Unexpected enhancement in biological activity of a GPCR ligand induced by an oligoethylene glycol substituent

Article abstract of DOI:10.1021/ja102640c

Polyethylene glycol (PEG) is widely used, and many biologically active molecules are modified with oligoethylene glycol substituents to enhance their half-lives in circulation. The pervasive use of PEG substituents is partly due to their presumed inertness. Our investigation of formyl peptide receptor (FPR)-mediated chemotaxis reveals that oligoethylene glycol substitution can enhance the ability of the peptide chemoattractant N-formyl-methionine-leucine- phenylalanine (fMLF) to activate signal transduction through FPR, a transmembrane G-protein-coupled receptor.

Full text of DOI:10.1021/ja102640c

Published on Web 06/10/2010
Unexpected Enhancement in Biological Activity of a GPCR Ligand Induced by
an Oligoethylene Glycol Substituent
Chutima Jiarpinitnun†,§ and Laura L. Kiessling*,†,‡
Departments of Chemistry and Biochemistry, UniVersity of Wisconsin-Madison, Madison, Wisconsin 53706
Received March 29, 2010; E-mail: kiessling@chem.wisc.edu
Scheme 1. Route to Formyl Peptides Designed To Bind to FPR
Polyethylene glycol (PEG) is often used to enhance key properties
of biologically active molecules.1,2 PEG substituents can augment the
efficacy of protein and peptide therapeutic agents by protecting them
from proteolytic degradation3 or decreasing their rate of clearance from
plasma.2 The widespread use of PEG for these purposes stems from
its low toxicity, excellent aqueous solubility, and low antigenicity.
These properties appear to be shared by ethylene glycol oligomers.
Indeed, oligoethylene glycol groups have been employed to tether
biological recognition elements (e.g., to form dimers4 or higher-order
oligomers5). Because their persistence length can be estimated,6
oligoethylene glycol moieties are attractive linkers in the construction
of potent multivalent ligands.4,5 Moreover, oligoethylene glycol
moieties on surfaces or in biomaterials resist nonspecific protein
binding.7 Thus, the widespread use of oligoethylene substituents also
stems from their presumed inertness. Here we present results demon-
strating that an oligoethylene glycol substituent can enhance the potency
of a ligand for a transmembrane G-protein-coupled receptor (GPCR).
Our studies originated from our interest in chemotactic signaling.
A key initiator of neutrophil chemotaxis is the formyl peptide receptor
(FPR). FPR belongs to the largest and most diverse family of integral
membrane signaling receptors, the GPCR family.8 FPR, which is
present at high levels on the surface of neutrophils and monocytes,
mediates chemotactic responses to N-formylated peptides, including
the canonical chemoattractant N-formyl-methionine-leucine-phenyla-
lanine (fMLF). Formylated peptides are produced from sources that
include the mitochondrial proteins of ruptured host cells and the
proteins of invading pathogens.9 The molecular details of FPR-ligand
complexes have not been elucidated to date; however, modeling of
the seven transmembrane R-helices10 suggests that the FPR binding
site can accommodate four to five amino acids.11 Structure-activity
relationship data indicate that formyl peptide derivatives with C-
terminal substituents can retain the activity of the parent compound.12,4a
Because we were interested in generating formyl peptide probes of
chemotactic signaling, we tested the consequences of adding C-terminal
linker substituents.
We used squarate-derived building block 4 and free peptide 1 to
assemble a series of derivatives possessing C-terminal substituents with
six (5), nine (6), or twelve (7) ethylene glycol units. The resulting
compounds were evaluated for their abilities to activate signaling in
FPR-transfected U937 cells, a monocytic cell line.14 Like neutrophils,
these cells can respond to even a shallow gradient of chemoattractant.15
To assay chemotactic responses, we employed a simplified multiwell
Boyden chamber assay, and the number of migrating cells was
determined by using a cell proliferation assay.16,17 All of the fNleLF
derivatives promote cell migration and therefore serve as attractants.
Their differential effects on chemotaxis, however, were surprising.
Specifically, the more hydrophilic ethylene glycol unit might be
expected to decrease the ability of fNleLF to bind to its transmembrane
receptor and thereby mitigate attractant activity. Unexpectedly, these
substituents had a dramatic positive effect on chemotaxis (Figure 1A).
Compound 5, with six ethylene glycol units, is a more powerful
attractant than the free N-formyl peptide. Compound 6 with nine
ethylene glycol units is even more potent. Indeed, compared with the
nonderivatized formyl peptide 1, compound 6 is >20-fold more active.
The trend, however, did not continue beyond nine units. Compound
7, which possesses an oligoethylene glycol substituent of 12 units, is
less active than compound 6. These results suggest that the oligoeth-
ylene glycol substituent is not inert but instead increases the chemo-
tactic activity by an extent that depends upon its length.
Precedent suggested that a tether based on oligoethylene glycol
would have little effect on signaling. To test this assumption, we
appended a series of ethylene glycol oligomers to the C-terminus of a
formyl peptide. The FPR ligand we employed, N-formyl-norleucine-
leucine-phenylalanine (fNleLF), is a chemoattractant.13 Though less
potent than fMLF, its chemical stability is superior. Specifically, the
methionine residue in fMLF can undergo oxidation, thereby complicat-
ing the synthesis and handling of its derivatives. In contrast, fNleLF-
based compounds are stable. To assemble the target compounds,
oligoethylene glycol building blocks 2 and 4 were synthesized.5a These
precursors could be conjugated to the peptidic chemoattractant to yield
a series of C-terminal-modified fNleLF derivatives (Scheme 1).
To test whether the differences in the cell migration assay depended
on FPR signaling, we evaluated the ability of the fNleLF derivatives
to elevate a key indicator of chemotactic signaling, namely, intracellular
calcium ion concentration ([Ca2+]i). As shown in Figure 1B, formyl
peptide 1 induced an increase in [Ca2+]i, and oliogethylene glycol
derivatives 5-7 likewise activated signaling. The most active fNleLF
derivatives in this assay were also the most potent attractants.
Specifically, compound 6, a powerful chemoattractant, caused the
greatest increase in [Ca2+]i; it was >10-fold more potent than formyl
peptide 1. These findings link the observed changes in chemotactic
activity to increases in intracellular signaling.
† Department of Chemistry.
‡ Department of Biochemistry.
§ Current address: Department of Chemistry and Center for Innovation in
Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
9
8844 J. AM. CHEM. SOC. 2010, 132, 8844–8845
10.1021/ja102640c  2010 American Chemical Society

C O M M U N I C A T I O N S
that other factors contribute to the differences in the cellular responses
they elicit. For example, the oligoethylene glycol substituent may
stabilize an active signaling conformation, increase the conformational
flexibility of FPR, or alter its oligomerization state. Regarding the latter,
several studies have shown that agonists can disrupt GPCR oligomer-
ization.20 An ethylene glycol group could serve in that capacity. It is
also interesting to note that the compound with the largest oligoethylene
glycol substituent, 7, was less active than compound 6. As the ethylene
glycol substituent becomes more sterically demanding, it may impede
binding.
In summary, ethylene glycol-substituted chemoattractants show
enhanced activities. The magnitude of the increase depends upon the
length of the oligoethylene glycol substituent. The physiological
importance of GPCRs is underscored by the many drugs that target
them. Our finding that an oligoethylene glycol unit can enhance the
activity of a chemotactic agonist provides a blueprint for generating
highly potent GPCR agonists.
Acknowledgment. This research was supported by the NIH (R01
GM055984). We thank Dr. E. R. Prossnitz for the FPR-transfected
U937 cell line and Dr. A. C. Lamanna for helpful scientific discussions.
Supporting Information Available: Synthetic methods and experi-
mental details. This material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 1. Effects of the formyl peptide derivatives. (A) Chemotactic responses
of FPR-transfected U937 cells to formyl peptides. Data shown are from three
separate experiments conducted in triplicate. The standard error is depicted.
(B) Change in intracellular Ca2+ concentration induced by formyl peptides.
Cells were loaded with the ratiometric dye Indo-1,18 and emission ratios were
measured using a Photon Technology International fluorimeter. The results
shown are from a representative experiment using formyl peptides 1 and 5-7
(10 nM). Experiments also were performed at different peptide concentrations
(see the SI).
References
(1) (a) Delgado, C.; Francis, G. E.; Fisher, D. Crit. ReV. Ther. Drug Carrier
Syst. 1992, 9, 249–304. (b) Roberts, M. J.; Bentley, M. D.; Harris, J. M.
AdV. Drug DeliVery ReV. 2002, 54, 459–476.
(2) Yamaoka, T.; Tabata, Y.; Ikada, Y. J. Pharm. Sci. 1994, 83, 601–606.
(3) Harris, J. M.; Chess, R. B. Nat. ReV. Drug DiscoVery 2003, 2, 214–221.
(4) (a) Miyazaki, M.; Kodama, H.; Fujita, I.; Hamasaki, Y.; Miyazaki, S.;
Kondo, M. J. Biochem. 1995, 117, 489–494. (b) Kramer, R. H.; Karpen,
J. W. Nature 1998, 395, 710–713. (c) Karellas, P.; McNaughton, M.; Baker,
S. P.; Scammells, P. J. J. Med. Chem. 2008, 51, 6128–6137.
(5) (a) Fan, E.; Zhang, Z.; Minke, W. E.; Hou, Z.; Verlinde, C. L. M. J.; Hol,
W. G. J. J. Am. Chem. Soc. 2000, 122, 2663–2664. (b) Solomon, D.; Kitov,
P. I.; Paszkiewicz, E.; Grant, G. A.; Sadowska, J. M.; Bundle, D. R. Org.
Lett. 2005, 7, 4369–4372. (c) Gestwicki, J. E.; Strong, L. E.; Borchardt,
S. L.; Cairo, C. W.; Schnoes, A. M.; Kiessling, L. L. Bioorg. Med. Chem.
2001, 9, 2387–2393. (d) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E.
Annu. Rep. Med. Chem. 2000, 35, 321–330. (e) Kim, Y.; Hechler, B.; Gao,
Z.-G.; Gachet, C.; Jacobson, K. A. Bioconjugate Chem. 2009, 20, 1888–
1898.
We conducted several additional experiments to determine whether
the responses observed were mediated through FPR. To test whether
the oligoethylene glycol unit alone can elicit chemotaxis or FPR-
mediated signaling, we synthesized compound 8 [see the Supporting
Information (SI)]. This compound neither promoted chemotaxis nor
signaling (see the SI). We also assessed whether the effects of 1 and
5-7 depended upon FPR by evaluating their ability to elicit chemotaxis
of FPR-negative U937 cells. None of the compounds induced
chemotactic activity in this cell line. These results indicate that the
observed activity arises from formyl peptide recognition by FPR.
The observed enhancements in chemotactic activity might stem
solely from C-terminal substitution and not from the nature of the
substituent. To test for this possibility, we synthesized formyl peptide
derivative 9 (see the SI), which possesses an alkyl linker with a length
comparable to that of the ethylene glycol substituent in 5. Though 5
is a more potent attractant than the unsubstituted 1, alkyl derivative 9
had no enhanced activity in either assay. These data indicate that the
unexpected increase in the activity of the formyl peptide derivatives
5-7 is due to the oligoethylene glycol substituent.
PEG substituents are known to exhibit a long-range protein repellent
effect. At short range, however, the interaction between PEG and
protein can become attractive and therefore facilitate binding.19 Thus,
oligoethylene glycol substituents might contribute to the binding affinity
of the ligands to the receptor. To test for this possibility, we performed
a competitive binding assay of oligoethylene glycol-substituented
fNleLF derivatives using commercially available fNleLFNleYK-FITC.
Our data indicate that oligoethylene glycol substitution could contribute
to binding affinity, as the dissociation constants (Kd) of oligoethylene
glycol substituents were found to be slightly lower (4-7-fold) than
that of the nonderivatized fNleLF (see the SI). Still, the affinity
differences for these formyl peptide derivatives are subtle, suggesting
(6) Knoll, D.; Hermans, J. J. Biol. Chem. 1983, 258, 5710–5715.
(7) (a) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M.
J. Am. Chem. Soc. 1991, 113, 12–20. (b) Prime, K. L.; Whitesides, G. M.
Science 1991, 252, 1164–1167. (c) Elbert, D. I.; Hubbell, J. A. Annu. ReV.
Mater. Sci. 1996, 26, 365–394.
(8) Murphy, P. M. Annu. ReV. Immunol. 1994, 12, 593–633.
(9) (a) Schiffmann, E.; Corcoran, B. A.; Wahl, S. M. Proc. Natl. Acad. Sci.
U.S.A. 1975, 72, 1059–1062. (b) Prossnitz, E. R.; Ye, R. D. Pharmacol.
Ther. 1997, 74, 73–102.
(10) Miettinen, H. M.; Mills, J. S.; Gripentrog, J. M.; Dratz, E. A.; Granger,
B. L.; Jesaitis, A. J. J. Immunol. 1997, 159, 4045–4054.
(11) Sklar, L. A.; Fay, S. P.; Selismann, B. E.; Freer, R. J.; Muthukumaraswamy,
N.; Mueller, H. Biochemistry 1990, 29, 313–316.
(12) Freer, R. J.; Day, A. R.; Muthukumaraswamy, N.; Pinon, D.; Wu, A.;
Showell, H. J.; Becker, E. L. Biochemistry 1982, 21, 257–263.
(13) Freer, R. J.; Day, A. R.; Becker, E. L.; Showell, H. J.; Schiffmann, E.;
Gross, E. Pept., Struct. Biol. Funct., Proc. Am. Pept. Symp., 6th 1979, 749–
751.
(14) Kew, R. R.; Peng, T.; Dimartino, S. J.; Madhavan, D.; Weinman, S. J.;
Cheng, D.; Prossnitz, E. R. J. Leukocyte Biol. 1997, 61, 329–337.
(15) Servant, G.; Weiner, O. D.; Herzmark, P.; Bella, T.; Sedat, J. W.; Bourne,
H. R. Science 2000, 287, 1037–1040.
(16) Boyden, S. V. J. Exp. Med. 1962, 115, 453–466.
(17) Jones, L. J.; Gray, M.; Yue, S. T.; Haugland, R. P.; Singer, V. L. J. Immunol.
Methods 2001, 254, 85–98.
(18) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 3440–
3450.
(19) (a) Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8399–
8404. (b) Israelachvili, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8378–
8379.
(20) (a) Cheng, Z. J.; Miller, L. J. J. Biol. Chem. 2001, 276, 48040–48047. (b)
Latif, R.; Graves, P.; Davies, T. F. J. Biol. Chem. 2002, 277, 45059–45067.
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