Cationic amino acid-engineered radiopeptides for breast cancer targeting

G-protein coupled estrogen receptors (GPER) represent clinically important target for detection and treatment of breast cancer owing to their high expression. Present study aimed to incorporate and determine the influence of cationic amino acids on the GPER-targeting tetrapeptide, Pro-Leu-Met-Ile (PLMI). Peptide variants were synthesized on solid phase by introducing nuclear localization sequence (KRSKK) at C and N-terminus of PLMI peptide respectively. The three peptides: DOTA-PLMI, DOTA-PLMIKRSKK and DOTA-KRSKKPLMI were radiolabeled with lutetium-177. [¹⁷⁷Lu]Lu-DOTA-PLMI, [¹⁷⁷Lu]Lu-DOTA-PLMIKRSKK and [¹⁷⁷Lu]Lu-DOTA-KRSKKPLMI could be obtained in high radiolabeling yield (> 97%). Cellular studies in SKOV3 (ER-, PR- and GPER+) cells revealed highest binding affinity (K_d: 13.24 ± 0.89 nM) for [¹⁷⁷Lu]Lu-DOTA-PLMIKRSKK along with enhanced nuclear localization and cellular internalization. Amongst the three radiopeptides, [¹⁷⁷Lu]Lu-DOTA-PLMIKRSKK exhibited significantly reduced cellular uptake (95% inhibition) during blocking studies in SKOV3 cells (ER-, PR- and GPER+) than in MCF7 (ER+, PR+ and GPER+) cells (80%) indicating high GPER-specificity. Cellular internalization was also observed to be higher in SKOV3 cells (89%) in comparison to MCF7 cells (75%). Confocal microscopy confirmed high nuclear localization (∼85%) of dye conjugated peptide (Cf-PLMIKRSKK). Biodistribution studies in healthy mice demonstrated no radioactive accumulation in major organs except kidneys. Present studies thus demonstrate strong influence of cationic amino acids at C-terminal, boosting the biological properties of GPER-targeting PLMI peptide.