Graziadio Alessandra, Zanda Matteo, Frau Simona, Fleming Ian N, Musolino Manuele, Dall'Angelo Sergio, Baldassarre Massimiliano, Piras Monica
Kosterlitz Centre for Therapeutics and Aberdeen Biomedical Imaging Centre, University of Aberdeen , Aberdeen, AB25 2ZD, Scotland, United Kingdom.
C.N.R. - I.C.R.M. , via Mancinelli 7, 20131 Milan, Italy.
Bioconjug Chem. 2016 May 18;27(5):1332-40. doi: 10.1021/acs.bioconjchem.6b00136. Epub 2016 May 2.
Cyclic CNGRC (cCNGRC) peptides are very important targeting ligands for Aminopeptidase N (APN or CD13), which is overexpressed on the surface of many cancer cells. In this work we have (1) developed an efficient solid-phase synthesis and (2) tested on purified porcine APN and APN-expressing human cells two different classes of cCNGRC peptides: the first carrying a biotin affinity tag or a fluorescent tag attached to the carboxyl Arg-Cys-COOH terminus and the second with the tags attached to the amino H2N-Cys-Asn terminus. Carboxyl-terminus functionalized cCNGRC peptides 3, 6, and 8 showed good affinity for porcine APN and very good capacity to target and be internalized into APN-expressing cells. In contrast, amino-terminus functionalized cCNGRC peptides 4, 5, and 7 displayed significantly decreased affinity and targeting capacity. These results, which are in agreement with the recently reported X-ray structure of a cCNGRC peptide bound to APN showing important stabilizing interactions between the unprotected cCNGRC amino terminus and the APN active site, indicate that the carboxyl and not the amino-terminus of cCNGRC peptides should be used as a "handle" for the attachment of toxic payloads for therapy or isotopically labeled functions for imaging and nuclear medicine.
环化CNGRC（cCNGRC）肽是氨肽酶N（APN或CD13）非常重要的靶向配体，APN在许多癌细胞表面过度表达。在这项工作中，我们（1）开发了一种高效的固相合成方法，并且（2）在纯化的猪APN和表达APN的人细胞上测试了两类不同的cCNGRC肽：第一类在羧基端的精氨酸-半胱氨酸-COOH末端连接生物素亲和标签或荧光标签，第二类在氨基端的H2N-半胱氨酸-天冬酰胺末端连接标签。羧基末端功能化的cCNGRC肽3、6和8对猪APN表现出良好的亲和力，并且具有很好的靶向和内化进入表达APN细胞的能力。相比之下，氨基末端功能化的cCNGRC肽4、5和7显示出显著降低的亲和力和靶向能力。这些结果与最近报道的与APN结合的cCNGRC肽的X射线结构一致，该结构显示未保护的cCNGRC氨基末端与APN活性位点之间存在重要的稳定相互作用，这表明cCNGRC肽的羧基端而非氨基端应用作连接治疗用有毒负载或用于成像和核医学的同位素标记功能的“手柄”。
