CHP Coordinated collagen degradation and synthesis constantly take place during natural tissue homeostasis; however, excessive collagen degradation is associated with numerous pathologic diseases. Our lab has previously developed a collagen hybridizing peptide (CHP), comprised of a repeating Glycine-Proline-Hydroxyproline motif [(GPO) n , n = 5 or 9], that can preferentially target the denatured collagen strands over native intact collagen 1,2,4 which has applications in diagnostics, targeted drug delivery and regenerative medicine. CHP hybridization occurs through native triple helical hybridization, similar to PCR primers hybridizing to DNA fragments. Due to its unique triple helix structure, collagen is highly resistant to most enzymes except matrix metalloproteinases (MMPs) and collagenases. CHPs that maintain this triple helical structure were found to have high serum stability 5 but monomeric CHPs, which can bind to denatured collagen, have yet to be tested. Therapeutic use of CHP derivatives will benefit from understanding their pharmacological properties. Here we report the serum stability of a series of monomeric CHP derivatives in mouse serum and an approach to increase their stability to enhance their longevity in vivo by reducing the rate of enzymatic degradation acting on the CHPs. -SH -SH + IR680-CHP Conjugate IR680-Albumin Conjugate High Serum Stability of Collagen Hybridizing Peptides Lucas L. Bennink a , Daniel J. Smith Ph.D. a , Yang Li Ph.D. a , Catherine A Foss Ph.D. b , Martin G. Pomper Ph.D. b and S. Michael Yu Ph.D. a a Department of Bioengineering, University of Utah, Salt Lake City UT . b Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore MD. Nitrobenzyl Group = Fluorescent Tag = (GPO) 9 Backbone = Structure of NB (GPO) 9 : Nitrobenzyl Keeps CHP Monomeric Materials and Methods Serum Stability Procedure: Peptide solution (50 μL, 1 mM) was incubated in an eppendorf tube containing 25% mouse serum and 70% 1X PBS for 24 hours. At each time point, a 100 μL sample was collected and proteins were precipitated out using ice cold ethanol before centrifugation. The supernatant was then analyzed by RP-HPLC to determine the amount of peptide remaining. HPLC Analysis: Peptide stability was determined by area under the curve (AUC) of the target peptide peak. The area from each time point was recorded as a percentage of the area compared to the 0 min time point . The data was collected in triplicate and graphically represented as an average with error bars. 70% PBS 25% Serum Add Peptide 50 mM Peptide Sample Extract 100 mL Add 200 mL ethanol Centrifuge Time Precipitate serum proteins Take out supernatant and add 900μL of 0.1% TFA to supernatant Analyze with HPLC Conclusions •Monomeric CHPs containing the (GPO) n sequence are resistant to endopeptidase activity, but are subject to a low level of N-terminal exopeptidase activity. • Degradation of the (GPO) n sequence by exopeptidase activity can be suppressed by N-terminal modification. With N-terminal modification, monomeric CHPs composed of (GPO) n repeats have high serum stability which is comparable to triple helical CHPs. •The IR680-Ahx- NB (GPO) 9 [uses NHS-amine chemistry for conjugation], has less protein interaction when compared to Ac-C(IR680)-Ahx- NB (GPO) 9 [uses maleimide-thiol chemistry] but still maintains similar in vivo behavior. Acknowledgements Work was supported by grants from NIAMS/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. 1. Yu, S. M. Curr. Opin. Chem. Biol. 2013, vol. 17(6): 968-975. 2. Li, Y.; Foss, C. A.; Summerfield, D. D.; Doyle, J. J.; Torok, C. M.; Dietz, H. C.; Pomper, M. G.; Yu, S. M. Targeting Collagen Strands by Photo-Triggered Triple-Helix Hybridization. PNAS 2012, 109 (37), 14767–14772. 3. Jenssen, H., & Aspmo, S. I. (2008). Peptide-Based Drug Design, 494(4), 177–186. doi:10.1007/978-1-59745-419-3 4. Li, Y., & Yu, S. M. (n.d.). Targeting and mimicking collagens via triple helical peptide assembly. Collagen-targeting molecules, 4,1–14. 5. Yasui, H.; Yamazaki, C. M.; 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), 705–713. References Results: Effects of N-Terminal Labeling on CHP Stability In Vitro A Figure 1. Peptide stability after 24 hours incubation in 25% mouse serum at 37 °C. (A) Stability profile for unlabeled peptides. (B) Stability profile for N- terminally labeled peptides. (C) Comparison between modified and unmodified peptides after 24 hr. In all cases (GPO) 9 is triple helical. B C Results: In Vivo Imaging Background Figure 3. NIRF-peptides with different linker chemistries (maleimide vs. amide) have similar in vivo binding patterns. These preliminary results show that the CHP conjugated to IR680 dye with either the maleimide-thiol chemistry or the NHS-amine chemistry has similar in vivo behavior. The images were taken from separate experiments but following the same protocol using athymic nude mice. Figure 2. Stability profile of IR680-peptides after 24 hours incubation in 25% mouse serum at 37 °C using different linker chemistries (maleimide-thiol vs NHS-amine). IR680-Ahx-(GPO) 9 Ac-C(IR680)-Ahx-(GPO) 9 (GPO) 9 NB (GPO) 9 S (G 9 P 9 O 9 ) (GPP) 9 (GPO) 5 Figure 3. Proposed mechanism of IR680-dye transfer from CHP to albumin through thiol exchange reaction. N O S O N O S O N O O Proline Specific Peptidases Unlabeled N-Terminally Labeled Rest of C-terminal segment APP DPPII DPPIV IPP Rest of C-terminal segment PE Figure 4. Proposed cleavage sites of relevant proline specific peptidases. This illustration shows possible positions of proline (●) and the potential peptidases that can cleave at that point in the sequence. PE is the only endopeptidase while all other proline specific peptidases shown are exopeptidases. APP- aminopeptidase P; DPPII- dipeptidyl peptidase II; DPPIV- dipeptidyl peptidase IV; PE- prolyl endopeptidase; IPP- iminopeptidase P.