Structure and activity of CPNGRC: a modified CD13/APN peptidic homing motif

Abstract

Asn-Gly-Arg peptides have been designed as vehicles for the delivery of chemotherapeutics, magnetic resonance imaging contrast agents, and fluorescence labels to tumor cells, and cardiac angiogenic tissue. Specificity is derived via an interaction with aminopeptidase N, also known as CD13, a cell surface receptor that is highly expressed in angiogenic tissue. Peptides containing the CNGRC homing sequence tethered to a pro-apoptotic peptide sequence have the ability to specifically induce apoptosis in tumor cells. We have now identified a modification to the Asn-Gly-Arg homing sequence motif that improves overall binding affinity to aminopeptidase N. Through the addition of a proline residue, the new peptide with sequence, CPNGRC, inhibits aminopeptidase N proteolytic activity with an IC(50) of 10 microM, a value that is 30-fold lower than that for CNGRC. Both peptides are cyclized via a disulfide bridge between cysteines. Steady-state kinetic experiments suggest that efficient aminopeptidase N inhibition is achieved through the highly cooperative binding of two molecules of CPNGRC. We have used NMR-derived structural constraints for the elucidation of the solution structures CNGRC and CPNGRC. Resulting structures of CNGRC and CPNGRC have significant differences in the backbone torsion angles, which may contribute to the enhanced binding affinity and demonstrated enzyme inhibition by CPNGRC.

Figure 1

(A) Hydrolysis of 2 mM pLeuNA as catalyzed by APN in the presence of bestatin, CNGRC and CPNGRC. The assay of activity was initiated with substrate after a 10 minute preincubation of enzyme and inhibitor. (B) Relative activity of APN in the presence of CNGRC (λ) or CPNGRC (○). Activity was quantified by the rate of hydrolysis of 0.20 mM pLeuNA at room temperature in the presence of increasing quantities of peptide.

Figure 1

(A) Hydrolysis of 2 mM pLeuNA as catalyzed by APN in the presence of bestatin, CNGRC and CPNGRC. The assay of activity was initiated with substrate after a 10 minute preincubation of enzyme and inhibitor. (B) Relative activity of APN in the presence of CNGRC (λ) or CPNGRC (○). Activity was quantified by the rate of hydrolysis of 0.20 mM pLeuNA at room temperature in the presence of increasing quantities of peptide.

Figure 2

Steady-state kinetic profile for APN in the presence of CPNGRC. Panels A and B illustrate velocity and double reciprocal plots of the same experimental data for CPNGRC. The concentrations of CPNGRC are 0 μM (λ), 10 μM (σ), 15 μM (ν), and 23 μM (◣). Panels C and D are replots of the intercepts (1/V_max) and slopes (K_m/V_max), respectively, obtained from the double reciprocal plots (panel B) exhibiting a quadratic dependence on the inhibitor concentration.

Figure 3

Steady-state kinetic profile for APN in the presence of CPNGRC-GG-(klaklak)₂. Panels A and B illustrate data obtained with CPNGRC-GG-klaklak₂ in velocity and double reciprocal format. The concentrations of CPNGRC-GG-klaklak₂ are 0 μM (λ), 10 μM (σ), 20μM (ν) and 50 μM (◣).

Figure 4

Loss of inhibitory potency when CPNGRC is incubated at elevated temperatures and pH. CPNGRC was incubated at 37°C and pH 8.5 for 2 hours (□) and then 24 hours (◁). The resultant peptide was assayed for its ability to inhibit APN catalyzed hydrolysis of pLeuNA, relative to peptide kept at pH 7.5 and at 4°C (◇). After 24 hours, the peptide shows almost no inhibition when compared to activity assays in the absence of peptide (○). The concentration of CPNGRC was 50 μM concentration in these assays.

Figure 5

Detection of SAH indicates formation of D_iso in CPD_isoGRC. The chromatograms of assay solutions of CPNGRC peptide that were kept at pH 7.5 on ice (bottom) or pH 8.5 at 37°C overnight (top). In the top panel, an arrow indicates the elution of SAH, which is a product of the methyltransferase reaction with D_iso.

Figure 6

Expanded regions amide proton, side chain regions of the TOCSY and ROESY NMR spectra of CPNGRC with a subset of peaks annotated. The data were collected at 500 MHz at 10°C.

Figure 7

Amide proton region of the ROESY spectra recorded for CNGRC and CPNGRC peptides. The absence of amide proton connections in the ROESY spectra of CNGRC probably reflect dynamics in the peptide, as the circular peptide cannot adopt an extended conformation.

Figure 8

Chemical structures of CNGRC and CPNGRC with dotted lines indicating selected observed crosspeaks in ROESY spectra.

Figure 9

Families of 5 lowest energy structures of (A) CNGRC and (B)CPNGRC obtained from simulated annealing calculations with incorporated NMR restraints. The presence of the proline in CPNGRC forces the peptide backbone into a more elongated ring than in CNGRC.

Figure 10

Ramachandran plot of the APN recognition elements in CNGRC (σ) and CPNGRC (λ) indicating rather large changes in backbone torsion angles. In parentheses next to the CPNGRC points are the ppm changes in chemical shift (CNGRC vs. CPNGRC), observed for the alpha carbon and alpha proton resonances, respectively.

Figure 11

Kinetic scheme for the inhibition of APN (E) by CPNGRC (I). IE and EI are distinct enzyme species with the inhibitor bound in 2 distinct sites.

Figure 12

Sigmoidal dose-response curve for CPNGRC. The velocity data from Figure 1B in the presence of CPNGRC was fit using a simple mixed inhibition (dotted line) or equation 5 (solid line) for the two site binding model. The R value for the simple mixed curve fitting is 0.950; whereas, the fitting