RGD-alpha-amanitin and isoDGR-alpha-amanitin conjugates were synthesized by joining integrin ligands to alpha-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified alpha(V)beta(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 alpha(V)beta(3) integrin expression: human glioblastoma U87 (alpha(V)beta(+)(3)), human lung carcinoma A549 (alpha(V)beta(3)-) and breast adenocarcinoma MDA-MB-468 (alpha(V)beta(3)-). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-alpha-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than a-amanitin. Apparently, for all these alpha-amanitin conjugates there is no correlation between the cytotoxicity and the expression of alpha V beta(3) integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-alpha-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 (alpha(V)beta(3)+, alpha(V)beta(5)+, alpha(V)beta(6)-, alpha(5)beta(1)+) and MDA-MB-468 (alpha(V)beta(3)-, alpha(V)beta(5)+, alpha(V)beta(6)+, alpha(5)beta(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 IC50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only alpha(V)beta(3), but also alpha(V)beta(5), alpha(V)beta(6), and alpha(5)beta(1). These data indicate that in this case the cyclo[DKP-isoDGR]-alpha-amanitin conjugates are possibly internalized by a process mediated by integrins different from alpha(V)beta(3) (e.g., alpha(V)beta(5)).
