Identification and in vitro characterization of C05-01, a PBB3 derivative with improved affinity for alpha-synuclein

• Published in: Brain research 2020-12, Vol.1749, p.147131-147131, Article 147131
• Main Authors: Miranda-Azpiazu, Patricia, Svedberg, Marie, Higuchi, Makoto, Ono, Maiko, Jia, Zhisheng, Sunnemark, Dan, Elmore, Charles S., Schou, Magnus, Varrone, Andrea
• Format: Article
• Language: English
• Subjects: Alpha synuclein; Autoradiography and in vitro binding assay; PBB3; Tissue microarrays
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/j.brainres.2020.147131

•C05-01 is an analogue of PBB3 displaying higher affinity for α-synuclein and was successfully labelled with 3H.•The binding of C05-01 to α-synuclein was evaluated using various techniques, such as fluorescence and autoradiographic evaluation in fresh frozen tissue, autoradiography in tissue microarrays (TMAs), and in vitro binding assays using human brain homogenates and fibrils.•[3H]C05-01 showed specific binding in tissue with α-synuclein pathology from PD and MSA patients, but also in tissue with amyloid-β and tau pathology.•[3H]C05-01 displayed off-target binding that could not be attributed to MAO-A, MAO-B, or Sigma 1 and that requires further examination and a need to improve the physicochemical properties and the relative affinity of C05-01 for further development as PET radioligand•TMAs are useful tools for evaluating the binding of test compounds to misfolded proteins. The neuropathological hallmark of Parkinsońs disease, multiple system atrophy and dementia with Lewy bodies is the accumulation of α-synuclein. The development of an imaging biomarker for α-synuclein is an unmet need. To date, no selective α-synuclein imaging agent has been identified, though initial studies suggest that the tau tracer [11C]PBB3 displays some degree of binding to α-synuclein. In this study, a series of compounds derived from the PBB3 scaffold were examined using fluorescence imaging and tissue microarrays (TMAs) derived from brain samples with different proteinopathies. One compound, C05-01, was selected based on its higher fluorescence signal associated with Lewy body aggregates compared with other PBB3 analogues. In vitro binding assays using human brain homogenates and recombinant fibrils indicated that C05-01 had higher affinity for α-synuclein (KD/Ki 25 nM for fibrils, Ki 3.5 nM for brain homogenates) as compared with PBB3 (KD 58 nM). In autoradiography (ARG) studies using fresh frozen human tissue and TMAs, [3H]C05-01 displayed specific binding in cases with α-synuclein pathology. C05-01 is the first PBB3 analogue developed as a potential compound targeting α-synuclein. Despite improved affinity for α-synuclein, C05-01 showed specific binding in AD tissue with Amyloid β and tau pathology, as well as relatively high non-specific and off-target binding. Additional efforts are needed to optimize the pharmacological and physicochemical properties of this series of compounds as ligands for α-synuclein. This study also showed that the construction of TMAs from diff