Introduction: Collagen is the most abundant protein in mammals and is a major component of the extracellular matrix (ECM)[1]. High levels of denatured collagen, a product of enzymatic collagen remolding activities, are often found in inflamed tissues in diseases such as tumors and arthritis. Our collagen hybridizing peptide (CHP), comprised of repeating Glycine-Proline-Hydroxyproline motifs [(GPO)n, where O= hydroxyproline and n= 5 or 9], specifically binds to denatured collagen strands over native collagen, by forming a hybrid triple helix structure with unfolded collagen strands[1]. Therapeutics conjugated to CHPs can be specifically delivered to areas with high collagen degradation, increasing the effective dosage at the lesion and reducing any adverse effects on healthy tissues. To exploit the binding specificity of CHP for targeted drug delivery, tissue engineering scaffolds, and diagnostic applications, the serum stability of monomeric CHP and its conjugates have been studied to gain understanding of their behaviors in vivo. CHP can self-assemble into a stable collagen triple helix which is resistant to most enzymatic degradation[2]. However, in order for CHPs to bind denatured collagen, they must be in a monomeric conformation. Here we report the stability of a series of monomeric CHP derivatives in mouse serum and an approach to increase their stability and enhance their longevity in vivo.
Materials and Methods: All peptides were synthesized by standard solid phase peptide synthesis procedures using Fmoc chemistry. Serum stability tests were performed in mouse serum at 37°C and the degradation profiles of the CHP conjugates were ascertained. RP-HPLC analysis was used and degradation was determined by area under the curve from the collected target peaks. Any additional fragment peaks were also collected. Each peak had their molecular weights determined by MALDI-ToF and possible sites of degradation were ascertained. CHP conformation was determined by circular dichroism.
Results and Discussion: Stability tests revealed how conformation, composition, sequence, length, and terminal modifications affected the serum stability of our CHP conjugates. Figure 1A shows the stability of derivatives tested.
CHPs with a triple-helical conformation [(GPO)9], which are highly resistant to enzymatic degradation, had much higher stability than their monomeric counterparts [NB(GPO)9 ]. The inclusion of charged residues [(GPO)4-PK-(GPO)4 ] or a reduction in length [(GPO)5] enhanced enzymatic recognition and degradation. Also, scrambled CHPs [SC(GPO)9] had lower stability than normal sequences with identical composition. Longer CHPs had greater stability than shorter ones. The addition of a bulky group (CF) to the N-terminus increased monomeric CHP stability by greatly reducing exopeptidase activity (Figure 1B).
Conclusion: Tailoring CHP structure for specific application requires full understanding of structure-stability relationship. By conjugating a bulky group (CF) to the N-terminus, exopeptidase activity was inhibited and the stability of monomeric CHPs was increased to a level that is comparable to triple helical CHPs. Additionally, we learned that endopeptidase activity can be reduced by strategic alteration of CHP’s amino acid sequence. This study provides the foundation for future work involving in vivo use of CHP in targeting denatured collagen, especially for diagnostic imaging and targeted drug delivery where stability and circulation time play a crucial role.
Work was supported by grants from NIASM/NIH (R01-AR060484 and R21-AR065124) and DOD (W81XWH-12-0555) awarded to S.M.Y. and by the Nano Institute of Utah: Nanotechnology Fellowship (University of Utah) awarded to L.L.B.; We would also like to thank our lab members for their editorial support in preparing the abstract
References:
[1] Li Y, Yu SM. Targeting and mimicking collagens via triple helical peptide assembly Collagen-targeting molecules. 4:1–14.
[2] Yasui H, Yamazaki CM, Nose H, Awada C, Takao T, Koide T. Potential of collagen-like triple helical peptides as drug carriers: Their in vivo distribution, metabolism, and excretion profiles in rodents. Biopolymers. 2013;100(6):705–13. doi:10.1002/bip.22234.
[3] Li Y, Foss CA, Summerfield DD, et al. Targeting collagen strands by photo-triggered triple-helix hybridization. PNAS. 2012. doi:10.1073/pnas.1209721109.