Synthesis and in vitro screening of uPAR-targeting peptides for SPECT imaging of aggressive tumors

Objectives The Urokinase Plasminogen Activator (uPA) protein and its receptor uPAR are involved in cell proliferation and migration; upregulation of the uPA/uPAR system is a marker of highly aggressive tumors. The objective of this work was to develop a Tc-based imaging probe for uPAR that can potentially be used as a clinical tool for non-invasively assessing the metastatic potential of tumors in vivo.

Methods Two amino acid sequences known to display high binding to uPAR were synthesized. A Re single amino acid chelate (ReSAAC) unit was included in each peptide backbone to assess the impact of incorporating a radiometal. The binding affinities of the resulting peptides were determined by flow cytometry. A SAAC-containing analogue of each peptide was synthesized and radiolabeled with ^99mTc; in vitro uptake of the labeled peptide was measured.

Results Flow cytometry data demonstrated that binding of FITC-uPA was inhibited by treating the cells with micromolar amounts of the linear ReSAAC-containing peptide (IC50 6.45 nM and 215 nM, respectively). Affinity of the sequence for uPAR was retained upon incorporation of the ReSAAC unit. The M-containing cyclic peptide exhibited low target-specific binding in both flow cytometry and cell uptake experiments. Several radiolabel-conjugates were substituted in place of MSAAC in order to optimize binding to uPAR.

Conclusions A family of ReSAAC ligands for uPAR were prepared and screened in vitro. It is apparent that peptides with a binding affinity > 2-fold higher than uPA are not suitable candidates for further investigation.