Oligoproline helices as structurally defined scaffolds for oligomeric G protein-coupled receptor ligands

Article abstract of DOI:10.1039/b923556f

Oligoprolines (OPs) are used as rigid backbone scaffolds for the design of oligomeric ligands that target specific G protein-coupled receptors. The OPs were designed to vary in length, the position and number of the ligand-functionalized residues incorporated. For all synthesized compounds a typical PP type II helix was evidenced by circular dichroism indicating that decoration of the helix with large ligands did not affect the helical conformation. Pharmacological evaluation revealed that oligomerization of an agonist with the use of an oligoproline scaffold showed an increase in potency when compared to the monomeric counterparts.

Full text of DOI:10.1039/b923556f

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Oligoproline helices as structurally defined scaffolds for oligomeric G
protein-coupled receptor ligands†
Kimberly M. Bonger,a Varsha V. Kapoerchan,a Gijsbert M. Grotenbreg,a Chris J. van Koppen,b
C. Marco Timmers,c Gijsbert A. van der Marela and Herman S. Overkleeft*a
Received 10th November 2009, Accepted 21st January 2010
First published as an Advance Article on the web 22nd February 2010
DOI: 10.1039/b923556f
Oligoprolines (OPs) are used as rigid backbone scaffolds for the design of oligomeric ligands that target
specific G protein-coupled receptors. The OPs were designed to vary in length, the position and number
of the ligand-functionalized residues incorporated. For all synthesized compounds a typical PP type II
helix was evidenced by circular dichroism indicating that decoration of the helix with large ligands did
not affect the helical conformation. Pharmacological evaluation revealed that oligomerization of an
agonist with the use of an oligoproline scaffold showed an increase in potency when compared to the
monomeric counterparts.
which multivalency is thought to be of importance for potency
Introduction
and/or selectivity.
Proline oligomers composed of three or more proline residues
In this respect, data has amassed recently that point towards
adopt well-defined helical structures,1 and are integral parts of
dimerization and oligomerization of G-protein coupled receptors
(GPCRs) as physiologically relevant in signal transduction.7
Oligomerization of weak or moderately selective GPCR ligands
therefore appears an attractive strategy to obtain compounds
with improved pharmacological activity.8 A case in point and
the focus of the here presented studies is the thienopyrimydyl
derivative, Org 43553 (Scheme 1),9 a potent agonist of the human
luteinizing hormone receptor (LHR) that also exerts considerable
agonistic activity on the human follicle-stimulating hormone
receptor (FSHR).9c Both these GPCRs are glycoprotein hormone
receptors that play a fundamental role in the human reproductive
system. LHR activation induces male testosterone production and
ovulation in females,10 whereas FSHR activation stimulates female
ovarian follicle growth and spermatogenesis in males.11
many proteins found in nature where they partake in protein
folding and protein–protein interactions.2 Artificial oligoproline
helices have found application as molecular rulers, for instance
to correlate the distance of a fluorophore and a quencher
molecule with the efficiency of resonance energy transfer.3 Related
research describes the design of amphiphilic molecules based
on oligoproline helices by the incorporation of modified (hy-
drophilic or hydrophobic) proline residues at specific sites of the
oligomers.4 Oligoprolines may exist in two helical forms that are
easily distinguished by circular dichroism (CD) measurements.
PP type II helices (PPII) are most common and are formed
when the amide bonds connecting the proline residues have the
trans configuration.1 PPII helices have three proline residues per
˚
turn with a rise of 3.1 A per residue. Oligoprolines assembled
from modified proline residues having an electron-withdrawing
substituent at C4 (fluorine,5 azide6) appear to equilibrate towards
cis amide bonds and the resulting PP type I (PPI) helices have 3.3
Synthesis and structural evaluation
We established that thienopyrimidine LHA exerts agonistic activ-
ity on the LHR and FSHR with a similar potency compared to
lead compound Org 43553 (Table 1).12 LHA contains an acetylenic
moiety that can be used in a reaction with a set of oligoprolines
(OPs) containing several 4-azidoproline (Azp) residues. The
azidoproline-containing helices were prepared by standard Fmoc
solid-phase peptide-synthesis by incorporation of one or more
copies of either (4R)-4-azidoproline or (4S)-4-azidoproline13 at
various positions in the sequence. Proline helices encompassing
one or more copies of the pharmacophore were readily assembled
by a copper(I)-catalysed Huisgen [2+3] cycloaddition of LHA
with the azidoproline oligomers. A representative example of
the synthesis of compound 9S-Azp and 9S-LHA2 is depicted in
Scheme 1. In this fashion, we prepared a panel of 26 (oligo)proline
derivatives of which the final compositions are depicted in
Table 1.
˚
proline residues per turn with a rise of 1.9 A per residue.
The secondary structure provided by oligoproline helices, which
are maintained in aqueous solutions in which they normally
dissolve well, indicates that oligoproline helices may be useful
scaffolds onto which two or more copies of a receptor ligand can
be grafted. The resulting dimeric and oligomeric structures may
find application in the study of ligand–receptor interactions in
aLeiden Institute of Chemistry, Department of Bio-organic Synthesis,
Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden,
The Netherlands. E-mail: h.s.overkleeft@chem.leidenuniv.nl; Fax: (+31)-
71-5274307
bDepartment of Molecular Pharmacology, Schering-Plough Research Insti-
tute, PO Box 20, 5340 BH, Oss, The Netherlands
cDepartment of Medicinal Chemistry, Schering-Plough Research Institute,
PO Box 20, 5340 BH, Oss, The Netherlands
The helical distribution of the compounds can be evidenced
by circular dichroism (CD) experiments. PPI type helices are
† Electronic supplementary information (ESI) available: Experimental
procedures and additional characterization. See DOI: 10.1039/b923556f
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1881–1884 | 1881
©

View Article Online
Scheme 1 A. Representative example for the preparation of ligands containing LHR agonist (LHA) by the copper catalyzed [2+3]-Huisgen cycloaddition.
᭺ represents proline; ᭹ represents LHA functionalized proline. B. Synthetic scheme of the oligoproline scaffolds. Reagents and conditions: i. 20%
piperidine/DMF, 0.5 h; ii. Fmoc-Pro-OH or Fmoc-Azp-OH, HCTU, DiPEA, NMP, 3 h; iii. Ac2O, DiPEA, DMF, 2 h; iv. 95% TFA/H2O, 1 h; v. Sodium
ascorbate (5 eq), CuSO4 (1 eq), tBuOH/CH3CN–H2O. C. Structure of lead compound Org 43553.
characterized by a minimum at 230 nm and a maximum at 212 nm
in their CD spectra, while a PPII type helix has its maximum at
225 nm and a minimum at 207 nm. CD spectra were recorded for
all compounds, both the ligand-functionalized derivatives from
Table 1 and their azidoproline containing precursors. In all cases a
spectrum indicative for PP type II helix was observed, independent
of the spacer length and substitution pattern when measured
in a 10% iPrOH/phosphate buffer (see for a representative CD
spectrum that of 9S-LHA2, Table 2).14 We also found that the
relative numbers populating the PPII conformation as opposed
to other (PPI) conformations differed from one compound to the
next. It is generally observed that compounds with more stable
PP type II helices have more intense ellipticities at 207 nm than
compounds that equilibrate faster with PP type I configuration.15
Compounds 5–9 all possess polyproline helices of similar lengths.
Within these series, a more intense ellipticity of the (4R)-
azidoproline containing helices was observed, compared to the
corresponding (4S)-Azp containing helices (Figure 1). This is
in agreement with the results described by Wennemers et al.6
The reverse trend is observed when the helices are substituted
with a LHA ligand. Here, the (4R)-LHAs have less intense
ellipticities than the (4S)-LHAs. This may indicate that the triazole
substituted proline may destabilize the PPII conformation for
the compounds containing (R)-configured 4-substituted proline
derivatives.
helices containing the LHA pharmacophores. It may be of interest
to investigate whether this is only due to the size of the substituent
or that other factors play a role.
Biological evaluation
Biological evaluation revealed that all compounds are potent
LHR agonists whereas their potency to agonize the FSHR
appeared at most rather modest (Table 1). Some general trends
are observed. For example, compounds bearing two copies of the
pharmacophore are two to five times more potent LHR agonists
compared to the compounds 1–4 that are equipped with a single
copy of the parent compound. The compounds incorporating
more than two pharmacophores (15R-LHA3 and 15S-LHA3)
are LHR agonists in the low nM range. Of importance is the
observation that all compounds are weaker LHR and FSHR
agonists compared to the parent compounds Org 43553 and
LHA. This trend is evident also in the oligoproline series 1–4
indicating the penalty of attaching an oligoproline moiety onto
the pharmacophore. Looking at the oligomeric series 5–14 there is
no clear selectivity trend discernible but we do note the high LHR
selectivity of 15S-LHA4 (FSHR/LHR = 188), a result that may
be attributed to the incorporation of multiple pharmacophores.
Also apparent, and pointing towards an explanation for the
enhancement in selectivity, is that whereas FSHR agonistic activity
for all compounds is at least a tenfold lower compared to the lead
compound (Org43533), agonistic activities towards LHR vary
much more. For instance, 15S-LHA4 agonizes the LHR with
an EC50 of 9 nM, which is only two- to threefold less potent
Another trend that can be seen in Table 2 is that the constructs
in where the substituents are grouped close together have more
intense ellipticities than those in which they are further apart
(compare compounds 5 to 9). This holds especially true for the
1882 | Org. Biomol. Chem., 2010, 8, 1881–1884
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Table 1 Mean agonistic potency (EC50) and selectivity for the LHR and FSHR. All compounds are full agonists on the LHR and partial agonists on
the FSHR. The mean EC50 are calculated from the -log EC50 values from two or three independent experiments performed in duplicate. The SD of pEC50
is generally lower than 0.2. ᭺ represents proline; ᭹ represents LHA functionalized proline
EC50/nM
FSHR
EC50/nM
LHR
FSHR/LHRa
Compound
FSHR LHR
FSHR/LHRa
Monomeric
Org 43553b
LHAc
1R-LHA
110
126
2109
3.7
0.9
32
28
137
67
1S-LHA
2S-LHA
3S-LHA
4S-LHA
>3000
>3000
>3000
>3000
76
40
2R-LHA
3R-LHA
4R-LHA
>3000
>3000
>3000
98
>31
>28
>69
110
114
84
>27
>26
>36
107
44
Dimeric
5R-LHA2
6R-LHA2
7R-LHA2
8R-LHA2
9R-LHA2
10R-LHA2
11R-LHA2
12R-LHA2
13R-LHA2
14R-LHA2
nd
nd
20
27
29
25
16
27
18
33
31
nd
71
56
51
54
76
82
84
68
65
5S-LHA2
6S-LHA2
7S-LHA2
8S-LHA2
9S-LHA2
10S-LHA2
11S-LHA2
12S-LHA2
13S-LHA2
14S-LHA2
2092
2362
2367
1898
1178
nd
26
32
30
27
20
nd
nd
nd
32
33
80
75
80
71
60
nd
nd
nd
84
36
1427
1499
1498
1335
1223
2193
1518
2267
1999
nd
nd
2707
1200
Oligomeric
15R-LHA4
1067
9
119
15S-LHA4
1764
9
188
a Selectivity observed for the LHR (EC50 FSHR/EC50 LHR). b Taken from reference 9c. c Taken from reference 12. nd = not determined
compared with Org43533. It may thus be tentatively concluded
that LHR agonism by ligand substituted oligoprolines depends on
the nature of the oligomers (number of ligands, distance between
these and possibly also the configuration of the 4-substituted
prolines employed) whereas FSHR agonism is compromised in any
case. Obviously, a more in-depth study employing more oligomers
is needed to reach a definite conclusion in this. An intriguing
question in this respect is also whether the oligomers bind to
the receptors as PP type II helices and thus whether geometric
restrictions in the oligomers make that two ligands can bind to
the LHR (or a dimer) at the same time but cannot do so, or to
a lesser extent, to the FSHR. One possibility to probe this would
be to introduce non-proline residues at predesigned sites in the
oligomers and establish the agonistic potency of these structures,
with an enhanced intrinsic conformational flexibility, towards the
LHR and FSHR.16 The results presented here clearly demonstrate
that oligoprolines equipped with two or more pharmacophores
are useful starting points in the design of bioactive compounds
with an improved selectivity profile.
receptor agonist by means of a copper (1)-catalyzed Huisgen
[2+3] cycloaddition. The resulting compounds adopted a PPII
helical conformation as judged by circular dichroism, indicating
that decoration of the helix with large agonists did not affect
the helical conformation. Pharmacological evaluation revealed
that functionalization of the lead compound with an oligoproline
scaffold resulted in a drop in potency for the LHR. However,
compared to the monomeric counterparts, a significant increase
in potency on the LHR was observed that is related to the increase
in LHA functionalized prolines on the helix. This result indicates
the potential of oligoproline helices substituted at multiple sites
with pharmacophores to obtain GPCR ligands to study receptor
dimerization in more detail. Although we did not succeed in
increasing the potency of the lead compound towards the LHR we
obtained a compound that combines a strong potency (only two-
threefold less compared to the lead compound) with a markedly
enhanced selectivity. Given that next to potency selectivity of
a compound towards the desired target is a highly important
parameter in medicinal chemistry we feel that our approach, in
which oligoprolines are used as scaffolds to bring together a
number of pharmacophores holds promise for the development
In summary, we prepared a set of (4R)- or (4S)-4-azidoproline
helices that were functionalized with a luteinizing hormone
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1881–1884 | 1883
©

View Article Online
Table 2 The intensity in ellipticity observed for dimeric azidoproline containing compounds 5–9-Azp and LHA functionalized proline compounds
5–9-LHA at 205 nm is given in the Table. All compounds were measured at 4E-5 M in 10% iPrOH/phosphate buffer pH 7.2. A representative CD-spectra
observed for the oligoproline compounds is depicted below. Here, compound 9S-LHA2 shows a minimum at 205 nm and maximum at 228 nm
Observed ellipticity at 205 nm (¥103 deg cm2 dmol-1)
(R)
(S)
(R)
(S)
5-Azp
6-Azp
7-Azp
8-Azp
9-Azp
-30.2
-28.2
-28.1
-32.7
-24.3
-28.5
-23.7
-27.7
-24.0
-11.9
5-LHA
6-LHA
7-LHA
8-LHA
9-LHA
nd
-60.2
-62.8
-49.1
-42.0
-42.1
-33.2
-29.6
-16.7
-10.0
of structurally and conformationally defined bi- and multivalent
ligands.
7 (a) M. B. Dalrymple, K. D. G. Pfleger and K. A. Eidne, Pharmacol.
Ther., 2008, 118, 359–371; (b) S. Terrillon and M. Bouvier, EMBO Rep.,
2004, 5, 30–34; (c) S. R. George, B. O’Dowd and S. P. Lee, Nat. Rev.
Drug Discovery, 2002, 1, 808–820.
8 (a) W. S. Messer, Jr., Curr. Pharm. Des., 2004, 10, 2015–2020; (b) S.
Halazy, Expert Opin. Ther. Pat., 1999, 9, 431–446.
Acknowledgements
9 (a) N. C. R. van Straten, G. G. Schoonus-Gerritsma, R. G. van Someren,
J. Draaijer, A. E. P. Adang, C. M. Timmers, R. Hanssen and C. A. A.
van Boeckel, ChemBioChem, 2002, 3, 1023–1026; (b) L. H. Heitman,
J. Oosterom, K. M. Bonger, C. M. Timmers, P. H. G. Wiegerinck and
A. P. IJzerman, Mol. Pharmacol., 2007, 73, 518–524; (c) C. J. van
Koppen, G. J. R. Zaman, C. M. Timmers, J. Kelder, S. Mosselman,
R. van de Lagemaat, M. Smit and R. Hanssen, Naunyn-Schmiedebergs
Arch. Pharmacol., 2008, 378, 503–514; (d) R. van de Lagemaat, C. M.
Timmers, J. Kelder, C. J. van Koppen, S. Mosselman and R. G. Hanssen,
Hum. Reprod., 2008, 24, 640–648; (e) R. G. J. M. Hanssen, and C. M.
Timmers, WO 03020729, 2003.
We are indebted to Dr A.J.W. Hsueh, Stanford University, for
providing human LHR and FSHR cDNA. We thank Monique
van Amstel and Robert Tan for expert technical assistance. This
work was supported by the Netherlands Organization for Scientific
Research (NWO).
References
1 (a) S. Kakinoki, Y. Hirano and M. Oka, Polym. Bull., 2005, 53, 109–
115; (b) N. Helbecque and M. H. Loucheuxlefebvre, Int. J. Pept. Protein
Res., 1982, 19, 94–101; (c) P. M. Cowan and S. McGavin, Nature, 1955,
176, 501–503.
10 M. Ascoli, F. Fanelli and D. L. Segaloff, Endocr. Rev., 2002, 23, 141–
174.
11 M. Simoni, J. Gromoll and E. Nieschlag, Endocr. Rev., 1997, 18, 739–
773.
2 (a) Z. S. Shi, K. Chen, Z. G. Liu and N. R. Kallenbach, Chem. Rev.,
2006, 106, 1877–1897; (b) A. Rath, A. R. Davidson and C. M. Deber,
Biopolymers, 2005, 80, 179–185; (c) B. J. Stapley and T. P. Creamer,
Protein Sci., 1999, 8, 587–595.
3 (a) H. Sahoo, D. Roccatano, A. Hennig and W. M. Nau, J. Am. Chem.
Soc., 2007, 129, 9762–9772; (b) L. Stryer and R. P. Haugland, Proc.
Natl. Acad. Sci. U. S. A., 1967, 58, 719–726.
12 K. M. Bonger, R. J. B. H. N. Van den Berg, A. D. Knijnenburg, L. H.
Heitman, C. J. Van Koppen, C. M. Timmers, H. S. Overkleeft and G. A.
Van der Marel, ChemMedChem, 2009, 4, 1189–1195.
13 (a) J. A. Go´mez-Vidal and R. B. Silverman, Org. Lett., 2001, 3, 2481–
2484; (b) L. L. Klein, L. P. Li, H. J. Chen, C. B. Curty, D. A. DeGoey,
D. J. Grampovnik, C. L. Leone, S. A. Thomas, C. M. Yeung, K. W.
Funk, V. Kishore, E. O. Lundell, D. Wodka, J. A. Meulbroek, J. D.
Alder, A. M. Nilius, P. A. Lartey and J. J. Plattner, Bioorg. Med. Chem.,
2000, 8, 1677–1696.
4 I. Geisler and J. Chmielewski, Bioorg. Med. Chem. Lett., 2007, 17,
2765–2768.
5 (a) J. C. Horng and R. T. Raines, Protein Sci., 2006, 15, 74–83; (b) J. A.
Hodges and R. T. Raines, Org. Lett., 2006, 8, 4695–4697; (c) M. L.
DeRider, S. J. Wilkens, M. J. Waddell, L. E. Bretscher, F. Weinhold,
R. T. Raines and J. L. Markley, J. Am. Chem. Soc., 2002, 124, 2497–
2505.
6 (a) M. Ku¨min, L. S. Sonntag and H. Wennemers, J. Am. Chem. Soc.,
2007, 129, 466–467; (b) L. S. Sonntag, S. Schweizer, C. Ochsenfeld and
H. Wennemers, J. Am. Chem. Soc., 2006, 128, 14697–14703.
14 It was recently shown that proline helices in where the N- and C- termini
are capped favor the PPII over the PPI conformation.M. Kuemin, S.
Schweizer, C. Ochsenfeld and H. Wennemers, J. Am. Chem. Soc., 2009,
131, 15474–15482.
15 (a) S. Kakinoki, Y. Hirano and M. Oka, Polym. Bull., 2005, 53, 109–
115; (b) M. Ku¨min, L. S. Sonntag and H. Wennemers, J. Am. Chem.
Soc., 2007, 129, 466–467.
16 We thank the referee for this relevant and interesting suggestion.
1884 | Org. Biomol. Chem., 2010, 8, 1881–1884
This journal is
The Royal Society of Chemistry 2010
©