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 alphavbeta3-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 alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alphavbeta3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alphavbeta3 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 (alphavbeta5, alphavbeta6, alphavbeta8, and alpha5beta1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-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.

FIGURE 1.

Competitive binding of NGR-Qdot with cyclic and linear NGR peptides to HUVECs. A, binding of NGR-Qdot, isoDGR-Qdot, ARA-Qdot, and Qdot (1:100) to HUVECs. B, competitive binding of NGR-Qdot and isoDGR-Qdot with NGR-2C and isoDGR-2C (500 μg/ml). C, competitive binding of NGR-Qdot with various doses of NGR-2C, NGR-2G or SGR-2C. Fluorescence microscopy assays were carried out as described under “Experimental Procedures.” Representative images of three independent experiments are shown (left). Quantification of staining intensity was performed by using the CellF Software (Olympus Soft Imaging Solutions GmbH, Germany) (right). Four images were analyzed for each condition. Magnification, ×400; scale bar, 50 μm; red, Qdot; blue, nuclear staining with DAPI.

FIGURE 2.

Differential stability of cyclic and linear NGR and isoDGR peptides. A, RP-HPLC of NGR-2C-TNF_1–11 (5 μg) and NGR-2G-TNF_1–11 (5 μg) after incubation at 37 °C in PBS. Dotted line, untreated peptide. Peak 1 corresponds to NGR-2C-TNF_1–11; peak height was proportional to the loaded material within the range of 1–50 μg. B, RP-HPLC of NGR-2C after incubation at 37 °C in human serum. The peptide was added to human serum (500 μg/ml, final concentration) and incubated for the indicated time. The sample was then ultrafiltered through a 5 kDa cut-off ultrafilter (Vivaspin 500, Sartorius, Italy). The permeate (50 μl) was analyzed by RP-HPLC. Peak 1 corresponds to NGR-2C. C, stability of NGR-2C-TNF_1–11, NGR-2G-TNF_1–11, and NGR-2C after incubation at 37 °C or at 4 °C in PBS, HEPES buffer, water, or human serum, as determined by RP-HPLC. D and E, MALDI-TOF MS analysis of non-acetylated and acetylated NGR-2G, NGR-2C, isoDGR-2G, and isoDGR-2C after incubation at 37 °C for 0 and 8 days in PBS. +0, +1, −17, and −18 correspond to the difference between the found and the expected molecular masses in daltons.

FIGURE 3.

Functional properties of different isoDGR peptides: αvβ3 integrin binding, inhibition of endothelial cell adhesion, and inhibition of tumor growth. A, competitive binding of isoDGR-Qdot with various doses of isoDGR-2C, isoDGR-2G, or SGR-2C to HUVECs. Representative images of three independent experiments are shown. Fluorescence microscopy assays were carried out as described under “Experimental Procedures.” Magnification, ×400; scale bar, 50 μm; red, Qdot; blue, nuclear staining with DAPI. B, binding of NGR/STV-HRP, isoDGR/STV-HRP, and ARA/STV-HRP to αvβ3 integrin. Complexes were diluted 1:500 in 25 mm Tris-HCl, pH 7.4, containing 150 mm sodium chloride, 1 mm magnesium chloride, 1 mm manganese chloride, 3% BSA (1:500), added to microtiter plates coated with αvβ3, and incubated for 2 h at room temperature. After washing, the binding was detected by chromogenic reaction with 3,3′,5,5′-tetramethylbenzidine chromogenic substrate. Mean ± S.E. of three independent experiments (each in duplicate). C, inhibition of EA.hy926 cells adhesion (upper panel) or HUVECs adhesion (lower panel) to isoDGR-TNF-coated plates by acetylated (ac) and non-acetylated isoDGR-2C and isoDGR-2G peptides. Cell adhesion assay was performed as described under “Experimental Procedures.” The representative results of three independent experiments (each in duplicate) is shown. D, anti-tumor effect of repeated administrations of ac-isoDGR-2C or ac-isoDGR-2G peptide (5 mg/kg, intraperitoneal) to WEHI-164 tumor-bearing mice. Animals were treated at the indicated times (arrows). Cumulative data of three independent experiments (16 mice/group in total) (mean ± S.E.) are shown. Two-tailed t test at day 13: *, p < 0.0003; n.s., not significant.

FIGURE 4.

Schematic representation of potential cyclic and linear NGR peptide degradation reactions. A, nucleophilic attack of the backbone NH center (blue) on the Asn side chain (red) of cyclic CNGRC leads to formation of a succinimide intermediate (−17 Da), which after hydrolysis may lead to formation of Asp and isoAsp (+1 Da). B, succinimide formation and hydrolysis can occur also with linear GNGRG. However in this case, the succinimide intermediate may also react with α-amino group leading to the formation of seven-membered ring or diketopiperazine (DKP).