Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting

Abstract

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified α_Vβ₃ receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of α_Vβ₃ integrin expression: human glioblastoma U87 (α_Vβ₃+), human lung carcinoma A549 (α_Vβ₃-) and breast adenocarcinoma MDA-MB-468 (α_Vβ₃-). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of α_Vβ₃ integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (α_Vβ₃+, α_Vβ₅+, α_Vβ₆-, α₅β₁+) and MDA-MB-468 (α_Vβ₃-, α_Vβ₅+, α_Vβ₆+, α₅β₁-) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC₅₀ was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only α_Vβ₃, but also α_Vβ₅, α_Vβ₆, and α₅β₁. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from α_Vβ₃ (e.g., α_Vβ₅).