(18)F-Labeled phosphopeptide-cell-penetrating peptide dimers with enhanced cell uptake properties in human cancer cells.
BACKGROUND
Phosphopeptides represent interesting compounds to study and elucidate cellular protein phosphorylation/dephosphorylation processes underlying various signal transduction pathways. However, studies of phosphopeptide action in cells are severely constrained by the negatively charged phosphate moiety of the phosphopeptide resulting in poor transport through the cell membrane. The following study describes the synthesis and radiopharmacological evaluation of two (18)F-labeled phosphopeptide-cell-penetrating peptide dimers. The polo-like kinase-1-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH was coupled to cell-penetrating peptides (CPPs), either sC18, a cathelicidin-derived peptide, or the human calcitonin derivative hCT(18-32)-k7.


METHODS
Radiolabeling was accomplished with the prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) using both, conventional and microfluidic-based bioconjugation of [(18)F]SFB to N-terminal end of phosphopeptide part of the peptide dimers. Cellular uptake studies in human cancer cell lines HT-29 and FaDu cells at 4 °C and 37 °C and small animal PET in BALB/c mice were utilized for radiopharmacological characterization.


RESULTS
Isolated radiochemical yields ranged from 2% to 4% for conventional bioconjugation with [(18)F]SFB. Significantly improved isolated radiochemical yields of up to 26% were achieved using microfluidic technology. Cellular uptake studies of radiolabeled phosphopeptide and phosphopeptide-CPP dimers indicate enhanced internalization of 50% ID/mg protein after 2 h for both phosphopeptide dimers compared to the phosphopeptide alone (<1% ID/mg protein). In vivo biodistribution of (18)F-labeled peptide dimers was determined with small animal PET revealing a superior biodistribution pattern of sC18-containing peptide dimer MQSpTPL-sC18 [(18)F]4.


CONCLUSIONS
([18)F]SFB labeling of the phosphopeptide-CPP dimers using a microfluidic system leads to an improved chemoselectivity towards the N-terminal NH(2) group compared to the conventional labeling approach. Cell-penetrating peptide sC18 can be considered as an ideal molecular shuttle for intracellular delivery of the Plk1-PBD-binding hexaphosphopeptide as demonstrated by its favourable radiopharmacological profile.
Institute of Radiopharmacy, Helmholtz Centre Dresden-Rossendorf, 01314 Dresden, Germany.
Susan
S
Richter
Vincent
V
Bouvet
Melinda
M
Wuest
Ralf
R
Bergmann
Joerg
J
Steinbach
Jens
J
Pietzsch
Ines
I
Neundorf
Frank
F
Wuest
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001565:Benzoates / Q000737:chemistry; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D019281:Dimerization; D005462:Fluorine Radioisotopes / Q000737:chemistry*; D019073:HT29 Cells; D006801:Humans; D007553:Isotope Labeling; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism*; D049268:Positron-Emission Tomography; D021381:Protein Transport; D019275:Radiopharmaceuticals / Q000737:chemistry / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry
D001565:Benzoates; D057846:Cell-Penetrating Peptides; D005462:Fluorine Radioisotopes; D010748:Phosphopeptides; D019275:Radiopharmaceuticals; D013388:Succinimides; C075713:N-succinimidyl-4-fluorobenzoate