Critical Role of Flanking Residues in NGR-to-isoDGR Transition and CD13/Integrin Receptor Switching

Abstract

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of ␣v␤3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the ␣-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind ␣v␤3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind ␣v␤3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (␣v␤5, ␣v␤6, ␣v␤8, and ␣5␤1), although with 10 -100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas ␣-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.Various peptides containing the Asn-Gly-Arg (NGR) motif have been discovered by peptide-phage library panning in tumor-bearing mice (1). The tumor-homing properties of these peptides rely on the interaction with aminopeptidase N (CD13), a membrane protease expressed by the tumor neovasculature (2, 3). Because of this property, these peptides have been exploited for ligand-directed delivery of various drugs and particles to tumor vessels, in the attempt to increase their antitumor activity (4). For instance, we have shown that peptides containing cyclic CNGRC and linear GNGRG motives can be used for delivering tumor necrosis factor ␣ (TNF) 3 (5-7), interferon ␥ (8 -10), and liposomal doxorubicin (11, 12) to tumor neovasculature, improving their therapeutic properties. The CNGRC-TNF conjugate, called NGR-TNF, is currently tested in phase II clinical studies (13-15). Other investigators have used the NGR motif embedded in similar or different molecular scaffolds for delivering chemotherapeutic drugs, antiangiogenic drugs, tissue factor, viruses, and other compounds to tumor vessels (1, 16 -32). Recently, a CNGRC peptide with an acetylated N-terminal ␣-amino group has been successfully exploited also for quantitative molecular magnetic resonance imaging of ...

Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

“…3B). Competitive binding experiments, performed either with the isoDGR peptide capable to bind the RGD binding pocket of integrins (12) or with the control ARA peptide, showed that the isoDGR peptide could abolish the binding of oxidized-aged Cp to the ␣v␤6 integrin (Student's t test, p Ͻ 0.0001) (Fig. 3C), supporting the role of Cp NGR deamidation in the acquisition of binding activity after aging.…”

Development of Cyclic NGR Peptides with Thioether Linkage: Structure and Dynamics Determining Deamidation and Bioactivity

“…Cyclic peptides of different ring sizes (15 < n < 18 atoms) were designed based on previous studies. 13,19,20 The smallest ring size (n=15) is that of 3, (2)) were prepared that were successfully applied previously for drug targeting. 33,34 An alternative synthetic route for 1 is proposed here.…”

“…43 However, the receptor recognition of isoDGR peptides highly depends on their structure. 13,44 Our objective was to evaluate the 3D structure, the chemo-stability as well as the in vitro interaction of these peptide models. For such study, cell adhesion was monitored as one of the most…”

“…30,31 By comparing c [CNGRC] and GNGRG, a cyclic and a linear peptide, not only the higher CD13 binding affinity of cyclic variant was detected but also its increased stability in PBS solution (half-lives were [6][7][8] h and 3-4 h, respectively at pH 7.3, 37°C) and in serum (5 h and 3 h, respectively) was measured. 13 However, chemo-stability of NGR-peptides under relevant biological conditions is not studied.…”

The tumor vessel targeting agent NGR-TNF controls the different stages of the tumorigenic process in transgenic mice by distinct mechanisms

“…This pattern of expression may due to the fact that during tumor progression endothelial cells and pericytes are differently exposed or susceptible to the angiogenic factors known to regulate CD13. 23 To investigate the interaction between the CNGRCG peptide and CD13 in vivo, the CNGRCGVRSSSRTPSDKY peptide, corresponding to the 17 N-terminal amino acids of the NGR-TNF, was conjugated to quantum dots (hereafter reported as NGR-Qd) 24 and injected in 9 weeks old RIP1-Tag2 mice, during the angiogenic switch stage. Specimens of fresh pancreas were then analyzed by ex vivo whole mount histology.…”