Bodero Lizeth, López Rivas Paula, Korsak Barbara, Hechler Torsten, Pahl Andreas, Müller Christoph, Arosio Daniela, Pignataro Luca, Gennari Cesare, Piarulli Umberto
Dipartimento di Scienza e Alta Tecnologia, Via Valleggio, 11, 22100, Como, Italy.
Dipartimento di Chimica, Università degli Studi di Milano, Via C. Golgi, 19, I-20133, Milan, Italy.
Beilstein J Org Chem. 2018 Feb 14;14:407-415. doi: 10.3762/bjoc.14.29. eCollection 2018.
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 αβ 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 αβ integrin expression: human glioblastoma U87 (αβ+), human lung carcinoma A549 (αβ-) and breast adenocarcinoma MDA-MB-468 (αβ-). 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 αβ 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 (αβ+, αβ+, αβ-, αβ+) and MDA-MB-468 (αβ-, αβ+, αβ+, αβ-) 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 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only αβ, but also αβ, αβ, and αβ. 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 αβ (e.g., αβ).
通过各种连接子和间隔物将整合素配体与α-鹅膏蕈碱连接，合成了RGD-α-鹅膏蕈碱和异DGR-α-鹅膏蕈碱缀合物。评估了这些缀合物抑制生物素化玻连蛋白与纯化的αβ受体结合的能力，其保留了良好的结合亲和力，与游离配体处于相同的纳摩尔范围内。在三种具有不同水平αβ整合素表达的细胞系中评估了缀合物的抗增殖活性：人胶质母细胞瘤U87（αβ+）、人肺癌A549（αβ-）和乳腺腺癌MDA-MB-468（αβ-）。在U87、MDA-MB-468以及部分A549癌细胞系中，发现带有可被溶酶体切割的Val-Ala连接子的环[DKP-异DGR]-α-鹅膏蕈碱缀合物比α-鹅膏蕈碱稍具更强的效力。显然，对于所有这些α-鹅膏蕈碱缀合物，细胞毒性与αβ整合素的表达之间没有相关性。为了确定环[DKP-异DGR]-α-鹅膏蕈碱缀合物增加的细胞毒性是否由整合素介导的结合和内化过程所控制，进行了竞争实验，其中在过量西仑吉肽存在的情况下，用U87（αβ+、αβ+、αβ-、αβ+）和MDA-MB-468（αβ-、αβ+、αβ+、αβ+）细胞测试缀合物，目的是阻断细胞表面的整合素。使用MDA-MB-468细胞系，在过量西仑吉肽存在的情况下，观察到缀合物的IC增加了五倍，已知西仑吉肽不仅能强烈结合αβ，还能结合αβ、αβ和αβ。这些数据表明，在这种情况下，环[DKP-异DGR]-α-鹅膏蕈碱缀合物可能通过不同于αβ（例如αβ）的整合素介导的过程内化。
