NGR Tumor-Homing Peptides: Structural Requirements for Effective APN (CD13) Targeting Alessandra Graziadio, † Matteo Zanda, †,‡ Simona Frau, † Ian N. Fleming, † Manuele Musolino, † Sergio Dall’Angelo, † Massimiliano Baldassarre,* ,† and Monica Piras* ,† † 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 * S Supporting Information ABSTRACT: 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 H 2 N-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. ■ INTRODUCTION Mammalian aminopeptidase N (APN or CD13) is a trans- membrane zinc-dependent metalloprotease involved in a variety of processes, including blood pressure regulation, cell migration, viral uptake, cell survival, and angiogenesis. 1 As an exopeptidase, APN cleaves amino acids from the N-terminal portion of peptides, preferentially recognizing neutral residues such as alanine, leucine, and phenylalanine. APN was shown to be overexpressed in many cancer cells and implicated in tumor progression and invasion. 2 Accordingly, APN knockdown or inhibition with bestatin 1, a natural substrate analogue, has been shown to significantly impair tumor growth in animal models. 2,3 APN is selectively recognized by peptides containing the Asn-Gly-Arg (NGR) sequence, a tumor-homing motif discovered by phage display technologies. 4,5 Owing to their tumor-homing properties, NGR-containing peptides, such as the cCNGRC 2 (Figure 1), have been conjugated to anticancer drugs to generate tumor-targeted therapeutics with enhanced efficacy and reduced off-target toxicity. 5,6 A noteworthy example is the NGR-TNF-α construct, a tumor-targeted bioconjugate consisting of human tumor necrosis factor α fused to the C-terminus of cCNGRC, which is currently undergoing clinical trials for the treatment of advanced solid tumors either as a single agent or in combination with standard chemotherapy (www.clinicaltrials.gov). 6,7 The tumor-homing peptide cCNGRC has also been used in molecular imaging probes for in vivo detection of APN expression in solid tumors. Various NGR peptides have been conjugated with signaling molecules for optical imaging, 8 MRI, 9 and nuclear imaging techniques, 10 and the final constructs tested in tumor xenograft models. Despite the potential of APN-directed tumor-homing strategies, the exact structural requirements necessary for APN-NGR recognition and tumor targeting with NGR peptides are still not completely clear. The crystal structure of porcine APN (which shares a high grade of homology with human APN) complexed with the c(CNGRC)G peptide has recently been reported, revealing important structural informa- tion for the development of high-affinity NGR analogues, as well as for the rational design of NGR-based bioconjugates used for site-directed delivery of anticancer drugs and cancer imaging agents. 11 X-ray crystallographic data showed that the NGR motif binds to the zinc-aminopeptidase active site forming specific interactions through the side chains of asparagine and arginine. The binding mode of the cyclic cCNGRC resembles that of APN natural substrates, 12 although not surprisingly the Received: March 10, 2016 Revised: April 12, 2016 Published: April 14, 2016 Article pubs.acs.org/bc © 2016 American Chemical Society 1332 DOI: 10.1021/acs.bioconjchem.6b00136 Bioconjugate Chem. 2016, 27, 1332-1340