title: Copper-64 radiolabelling of the C2A domain of synaptotagmin I using a functionalised bis(thiosemicarbazone): A pre- and post-labelling comparison.
abstract: Dysregulation of apoptosis and necrosis is central to many diseases and non-invasive imaging of cell death is an important clinical objective to stage disease or to monitor treatment progress. The C2A domain of rat synaptotagmin I binds to phosphatidylserine (PS) exposed during cell death and modification to its lysine residues has been shown to disrupt PS binding. Site-specifically labelled (99m)Tc(CO)3-C2AcH and (68)Ga-C2Ac have previously been investigated for single photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging, respectively. We wished to design a (64)Cu-labelled counterpart due to the longer half-life of (64)Cu. Since the calcium binding sites in C2A may interfere with copper binding we sought a high affinity, fast labelling chelator. We synthesised a maleimide functionalised bis(thiosemicarbazone), H2ATSE/AMal, for the site-specific copper-64 radiolabelling of thiol-functionalised C2Ac. When radiolabelling was performed by incubation of the ligand-protein conjugate (post-labelling approach), analysis of the resultant (64)CuATSE/AMal-C2Ac revealed that the C2Ac was able to compete for radiocopper with the chelator. In contrast, the pre-labelled (64)CuATSE/AMal-C2Ac conjugate revealed good stability in serum and maintained target affinity in a red blood cell binding assay. The results suggest that due to the intrinsic copper binding properties of the protein, a pre-labelling approach is preferred for the C2Ac domain of synaptotagmin I when copper is the desired radioisotope.
authors: Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom; Division of Imaging Sciences & Biomedical Engineering, King's College London, St. Thomas' Hospital, London, United Kingdom.
authors: Rebekka
authors: R
authors: Hueting
authors: Richard
authors: R
authors: Tavaré
authors: Jonathan R
authors: JR
authors: Dilworth
authors: Gregory E
authors: GE
authors: Mullen
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium; D003301:Copper Radioisotopes / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D015394:Molecular Structure; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D050863:Synaptotagmin I / Q000737:chemistry* / Q000378:metabolism; D013882:Thiosemicarbazones / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003301:Copper Radioisotopes; D010718:Phosphatidylserines; D019275:Radiopharmaceuticals; D050863:Synaptotagmin I; D013882:Thiosemicarbazones; D002118:Calcium
keywords: C2A; Copper bis(thiosemicarbazone); Copper-64