NGR Tumor-Homing Peptides: Structural Requirements for Effective APN (CD13) Targeting

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 functionalised cCNGRC peptides 3, 6, 8 showed good affinity for porcine APN and very good capacity to target and be internalised into APN-expressing cells. In contrast, amino-terminus functionalised cCNGRC peptides 4, 5, 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 stabilising 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 labelled functions for imaging and nuclear medicine.