elderly controls. Non-specific binding was measured with excess (5 µM) of cold blocker (GMC-044).
Table A8. Specific 25 nM [3H]GMC058 binding (fmol/mg tissue equivalent) from autoradiography of brain tissue from different proteinopathies. Binding was quantified from gray (GM) and white matter (WM) regions of interest (ROI) in all cases. Bars represent the average ± s.e.m. of two technical replicates quantified from each case. MSA = multiple-system atrophy; CBD = cortical-basal degeneration; PSP = progressive supranuclear palsy; NDE = non-dementia elderly controls. Non-specific binding was measured with excess (5 µM) of cold blocker (GMC-044).
Specific Binding
(fmol/mg Tissue Equivalent)
Region
PathologyCaseRegionsWMGM
MSAMSA3Cerebellum2126
MSA4 4923
CBDCBD1 block 1SFG2950
CBD1 block 2 13032
PSPPSP1GP32102
PSP2 −1118
NDEC03Cingulate cortex−215
C04Caudate−18
C04Globus Pallidus010
Co4Putamen412

Appendix B

Cells 14 00869 g0a1
Figure A1. Representative images from a pilot autoradiography experiment on post-mortem sections from the cingulate cortex of one healthy human subject. Testing specific binding of: [3H]GMC-058: 25 nm, 50 nM; [3H]GMC-073: 10 nM, 20 nM; [3H]GMC-098: 10 nM, 20 nM. Non-specific binding (NSB) was measured with excess (5 µM) of blocker GMC044. GMC044 displaced ~65% of [3H]GMC-058 binding and ~50% of [3H]GMC-073 and [3H]GMC-078. Line scale: 10 mm.
Figure A1. Representative images from a pilot autoradiography experiment on post-mortem sections from the cingulate cortex of one healthy human subject. Testing specific binding of: [3H]GMC-058: 25 nm, 50 nM; [3H]GMC-073: 10 nM, 20 nM; [3H]GMC-098: 10 nM, 20 nM. Non-specific binding (NSB) was measured with excess (5 µM) of blocker GMC044. GMC044 displaced ~65% of [3H]GMC-058 binding and ~50% of [3H]GMC-073 and [3H]GMC-078. Line scale: 10 mm.
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Figure A2. [3H]GMC-058 saturation binding studies. Graphs showing total and non-specific binding of [3H]GMC-058 in homogenates from AD, PSP, CBD, MSA-P, PD, and control patients using concentrations of 0.9 to 80nM.
Figure A2. [3H]GMC-058 saturation binding studies. Graphs showing total and non-specific binding of [3H]GMC-058 in homogenates from AD, PSP, CBD, MSA-P, PD, and control patients using concentrations of 0.9 to 80nM.
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Figure A3. Representative ROIs selected in tissue sections from patients with multiple-system atrophy (MSA), Parkinson’s disease (PD), Lewy-body disease (LBD), and controls (CO) on which analysis was done for Figure 3 and Table A7. The images illustrate an overlay of the toluidine blue stain with the autoradiogram image from the analysis software, where the ROI delineation is visible. The ROIs delineated on the white matter (WM) and gray matter (GM) are indicated with an arrow. Line scale: 10 mm.
Figure A3. Representative ROIs selected in tissue sections from patients with multiple-system atrophy (MSA), Parkinson’s disease (PD), Lewy-body disease (LBD), and controls (CO) on which analysis was done for Figure 3 and Table A7. The images illustrate an overlay of the toluidine blue stain with the autoradiogram image from the analysis software, where the ROI delineation is visible. The ROIs delineated on the white matter (WM) and gray matter (GM) are indicated with an arrow. Line scale: 10 mm.
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Figure A4. Representative ROIs selected in tissue microarray (TMA) autoradiography on which analysis was done for Table 3, Figure A5 and Figure A6. In the microarrays from PD (substantia nigra), the cores were selected based on the pathological evidence of α-syn inclusions, with no A-β plaques or pTau tangles. In the MSA cases, the ROIs were delineated on the areas where the binding of [3H]GMC-058 was observed and corresponded to areas with α-syn inclusions. The numbers are identifiers of the ROIs generated by the analysis software.
Figure A4. Representative ROIs selected in tissue microarray (TMA) autoradiography on which analysis was done for Table 3, Figure A5 and Figure A6. In the microarrays from PD (substantia nigra), the cores were selected based on the pathological evidence of α-syn inclusions, with no A-β plaques or pTau tangles. In the MSA cases, the ROIs were delineated on the areas where the binding of [3H]GMC-058 was observed and corresponded to areas with α-syn inclusions. The numbers are identifiers of the ROIs generated by the analysis software.
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Figure A5. Quantitative analysis from [3H]GMC-058 saturation binding autoradiography in tissue microarrays (TMAs). (A) Non-specific binding plotted as % of total binding. (B) Specific binding in fmol/mg tissue equivalent. Bars represent the average ± s.e.m. of the respective number of cases as described in Table 2. NDE = non-dementia elderly controls; MSA = multiple-system atrophy; CAA = Cerebral amyloid angiopathy; AD = Alzheimer’s disease; CBD = corticobasal degeneration; PSP = progressive supranuclear palsy; PD = Parkinson’s disease.
Figure A5. Quantitative analysis from [3H]GMC-058 saturation binding autoradiography in tissue microarrays (TMAs). (A) Non-specific binding plotted as % of total binding. (B) Specific binding in fmol/mg tissue equivalent. Bars represent the average ± s.e.m. of the respective number of cases as described in Table 2. NDE = non-dementia elderly controls; MSA = multiple-system atrophy; CAA = Cerebral amyloid angiopathy; AD = Alzheimer’s disease; CBD = corticobasal degeneration; PSP = progressive supranuclear palsy; PD = Parkinson’s disease.
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Figure A6. Specific binding (fmol/mg tissue equivalent) of 25 nM [3H]GMC-058, measured in TMAs from cases with different proteinopathies. NDE = non-dementia elderly controls; MSA = multiple-system atrophy; CAA = cerebral amyloid angiopathy; AD = Alzheimer’s disease; CBD = corticobasal degeneration; PSP = progressive supranuclear palsy; PD = Parkinson’s disease. **** p < 0.0001; *** p < 0.001; ** p < 0.01, compared to NDE control, obtained by one-way ANOVA with Dunnett’s multiple comparisons test. Bars represent the average ± s.e.m. of the respective number of cases as described in Table 2. In non-dementia controls (NDE) and liver tissue the values represent displaceable (off-target) binding.
Figure A6. Specific binding (fmol/mg tissue equivalent) of 25 nM [3H]GMC-058, measured in TMAs from cases with different proteinopathies. NDE = non-dementia elderly controls; MSA = multiple-system atrophy; CAA = cerebral amyloid angiopathy; AD = Alzheimer’s disease; CBD = corticobasal degeneration; PSP = progressive supranuclear palsy; PD = Parkinson’s disease. **** p < 0.0001; *** p < 0.001; ** p < 0.01, compared to NDE control, obtained by one-way ANOVA with Dunnett’s multiple comparisons test. Bars represent the average ± s.e.m. of the respective number of cases as described in Table 2. In non-dementia controls (NDE) and liver tissue the values represent displaceable (off-target) binding.
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Figure A7. Representative high magnification images from immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058 performed on TMA sections from cases with MSA and Lewy-body disease. ARG signal (silver grains on the left) correspond to α-syn inclusions in MSA (oligodendrocytic cytoplasmic inclusions) and PD (Lewy bodies) and is reduced after co-incubation of 5 µM GMC-044.
Figure A7. Representative high magnification images from immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058 performed on TMA sections from cases with MSA and Lewy-body disease. ARG signal (silver grains on the left) correspond to α-syn inclusions in MSA (oligodendrocytic cytoplasmic inclusions) and PD (Lewy bodies) and is reduced after co-incubation of 5 µM GMC-044.
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Figure A8. Representative high magnification images from immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058 performed on TMA sections from the substantia nigra of one case with Parkinson’s disease. The circled Lewy bodies are the same identified with ARG and IHC.
Figure A8. Representative high magnification images from immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058 performed on TMA sections from the substantia nigra of one case with Parkinson’s disease. The circled Lewy bodies are the same identified with ARG and IHC.
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Figure A9. Representative high magnification images from immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058 performed on TMA sections from one case with Alzheimer’s disease (AD) and one case with progressive supranuclear palsy (PSP). The ARG signal corresponds to dense A-β plaques in AD and p-tau inclusions in PSP.
Figure A9. Representative high magnification images from immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058 performed on TMA sections from one case with Alzheimer’s disease (AD) and one case with progressive supranuclear palsy (PSP). The ARG signal corresponds to dense A-β plaques in AD and p-tau inclusions in PSP.
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Figure A10. Representative ROIs selected in tissue sections from patients with multiple-system atrophy (MSA), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and non-dementia controls (CO) on which analysis was done for Figure 5 and Table A8. ROIs were delineated based on toluidine blue staining for anatomical reference.
Figure A10. Representative ROIs selected in tissue sections from patients with multiple-system atrophy (MSA), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and non-dementia controls (CO) on which analysis was done for Figure 5 and Table A8. ROIs were delineated based on toluidine blue staining for anatomical reference.
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Figure 1. Chemical structure of [3H]GMC-058.
Figure 1. Chemical structure of [3H]GMC-058.
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Figure 2. [3H]GMC-058 saturation binding assays in (a,a1) AD, (b,b1) PSP, (c,c1) CBD, (d,d1) MSA-P, (e) PD and (f,f1) control brain homogenates. [3H]GMC-058 saturation binding assays were carried out in brain tissue homogenates from AD, PSP, CBD, MSA-P, PD, and control cases using concentrations of 0.9 to 80 nM. Non-specific binding was determined using 10 μM of unlabeled GMC-058. Scatchard plots indicating Bmax and KD values. Error bars represent the mean ± SD for two experiments in triplicates. Bmax = maximum number of binding sites; KD = dissociation constant.
Figure 2. [3H]GMC-058 saturation binding assays in (a,a1) AD, (b,b1) PSP, (c,c1) CBD, (d,d1) MSA-P, (e) PD and (f,f1) control brain homogenates. [3H]GMC-058 saturation binding assays were carried out in brain tissue homogenates from AD, PSP, CBD, MSA-P, PD, and control cases using concentrations of 0.9 to 80 nM. Non-specific binding was determined using 10 μM of unlabeled GMC-058. Scatchard plots indicating Bmax and KD values. Error bars represent the mean ± SD for two experiments in triplicates. Bmax = maximum number of binding sites; KD = dissociation constant.
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Figure 3. Representative images from autoradiography with [3H]GMC-058 (25 nM) on fresh-frozen tissue sections from patients with multiple-system atrophy (MSA), Parkinson’s disease (PD), Lewy-body disease (LB), and controls. Non-specific binding was measured with excess (5 µM) of blocker (GMC-044). Line scale: 10 mm.
Figure 3. Representative images from autoradiography with [3H]GMC-058 (25 nM) on fresh-frozen tissue sections from patients with multiple-system atrophy (MSA), Parkinson’s disease (PD), Lewy-body disease (LB), and controls. Non-specific binding was measured with excess (5 µM) of blocker (GMC-044). Line scale: 10 mm.
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Figure 4. Representative high magnification images from α-synuclein (α-syn), amyloid beta (Aβ) and, phospho-tau (p-tau) immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058. (a–d) In two representative LBD (a,b) and MSA cases (c,d), the ARG signal (yellow arrows) corresponds to Lewy bodies (a) and oligodendrocytic inclusions (c). (e,f) In CAA cases, the ARG signal corresponds to dense perivascular Aβ deposits (e). (g–j) In PSP (g,h) and CBD (j,k), the ARG signal seems to localize in areas of p-tau positive inclusions (g,i). (k,l) In AD the case with abundant Aβ plaques and p-tau inclusions (k), a weak ARG signal was observed (l).
Figure 4. Representative high magnification images from α-synuclein (α-syn), amyloid beta (Aβ) and, phospho-tau (p-tau) immunohistochemistry (IHC) and emulsion autoradiography (ARG) with 25 nM [3H]GMC-058. (a–d) In two representative LBD (a,b) and MSA cases (c,d), the ARG signal (yellow arrows) corresponds to Lewy bodies (a) and oligodendrocytic inclusions (c). (e,f) In CAA cases, the ARG signal corresponds to dense perivascular Aβ deposits (e). (g–j) In PSP (g,h) and CBD (j,k), the ARG signal seems to localize in areas of p-tau positive inclusions (g,i). (k,l) In AD the case with abundant Aβ plaques and p-tau inclusions (k), a weak ARG signal was observed (l).
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Figure 5. Representative images from autoradiography with [3H]GMC-058 (25 nM) and corresponding immunohistochemistry (IHC) heatmap showing the density of α-synuclein (α-syn) or phospho-tau (p-tau) immunoreactivity on fresh-frozen tissue sections from patients with (A) multiple-system atrophy (MSA); (B) cortical-basal degeneration (CBD); (C) progressive supranuclear palsy (PSP); and (D) non-dementia controls. Non-specific binding was measured with excess (5 µM) of cold blocker (GMC-044). (E) Specific binding (fmol/mg) quantified from gray and white matter regions of interest in all cases. Bars represent the average ± s.e.m. of two technical replicates quantified from each case. Line scale: 10 mm. C04 was negative for pTau, α-Syn, and Aβ; therefore a Toluidine blue stain is shown in (D).
Figure 5. Representative images from autoradiography with [3H]GMC-058 (25 nM) and corresponding immunohistochemistry (IHC) heatmap showing the density of α-synuclein (α-syn) or phospho-tau (p-tau) immunoreactivity on fresh-frozen tissue sections from patients with (A) multiple-system atrophy (MSA); (B) cortical-basal degeneration (CBD); (C) progressive supranuclear palsy (PSP); and (D) non-dementia controls. Non-specific binding was measured with excess (5 µM) of cold blocker (GMC-044). (E) Specific binding (fmol/mg) quantified from gray and white matter regions of interest in all cases. Bars represent the average ± s.e.m. of two technical replicates quantified from each case. Line scale: 10 mm. C04 was negative for pTau, α-Syn, and Aβ; therefore a Toluidine blue stain is shown in (D).
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Table 1. Main in vitro and in vivo characteristics of α-syn PET radioligands evaluated in patients with synucleinopathies.
Table 1. Main in vitro and in vivo characteristics of α-syn PET radioligands evaluated in patients with synucleinopathies.
RadioligandIC50 or KDSynucleinopathies
Evaluated **
α-synAβpTau
[18F]ACI-1258933.5 ± 17.4 nM (sporadic PD); 28 nM (MSA)317 nM (AD)PD ***, MSA (+), DLB
[18F]SPAL-T-062.5 nM (MSA)Not reportedMSA (+)
[18F]C05-05IC50 *: 1.5 nM (DLB);IC50 * in AD tissue: 12.9 nMPD/DLB (+) and MSA (+)
* = only 50% of the total binding was displaced in homologous competition experiments. ** = (+) indicates the cases in which specific binding higher than controls was measured. *** = two participants with duplications of the SNCA gene were included in the study.
Table 2. Demographic data of cases from the Netherlands Brain Bank selected for autoradiography studies using fresh-frozen tissue.
Table 2. Demographic data of cases from the Netherlands Brain Bank selected for autoradiography studies using fresh-frozen tissue.
Case n.DiagnosisAgeSexPMI (h)Brain Region
PD1Parkinson’s disease69M7Cingulate gyrus
PD2Parkinson’s disease77M5Cingulate gyrus
PD3Parkinson’s disease75M6Cingulate gyrus
LBV1Lewy-bodies variant83M5Cingulate gyrus
LBV2Lewy-bodies variant54M5Cingulate gyrus
MSA1Multi-system atrophy66M5Cerebellum
MSA2Multi-system atrophy69F5Cerebellum
CBD1Corticobasal degeneration58F7Superior frontal gyrus
C01Non-dementia control81F4Cingulate gyrus
C02Non-dementia control51M8Cingulate gyrus
C03Non-dementia control79F11Cingulate gyrus
Table 3. Demographic data of cases from the UK Brain Bank selected for autoradiography studies using fresh-frozen tissue.
Table 3. Demographic data of cases from the UK Brain Bank selected for autoradiography studies using fresh-frozen tissue.
Case n.DiagnosisAgeSexPMI (h)Brain Region
MSA3Multiple-system atrophy58F10Cerebellum
MSA4Multiple-system atrophy—cerebellar form52M33Cerebellum
PSP1Progressive supranuclear palsy66M8Globus pallidus
PSP2Progressive supranuclear palsy77F11Globus pallidus
C04Control68M7.5Caudate, putamen and globus pallidus
Table 4. Specific binding (fmol/mg tissue equivalent) of 25 nM [3H]GMC-058, measured in TMAs from cases with different proteinopathies. In non-dementia controls (NDE) and liver tissue, the values refer to displaceable (off-target) binding.
Table 4. Specific binding (fmol/mg tissue equivalent) of 25 nM [3H]GMC-058, measured in TMAs from cases with different proteinopathies. In non-dementia controls (NDE) and liver tissue, the values refer to displaceable (off-target) binding.
TMAsNDELiver ControlMSACAAADCBDPSPPD
N=84134226
Mean (±SEM)7057166 **131 **127 **204 ***173 ***129 ***
(4)(7)(19) †(12)(15)(38)(2)(7)
NDE = non-dementia elderly controls; MSA = multiple-system atrophy; CAA = cerebral amyloid angiopathy; AD = Alzheimer’s disease; CBD = corticobasal degeneration; PSP = progressive supranuclear palsy; PD = Parkinson’s disease. *** p < 0.0001; ** p < 0.01, compared to NDE control, obtained via one-way ANOVA with Dunnett’s multiple comparisons test. † SEM from 10 technical replicates of n = 1 MSA case.
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Varrone, A.; Sousa, V.C.; Mugnaini, M.; Biesinger, S.; Nordvall, G.; Kingston, L.; Guzzetti, I.; Elmore, C.S.; Sunnemark, D.; Saturnino Guarino, D.; et al. Development and In Vitro Characterization of [3H]GMC-058 as Radioligand for Imaging Parkinsonian-Related Proteinopathies. Cells 2025, 14, 869. https://doi.org/10.3390/cells14120869

AMA Style

Varrone A, Sousa VC, Mugnaini M, Biesinger S, Nordvall G, Kingston L, Guzzetti I, Elmore CS, Sunnemark D, Saturnino Guarino D, et al. Development and In Vitro Characterization of [3H]GMC-058 as Radioligand for Imaging Parkinsonian-Related Proteinopathies. Cells. 2025; 14(12):869. https://doi.org/10.3390/cells14120869

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Varrone, Andrea, Vasco C. Sousa, Manolo Mugnaini, Sandra Biesinger, Gunnar Nordvall, Lee Kingston, Ileana Guzzetti, Charles S. Elmore, Dan Sunnemark, Dinahlee Saturnino Guarino, and et al. 2025. "Development and In Vitro Characterization of [3H]GMC-058 as Radioligand for Imaging Parkinsonian-Related Proteinopathies" Cells 14, no. 12: 869. https://doi.org/10.3390/cells14120869

APA Style

Varrone, A., Sousa, V. C., Mugnaini, M., Biesinger, S., Nordvall, G., Kingston, L., Guzzetti, I., Elmore, C. S., Sunnemark, D., Saturnino Guarino, D., Finnema, S. J., & Schou, M. (2025). Development and In Vitro Characterization of [3H]GMC-058 as Radioligand for Imaging Parkinsonian-Related Proteinopathies. Cells, 14(12), 869. https://doi.org/10.3390/cells14120869

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