1. 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 collagen1,2,4 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 stability5 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. Benninka, 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
aDepartment of Bioengineering, University of Utah, Salt Lake City UT .
bDepartment 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
Amide-linked
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(G9P9O9)
(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.