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

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 αVβ3 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 αVβ3 integrin expression: human glioblastoma U87 (αVβ3+), human lung carcinoma A549 (αVβ3−) and breast adenocarcinoma MDA-MB-468 (αVβ3−). 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 αVβ3 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 (αVβ3+, αVβ5+, αVβ6−, α5β1+) and MDA-MB-468 (αVβ3−, αVβ5+, αVβ6+, 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 αVβ3, but also αVβ5, αVβ6, 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 αVβ3 (e.g., αVβ5).