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 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).

Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery

“…The linker type has only a minimal effect, conjugates with uncleavable linker displayed a slightly reduced affinity, presumably because of the shorter linker between the ligand and the drug. Similar results were reported for analogous conjugates of the cyclo [DKP‐RGD] and cyclo [DKP‐ iso DGR] ligands and paclitaxel or α‐amanitin . In addition, high selectivity for α v β 3 integrin was reported for cyclo [DKP‐RGD]‐paclitaxel conjugates involving various lysosomally cleavable Val−Ala or GFLG linkers .…”

“…The resulting alkyne‐functionalized activated linker 8 was conjugated to cryptophycin‐55 glycinate by forming a carbamate to give compound 9 . The PEG4‐containing azido‐functionalized cyclo [DKP‐RGD] and cyclo [DKP‐ iso DGR] ligands were prepared as previously reported . Final conjugation was achieved by copper(I)‐catalyzed azide−alkyne cycloaddition (CuAAC “click” reaction) between compound 9 and cyclo [DKP‐RGD]‐PEG4‐azide or cyclo [DKP‐ iso DGR]‐PEG4‐azide, respectively.…”

“…In this regard, it has to be noted that the α v β 3 ‐negative M21‐L cell line seems to be more sensitive to cryptophycin and cryptophycin conjugates. It was previously reported that the cytotoxicity of cyclo[DKP‐ iso DGR]‐α‐amanitin conjugates was not integrin‐specific when tested on human glioblastoma U‐87 (α v β 3 +), breast adenocarcinoma MDA‐MB‐468 (α v β 3 −), and it was hypothesized that the internalization process was mediated by integrins different from α v β 3 (e. g. α v β 5 ) . This observation prompted us to select the isogenic M21 and M21‐L cell lines for the in vitro evaluation of cryptophycin conjugates.…”

Size‐Dependent Cellular Uptake of RGD Peptides

“…For example, studies by us revealed colocalization of an RGD‐cryptophycin‐52 conjugate with lysosomes in WM‐115 cells (overexpressing α v β 3 ) and in contrast, an RGD‐cryptophycin‐55 conjugate was internalized both by M21 (overexpressing α v β 3 ) and M21L (α v knockout) cells . Likewise, cytotoxic RGD‐α‐amanitin conjugates did not provide specific uptake . It was further demonstrated that a monomeric RGD‐doxorubicin conjugate afforded good binding affinities toward α v β 3 in a cell adhesion assay, but was not internalized .…”

Synthesis and Biological Evaluation of RGD and isoDGR–Monomethyl Auristatin Conjugates Targeting Integrin αVβ3

“…Conjugates of the functionalized integrin ligands 5 and 6 with the antimitotic drug paclitaxel (PTX) were synthesized featuring a self‐immolative spacer and the lysosomally cleavable Val‐Ala (VA) linker . More recently, ligands 5 and 6 were bound to the RNA polymerase II inhibitor α‐amanitin via a 6′‐ether, giving cyclo [DKP‐RGD]‐VA‐α‐amanitin and cyclo [DKP‐ iso DGR]‐VA‐α‐amanitin conjugates . Cell viability assays indicated a slightly increased cytotoxicity of the cyclo [DKP‐ iso DGR]‐VA‐α‐amanitin conjugate relative to the free drug …”