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Synthesis, gallium labelling and characterization of P04087, a functionalized phosphatidylserine-binding peptide.

ABSTRACT: Background:Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis and thrombus. The hexapeptide PGDLSR was described as a selective and high affinity ligand for PS. In this work, we synthesized and evaluated a gallium labelled-PGDLSR peptide as a potential and selective radiopharmaceutical for nuclear imaging. PGDLSR-?-alanine-NODAGA (P04087) was prepared using Fmoc-based synthesis and then chelated with cold gallium, 68Ga and 67Ga. The affinity of Ga-P04087 for PS was evaluated by a competitive binding assay using biotinylated AnnexinV. The in vitro stability of the radiotracer was checked at room temperature and after incubation in human serum at 37 °C with and without a metalloprotease inhibitor. The in vivo binding of 67Ga-P04087 to phosphatidylserine was evaluated in a rat model of infective endocarditis. Results:PGDLSR was successfully prepared with a yield of 31%. P04087 was obtained with a yield of 28% and in high chemical purity (>95%). The radiochemical purities of 67Ga-P04087 and 68Ga-P04087 exceeded 98% in all cases. IC50 of P04087 and Ga-P04087 were in the same order of magnitude (10-7M). The radiolabelled product was stable for 24 h at room temperature, but was very rapidly degraded in human serum in the absence of a protease inhibitor, which had a stabilizing effect. No focal uptake could be detected visually in the cardiac area on SPECT images. On autoradiography however, a focal uptake of 67Ga-P04087 in the valve area was present and histological slices demonstrated localization of peptide binding at the peripheral layer of vegetations. Conclusion:In spite of the preservation of the peptide affinity to the PS after its conjugation to the NODAGA chelator, and of the presence of 67Ga-P04087 uptake on autoradiography, the absence of detectable foci in vivo in the valve area may be attributed to both the low intensity of the signal and the presence of background activity originating from blood pool and surrounding tissues in the living animals. Further modifications are necessary to design a radiolabeled peptide with higher binding potencies to PS while possessing enhanced metabolic stability in vivo.

SUBMITTER: Ben Azzouna R

PROVIDER: S-EPMC5835976 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Synthesis, gallium labelling and characterization of P04087, a functionalized phosphatidylserine-binding peptide.

Ben Azzouna Rana R Guez Alexandre A Benali Khadija K Al-Shoukr Faisal F Gonzalez Walter W Karoyan Philippe P Rouzet François F Le Guludec Dominique D

EJNMMI radiopharmacy and chemistry 20170207 1

<h4>Background</h4>Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis and thrombus. The hexapeptide PGDLSR was described as a selective and high affinity ligand for PS. In this work, we synthesized and evaluated a gallium labelled-PGDLSR peptide as a potential and selective radiopharmaceutical for nuclear imaging. PGDLSR-β-alanine-NODAGA (P04087) was prepared using Fmoc-based synthesis and then chelated with cold gallium, <su ...[more]

PMID: 29527564

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Project description:BackgroundThe dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure.MethodsFor this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression.ResultsThe biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice.ConclusionsWe have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.

Dataset Information

Synthesis, gallium labelling and characterization of P04087, a functionalized phosphatidylserine-binding peptide.

Publications

Synthesis, gallium labelling and characterization of P04087, a functionalized phosphatidylserine-binding peptide.

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