ALBERT

Description: 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 V β 3 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 V β 3 integrin expression: human glioblastoma U87 (α V β 3 +), human lung carcinoma A549 (α V β 3 −) and breast adenocarcinoma MDA-MB-468 (α V β 3 −). 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 V β 3 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 V β 3 +, α V β 5 +, α V β 6 −, α 5 β 1 +) and MDA-MB-468 (α V β 3 −, α V β 5 +, α V β 6 +, α 5 β 1 −) 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 IC 50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only α V β 3 , but also α V β 5 , α V β 6 , and α 5 β 1 . 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 V β 3 (e.g., α V β 5 ). Beilstein J. Org. Chem. 2018, 14, 407–415. doi:10.3762/bjoc.14.29