Synthesis and evaluation of radioactive/fluorescent peptide probes for imaging of legumain activity
ABSTRACT
Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some
cancers and involved in cancer migration, invasion, and metastasis. We
have developed radioiodine- ([125I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu
nonamer via a legumain-cleavable linker, to function as peptide probes
that measure and monitor legumain activity. Non-cleavable probes of
FL-NCP and [125I]I-NCP were similarly prepared and evaluated
as negative control probes by altering their non-cleavable sequence.
Model peptides with the legumain-cleavable or non-cleavable sequence
(LCP and NCP, respectively) reacted with recombinant human legumain, and
only LCP was digested by this enzyme. [125I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [125I]I-NCP
(11.2 ± 0.44% vs 1.75 ± 0.06% dose/mg). The accumulation of FL-LCP in
the HCT116 cells was rather low (4.75 ± 0.29% dose/mg protein), but not
significantly different from the levels of FL-NCP. It is possible that
low concentrations of [125I]I-LCP (40 pM) can be effectively
internalized after legumain cleavage. On the other hand, the cellular
uptake of much higher concentrations of the FL-LCP derivative (1 mM) may
be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [125I]I-LCP
was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood
and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively,
indicating that the [125I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [125I]I-LCP
has been demonstrated to be an effective scaffold for the development
of nuclear medicine imaging probes to monitor legumain activity in
living subjects.