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Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search

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Abstract

Purpose

Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates.

Methods

Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson’s disease (PD) and Alzheimer’s disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21.

Results

In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aβ42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.).

Conclusion

Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aβ and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

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The data needed to evaluate the conclusions of the paper are present in the paper and/or the Supplementary Materials.

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Acknowledgments

This research was funded by the Michael J. Fox Foundation and NIH grant U19-NS110456. M.G.L. was supported by an Age Related Neurodegenerative Disease Training Grant fellowship (NIH T32-AG000255). H.J.K. was supported by an NIH Predoctoral Fellowship (NIH F31-AG069390).

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Authors and Affiliations

  1. Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Bieneke Janssen, Guilong Tian, Zsofia Lengyel-Zhand, Chia-Ju Hsieh, Aladdin Riad, Kuiying Xu, Catherine Hou, Chi-Chang Weng, Vinayak V. Pagar & Robert H. Mach

  2. Department of Chemistry, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Marshall G. Lougee, Vinayak V. Pagar, John J. Ferrie & E. James Petersson

  3. Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA

    Brian J. Lopresti & Chester A. Mathis

  4. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Hee Jong Kim & Benjamin A. Garcia

  5. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Kelvin Luk

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Janssen, B., Tian, G., Lengyel-Zhand, Z. et al. Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search. Mol Imaging Biol 25, 704–719 (2023). https://doi.org/10.1007/s11307-023-01814-9

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