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ORIGINAL ARTICLE
Effect of helix-promoting strategies on the biological activity
of novel analogues of the B-chain of INSL3
Fazel Shabanpoor ÆRichard A. Hughes ÆSuode Zhang ÆRoss A. D. Bathgate Æ
Sharon Layfield ÆMohammed Akhter Hossain ÆGeoffrey W. Tregear Æ
Frances Separovic ÆJohn D. Wade
Received: 23 October 2008 / Accepted: 17 November 2008 / Published online: 7 December 2008
ÓSpringer-Verlag 2008
Abstract Insulin-like 3 (INSL3) is a novel circulating
peptide hormone that is produced by testicular Leydig cells
and ovarian thecal and luteal cells. In males, INSL3 is
responsible for testicular descent during foetal life and
suppresses germ cell apoptosis in adult males, whereas in
females, it causes oocyte maturation. Antagonists of INSL3
thus have significant potential clinical application as con-
traceptives in both males and females. Previous work has
shown that the INSL3 receptor binding region is largely
confined to the B-chain central a-helix of the hormone and
a conformationally constrained analogue of this has modest
receptor binding and INSL3 antagonist activity. In the
present study, we have employed and evaluated several
approaches for increasing the a-helicity of this peptide in
order to better present the key receptor binding residues
and increase its affinity for the receptor. Analogues of
INSL3 with higher a-helicity generally had higher receptor
binding affinity although other structural considerations
limit their effectiveness.
Keywords INSL3 RXFP2 Lactam-constraint
Disulfide-constraint Helicity Peptide
Introduction
Insulin-like peptide 3 (INSL3) was discovered in the early
1990s (Adham et al. 1993) and shown to belong to the
insulin–relaxin superfamily of polypeptide hormones. It was
originally named Leydig cell insulin-like peptide (Ley-IL)
because it was found in the Leydig cells of the testis
(Burkhardt et al. 1994) and has also been referred to as RLF
(relaxin-like factor) due to its relaxin-like activity in a mouse
interpubic ligament bioassay (Bu
¨llesbach and Schwabe
1995). In the male, INSL3 acts as a marker for fully differ-
entiated adult-type Leydig cells (Ivell and Einspanier 2002)
and is also expressed by ovarian follicles and in the corpus
luteum in the female but at lower levels compared to the male
(Roche et al. 1996; Tashima et al. 1995).
INSL3 is a circulating hormone which has important
reproductive and non-reproductive roles. During foetal life
it is principally involved in mediation of the transabdom-
inal phase of testicular decent as INSL3 or its receptor,
RXFP2, knockout male mice have been shown to have a
similar phenotype in which both are cryptorchid, i.e. they
retain their testes in the abdominal cavity, which leads to
impaired spermatogenesis and infertility (Bachelot et al.
2000; Bogatcheva et al. 2003; Feng et al. 2004; Foresta and
Ferlin 2004; Nef and Parada 1999; Spiess et al. 1999;
Zimmermann et al. 1999). In adults, the INSL3 and RXFP2
system acts as a paracrine factor in mediating gonadotropin
actions (Kawamura et al. 2004). Luteinizing hormone
(LH), which is released by the anterior pituitary gland,
stimulates INSL3 transcripts in ovarian theca and testicular
Leydig cells. INSL3 successively binds RXFP2 expressed
F. Shabanpoor S. Zhang R. A. D. Bathgate S. Layfield
M. A. Hossain G. W. Tregear J. D. Wade (&)
Howard Florey Institute, University of Melbourne,
Melbourne, VIC 3010, Australia
e-mail: john.wade@florey.edu.au
F. Shabanpoor F. Separovic J. D. Wade
School of Chemistry, University of Melbourne,
Melbourne, VIC 3010, Australia
R. A. Hughes
Department of Pharmacology, University of Melbourne,
Melbourne, VIC 3010, Australia
R. A. D. Bathgate
Department of Biochemistry and Molecular Biology,
University of Melbourne, Melbourne, VIC 3010, Australia
123
Amino Acids (2010) 38:121–131
DOI 10.1007/s00726-008-0219-2
in germ cells to activate the inhibitory G protein, thus
leading to decreases in cAMP production. This, in turn,
leads to the initiation of meiotic progression of arrested
oocytes in preovulatory follicles in vitro and in vivo and
suppresses male germ cell apoptosis in vivo (Kawamura
et al. 2004).
A recent study has shown that in males the INSL3/
RXFP2 signalling system is also involved in bone metab-
olism as RXFP2
-/-
knockout mice showed a considerable
reduction in their bone mass, mineralizing surface and bone
formation compared to wild type mice (Ferlin et al. 2008).
This study also showed that 64% of young men with
RXFP2 mutations had significant reduction in bone mass
density, a sign of osteoporosis (Ferlin et al. 2008). INSL3
may also play a role in the pathobiology of some forms of
human cancers, such as thyroid carcinoma, as its expres-
sion is upregulated in hyperplastic and neoplastic human
thyrocytes (Klonisch et al. 2005).
INSL3 is expressed as a preprohormone with an
N-terminal signal peptide for secretion, a B-chain, a
C-peptide, and a C-terminal A-chain. The preprohormone
is subsequently processed into a mature peptide through
cleavage of the signal peptide and formation of two inter-
chain and an intra-A-chain disulfide bond followed by
proteolytic removal of the C-peptide (Adham et al. 1993;
Hsu 2003). Mature human INSL3 consists of an A- and
B-chain of 26 and 31 amino acids, respectively, and its
tertiary structure has recently been solved using solution
NMR spectroscopy (Rosengren et al. 2006) (Fig. 1a).
INSL3 adopts a core structure similar to that found in
insulin and relaxin, especially in the region confined by
the disulfide bonds.
To determine the residues involved in receptor binding,
recent structure–activity studies by our group using single
Ala substitution have shown that substituting Arg
B16
and
Val
B19
significantly reduced receptor binding affinity
(Rosengren et al. 2006). On the other hand, multi-Ala
substitution showed that His
B12
and Arg
B20
have a strong
synergistic effect with Arg
B16
, suggesting that His
B12
and
Arg
B20
may play a role in the initial step of receptor rec-
ognition, involving electrostatic interactions between basic
residues of the peptide and acidic residues on the receptor
(Rosengren et al. 2006). In addition to these residues,
Trp
B27
toward the C-terminus of the B-chain has also been
shown to be crucial for binding of INSL3 as the mutation
or deletion of Trp
B27
leads to loss of receptor binding
affinity (Bu
¨llesbach and Schwabe 1999; Rosengren et al.
2006). These B-chain residues collectively form a receptor
binding motif (H
B12
,R
B16
,V
19
,R
20
and W
27
). A-chain
N-terminal truncation studies of INSL3 have shown that
truncation of the INSL3 peptide to Cys
A10
results in a
peptide with high receptor binding affinity but which is
devoid of signalling activity, i.e. an antagonist (Bu
¨llesbach
and Schwabe 2005; Hossain et al. 2008).
Despite knowledge of the region of the peptide that is
involved in receptor signalling, there is no clear under-
standing of the mechanism of receptor activation. A
recent study has shown that the mechanism of receptor
activation by INSL3 is independent of the amino acid side
chains and is a function of certain peptide bonds at the
N-terminus of the A-chain (Bu
¨llesbach and Schwabe
2007). These authors proposed the backbone amide bond
around Arg
A8
and Tyr
A9
to be crucial for receptor acti-
vation, as the replacement of these residues with alanine
does not affect signalling whereas their deletion or
replacement with D-Pro has no impact on receptor binding
but severely retards receptor activation (Bu
¨llesbach and
Schwabe 2007). In contrast, a more recent study on a
relaxin-2, which also binds to INSL3 receptor (RXFP2),
has shown that there are other residues in the A-chain
which are involved in receptor activation. These authors
have shown that K
A17
is an important residue for receptor
activation as its mutation to alanine enhances RXFP2-
activation activity of relaxin-2 as a result of inducing
active conformational transformation. On the other hand,
the replacement of this residue with a polar or negatively
charged residue reduces the receptor activation activity of
relaxin-2 (Park et al. 2008).
W27
V19 R20
R16
H12
A
W27
V19 R20
R
16
H12
B
B-chain
A-chain
Fig. 1 a Solution NMR
structure of native human
INSL3 showing the important
receptor binding residues
(H
12
,R
16
,V
19
,R
20
and W
27
).
bAnalogue 30 (Table 1)in
which a truncated INSL3
A-chain (from residue Cys
A15
to
Cys
A24
) is linked via a disulfide
bond to the truncated B-chain
122 F. Shabanpoor et al.
123
INSL3, due to its role in germ cell maturation in adults,
has enormous potential as a clinical agent in the area of
fertility management; in particular, antagonists of this
peptide may have significant clinical promise for use as
both a male and female contraceptive. As discussed above,
INSL3 has been shown to bind to its receptor using the
residues primarily located on the a-helical region of the
B-chain. In an attempt to develop mimetics of INSL3
B-chain with high receptor binding affinity and antago-
nistic activity, our group recently designed and synthesized
shortened analogues of the INSL3 B-chain that had
antagonistic activity in vitro (Del Borgo et al. 2006;
Shabanpoor et al. 2007). In vivo administration of one of
these antagonists into the testes of rats resulted in a sub-
stantial decrease in testis weight probably due to the
inhibition of germ cell survival (Del Borgo et al. 2006).
However, these peptides have receptor binding affinities
within the micromolar range compared to the nanomolar
affinity of the native INSL3. This is due, in part, to the lack
of INSL3-like native a-helical structure in these peptides,
which is thought to be important for the presentation of
binding residues in the correct orientation to the binding
pocket of the receptor. Therefore, the aim of this study is
to systematically examine known methods, including
introduction of disulfide and lactam constraints or a-helix-
inducing residues and N-caps, to induce additional
a-helicity in the B-chain mimetics of INSL3 and to eval-
uate their effectiveness as INSL3 antagonists.
Materials and methods
9-Fluroenylmethoxycarbonyl (Fmoc) protected L-a-amino
acids, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU), N,N-dimethylformamide
(DMF), piperidine and trifluoroacetic acid (TFA) were
obtained from Auspep (West Melbourne, Australia). Fmoc-
Aib-OH, Fmoc-Asp(O-2-PhiPr)-OH, Fmoc-Dab(Mtt)-OH,
Fmoc-Glu(O-2-PhiPr)-OH, Fmoc-Lys(Mtt)-OH and PyBOP
were obtained from Novabiochem (Melbourne, Australia).
Fmoc-PAL-PEG-PS and Fmoc-L-Ala-PEG-PS resins
with substitution of 0.20 mmol/g were purchased from
Applied Biosystems (Melbourne, Australia). Methanol,
diethylether, dichloromethane (Merck, Melbourne, Austra-
lia); 3,6-dioxa-1,8-octanedithiol (DODT), triisopropylsilane
(TIPS), diisopropylethylamine (DIPEA), 1,2,4,5-benzene-
tetracarboxylic dianhydride (Sigma-Aldrich, Sydney,
Australia); 2,20-dipyridyl disulfide (DPDS), (Fluka-
Switzerland); acetonitrile and NH
4
HCO
3
, (NH
4
)
2
CO
3
(BDH
Laboratory Supplies, Poole, UK); and trifluoromethanesul-
fonic acid (TFMSA) (MP Biomedicals, Sydney, Australia).
Dulbecco’s modified Eagles’ medium (DMEM), RPMI 1640
medium, 2 mM L-glutamine, foetal calf serum and penicil-
lin/streptomycin were all obtained from Trace Biosciences
(Sydney, Australia). All other reagents were obtained from
Sigma-Aldrich (Sydney, Australia).
Molecular modelling
All molecular modelling was performed using SYBYL
molecular modelling software (Tripos, version 7.0, St
Louis, MO, USA) on a Silicon Graphics O
2
workstation.
Design of disulfide constrained mimetics
All the single chain disulfide constrained mimetics were
designed as described previously (Shabanpoor et al. 2007).
Briefly, using the NMR structure of native human INSL3
as a template, the A-chain was deleted and a disulfide bond
was inserted between b-carbon atoms of residues less than
10 A
˚apart on the strand and a-helical segments of the
B-chain. The two chain disulfide constrained analogue 30
(Fig. 1b) was designed by truncating the B-chain strand
from N-terminus up to Leu
B9
and from the C-terminus until
Trp
B27
, and the A-chain was truncated from N-terminus
until Cys
A15
and from C-terminus until Cys
A24
. In native
INSL3, Cys
A24
forms a disulfide bond with Cys
B22
so,
hence, there was no need for creation of a disulfide bond.
Cys
B10
, which pairs with Cys
A11
in an inter-chain disulfide
bond, was mutated to serine (Ser
B10
). On the other hand,
Cys
A15
forms an intra-A-chain disulfide bond with Cys
A10
and in order to from the second disulfide bond, Leu
B9
,
which points toward Cys
A15
, was mutated to Cys
B9
and
then a disulfide bond was created between this pair of
cysteines. Finally, the two-chain disulfide constrained
analogue was energy minimized in vacuo as described
previously (Shabanpoor et al. 2007) using the Powell
method with the Tripos force field, Gasteiger-Marsili
charges and termination at a root mean square (RMS)
gradient of less than 0.05 kcal/mol per A
˚.
Design of i to i ?4 lactam constrained mimetics
In designing lactam constrained mimetics, we first
inspected the NMR structure of the INSL3 B-chain for an
optimum place to introduce the lactam. Phe
B14
and Leu
B18
,
spaced iand i?4 on one face of the helix opposite to the
side where the key receptor binding residues were located,
was observed to be a suitable place to introduce a lactam
constraint. We designed a series of lactam constrained
analogues of INSL3 B-chain where we truncated the
B-chain from the C-terminus until Trp
B27
and to Pro
B1
at
the N-terminus. Some of the lactam constrained analogues
were truncated further from the N-terminus to Gly
B11
.
Effect of helix-promoting strategies on the biological activity 123
123
Following this truncation, Gly
B11
was mutated to Ala
B11
,
then Phe
B14
was mutated to Lys
B14
or Dab
B14
, and Leu
B18
was mutated to Glu
B18
or Asp
B18
(Fig. 3). Finally, an amide
bond was created between the e-amino group of the Lys or
Dab side-chain and carbonyl group of either the Glu or Asp
side-chain, and the resultant analogues were energy mini-
mized as described earlier.
Incorporation of a-helix-inducing residues and N-caps
The INSL3 B-chain was truncated from the N-terminus up
to Gly
B11
, which was then mutated to a more helix-
favouring residue, Ala
B11
. Ala
B17
was mutated to a more
helix-inducing residue, a-aminoisobutyric acid (Aib).
Valine residues along B-chain helix were mutated to either
Ala or Aib. The N-cap, 2,4,5 benzenecarboxylate, which is
known to stabilize helices by acting as a surrogate H-bond
acceptor (Mimna et al. 2007), was coupled to the N-ter-
minus of the INSL3 B-chain helix.
Solid-phase peptide synthesis
In order to increase the enzymatic stability of the ana-
logues, all linear precursor peptides were synthesized as
C-terminal amides (Werle and Bernkop-Schnu
¨rch 2006)on
PAL-PEG-PS resin with 0.19–0.22 mmol/g loadings using
Fmoc chemistry. The side chain protected amino acids used
were: Arg(Pbf), Asp(OPip), Cys(Trt), Cys(Acm), Cys(tBu),
Glu(OPip), Glu(OtBu), His(Trt), Lys(Boc), Lys(Mtt) and
Trp(Boc). Peptides were synthesized on either a Pioneer
peptide synthesizer (PerSeptive Biosystems, MA, USA)
using continuous flow methodology or a microwave
peptide synthesizer (CEM, Liberty, Matthews, USA). In
continuous flow syntheses, the coupling of Fmoc protected
L-a-amino acids was accomplished using HBTU
(0.3 mmol) and DIPEA in DMF (5 ml) for 30 min and
Fmoc protecting groups were removed by treating the
resin-attached peptide with piperidine (20% v/v) in DMF
for 20 min. For microwave-assisted syntheses, a fivefold
excess of amino acid and HBTU and a tenfold excess of
DIEA were used, and the coupling and deprotection were
carried out at 75°C using 25 W microwave power for
5 min and 60 W microwave power for 3 min, respectively.
The single chain disulfide-constrained peptides were
synthesized as described previously (Shabanpoor et al.
2007). Analogues 30 and 31 (Table 1) with two inter-chain
disulfide bonds were synthesized with two Cys(Trt)s and
two Cys(Acm)s, one of each in either chain. The formation
of a disulfide bond between the two Cys(Trt) was carried
out by dissolving the A and B-chains in an equimolar ratio
in 0.1 M NH
4
CO
3
, adding 300 ll of 100 mM DPDS and
stirring the reaction mixture for 30 min. The second inter-
chain disulfide bond was formed by first dissolving the
peptide in acetic acid (2 mg/ml) followed by the addition
of 60 mM HCl (0.1 ml/mg) and 20 mM I
2
(42 eq/Acm).
The reaction mixture was stirred at room temperature for
1 h and the progression of the reaction was monitored by
HPLC.
The all-linear form of the lactam-constrained peptides
were synthesized at the 0.1-mmol scale on PAL-PEG-PS
resin (substitution 0.20 mmol/g) using a microwave-assis-
ted peptide synthesizer and the conditions described above.
The formation of an amide bond between the side chains of
two residues, Lys or Dab and Glu or Asp, was carried out
on-resin. The phenylisopropyl ester (OPip) of aspartic and
glutamic acids and methyltrityl (Mtt) group of lysine and
Dab were removed by treating the peptide resin with 3%
TFA/5% TIPS in DCM (2 930 min) (Shepherd et al.
2006). The on-resin cyclization was carried out in three
different ways. In the first instance, we attempted to cyclize
the peptide on-resin using a standard protocol of coupling
with 3 equivalents of HBTU and 3.5 equivalents of DIPEA
in 3 ml of DMF overnight. Second, the resin-bound peptide
was treated with PyBOP/HOAt/DIPEA (3:3:3.5) in 3 ml of
DMF/DMSO/NMP (1:1:1) overnight. Finally, the cycliza-
tion was carried out in a microwave-assisted peptide
synthesizer using HBTU (3 eq) DIPEA (3.5 eq) for 10 min
at 75°C, 25 W.
The syntheses of peptides with helicogenic residues and
N-caps were carried out in the same way as for the disulfide
constrained mimetics. The N-terminus was either capped
with acetic anhydride (10 eq) or 1,2,4,5-benzene-tetra-
carboxylic dianhydride (10 eq) in DMF in the presence of
DIEA (10 eq).
The cleavage of peptides was carried out using a TFA:
H
2
O:DODT:TIS (94:2.5:2.5:1, 20 ml) mixture for 90 min.
Cleaved peptides were precipitated in ice-cold diethyl ether,
centrifuged at 3,000 rpm for 3 min; the pellet was washed by
resuspending it in ice-cold diethylether and centrifuging it
again for three times. Peptides were analysed and purified by
RP-HPLC on Waters XBridge
TM
columns (4.6 9250 mm,
C18, 5 lm) and (19 9150 mm, C18, 5 lm), respectively,
using H
2
O with 0.1% TFA as solvent A and acetonitrile with
0.1% TFA as solvent B, with a gradient of 1% change in
buffer B per min over 30 min. Peptide 24 (Table 1) N-cap-
ped with 1,2,4,5-benzene-tetracarboxylic dianhydride was
dissolved in 1 M (NH
4
)
2
CO
3
and lyophilized before HPLC
analysis and purification.
Matrix-assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF/TOF MS, Bruker Dal-
tonics, Germany) was used to characterize the peptides at
each intermediate step using sinapinic acid, a-cyano-4-
hydroxy-cinnamic acid and 2,5-dihydroxy benzoic acid
(Bruker Daltonics, Germany) as matrices, based on the
molecular size of a peptide. The matrices were made up in
50% acetonitrile containing 0.05% TFA. The peptide
124 F. Shabanpoor et al.
123
Table 1 Primary amino acid sequence, monoisotopic mass, calculated and theoretical a-helicity in PBS and 20% TFE, and binding affinity (pK
i
,
n=3) of INSL3 analogues
Peptide No Sequence pKi
Calcul Exper PBS 20% TFE Theo Mean ± SEM (n=3)
H-PTPEMREKLCGHHFVRALVRVCGGPRWSTEA-OH
H-AAATNPARYCCLSGCTQQDLLTLCPY-OH
1 Ac-TPEMREKLSGHHFVRALVRVSGGPRW-NH23044.5 3045 10 55 42 5.31 ± 0.24
2H2N-CPEMREKLSGHHFVRALVRCSGGPRW-NH23009.6 3009.7 8 37 42 6.09 ± 0.05
3Ac-
CPEMREKLSGHHFVRALVRCSGGPRW-NH2
3050.5 3050.9 8 30 42 6.41 ± 0.11
4Ac-
CPEMREKLSGAHFVRALVRCSGGPRW-NH2
2982.5 2982.8 10 32 42 5.1 ± 0.09
5Ac-CPEMREKLSGHHFVAALVRCSGGPRW-NH2
2965.4 2965.6 11 35 42 <4
6Ac-
CPEMREKLSGHHFVRALARCSGGPRW-NH2
3018.5 3019 8 28 42 5.07 ± 0.06
7Ac-CPEMREKLSGHHFVRALVACSGGPRW-NH2
2965.4 2965.6 9 35 42 5.93 ± 0.06
8Ac-CPEMREKLSGHHFVRALVRCSGGPRA-NH22934 2934.6 8 34 42 NB
9Ac-CPEMREKLSGHHFVAALVRCSGGPRBAl-NH23068 3068.3 - - - 5.65 ± 0.08
10 Ac-CPEMREKLSGHHFVRALARCSGGPR(1NAL)-NH23061 3061 - - - 5.63 ± 0.03
11 Ac-CPEMREKLSGHHFVRALVRCSAAARW-NH23052.6 3052.6 8 41 42 5.74 ± 0.20
12
Ac-CPEMREKLSGHHCVRACVRCSGGPRW-NH22989.5 2989.4 5 12 42 < 4
13 Ac-TPEMREKLSGHHCVRACVRVSGGPRW -NH22986.4 2987 8 25 42 NB
14 Ac-TPEMREKLSGHHDVRAKVRVSGGPRW-NH23010.5 3010.9 17 47 42 5.82 ± 0.08
15 Ac-TPEMREKLSGHHEVRAKVRVSGGPRW -NH23023.5 3023.8 18 58 42 6.09 ± 0.08
16 Ac-TPEMREKLSGHHEVRADabVRVSGGPRW -NH22996.5 2966.8 11 14 42 <5
17 Ac-AHHKVRADVRVSGGPRW -NH21952.2 1952.4 17 36 65 <5
18 Ac-AHHDVRAKVRVSGGPRW -NH21952.2 1952 8 27 65 <5
19 Ac-AHHDVRAKVRVSGGPRW -NH21970.1 1970.1 7 22 65 <5
20 Ac-AHHKVRAEVRVSGGPRW -NH21965.2 1965.7 4 17 65 <5
21 Ac-AHHEVRAKVRVSGGPRW -NH21965.2 1965.7 17 48 65 <5
22 Ac-AHHEVRAKVRVSGGPRW -NH21983.2 1983.7 7 23 65 <5
23 H
2
N-AHHFVRALVRVSGGPRW-NH
2
1944.1 1944.04 7 49 65 <5
24 Ac-AHHFVRALVRVSGGPRW -NH
2
1986.1 1986.8 8 46 65 <5
25 Da-AHHFVRALVRVSGGPRW -NH
2
2181.1 2181.3 6 41 65 <5
26 Ac-AHHFVRAibLVRVSGGPRW -NH
2
2001.5 2001.1 7 46 65 <5
27 Ac-GHHFAibRALVRAib SGGPRW-NH
2
1944 1944.9 7 21 65 <5
28 Ac-GHHFAibRAibLVRAibSGGPRW-NH
2
1958 1959.2 7 18 65 5.5 ± 0.12
29 Ac-AHHFV(A)RALVRV(A)SGGPRW-NH
2
1930.2 1930.7 7 43 65 <5
Ac-CTQQDLLTLC-NH2
CSAHHFVRALVRVCGGPRW
H-PTPEMREKLCGHHFVRALVRVCGGPRWSTEA-OH
H-PTPEMREKLCGHHFVRALVRVCGGPRW STEA-OH
020.0 ± 41.713328.0733073303 58
9.27 ± 0.06
-
%α-helix [M+H]
7029.52 32 53 42 8.43 ± 0.06
3339268.29263LSNIn -
5.920713
NB No binding
Effect of helix-promoting strategies on the biological activity 125
123
content was determined using vapour-phase acid hydrolysis
in 6 M HCl containing 2% phenol at 110°C for 24 h. The
individual amino acids were converted to stable, fluores-
cent derivatives using Waters AccQ.Tag kit (Waters,
Sydney, Australia). The derivatized amino acids were
separated using a Shim-Pak XR-ODS (3 975 mm,
2.2 lm) column on a Shimadzu RP-HPLC system (Shi-
madzu, VIC, Australia). The concentrations of individual
amino acids were standardized against an internal standard
(norvaline) at a concentration of 100 pmol/ll sample
injected.
Circular dichroism spectroscopy
The peptides were made up to a concentration of 0.1 lMin
phosphate buffered saline (PBS: 10 mM potassium phos-
phate buffer containing 137 mM NaCl pH 7.4). The far UV
circular dichroism (CD) spectra of peptides were acquired
using a JASCO model J815 spectropolarimeter between the
wavelengths of 195–250 nm at room temperature with a
resolution of 0.1 nm, bandwidth of 0.1 nm and a cell of
0.1 cm path length (P). The recorded spectra in millidegrees
of ellipticity (h) were converted to mean residue ellipticity
(MRE) in degcm
2
dmol
-1
. The CD spectra data were first
transformed from machine unit hto delta epsilon (De) using
GraphPad PRISM 4 (GraphPad Inc., San Diego, USA) with
a user defined formula (De=h9(0.1 9MRW)/
(P 9C)93,298) (C: Peptide concentration, MRW: Pep-
tide molecular weight/number of residues). The converted
values were then submitted to the DichroWeb server (Lees
et al. 2006; Lobley and Wallace 2001; Lobley et al. 2002;
Whitmore and Wallace 2004) for the calculation of sec-
ondary structure using the CDSSTR (Compton and Johnson
1986) and K2D (Andrade et al. 1993) analysis algorithms.
Ligand binding assay
Human embryonic kidney (HEK)-293T cells stably trans-
fected with RXFP2 and europium-labelled INSL3 were
used in whole cell binding assays. Cells were plated out at
a density of 80,000 cells per 200 ll per well in 96 well
Isoplate with white wall and clear bottom precoated with
poly-L-lysine. Competition binding experiments were car-
ried out as described previously (Shabanpoor et al. 2008)
with 300 pM of Eu-DTPA-INSL3 (K
d
: 0.892 nM) in
presence of increasing concentrations of peptide analogues.
Non-specific binding was determining with an excess
(500 nM) of unlabelled INSL3. Each concentration point
was performed in triplicate and the data expressed as the
mean ±SEM (standard error of mean) of the percentage of
total specific binding of triplicates from at least three
independent experiments. Curves were fitted using a one-
site binding model in GraphPad PRISM 4 (GraphPad Inc.,
San Diego, USA). The inhibition constants (K
i
) were
determined from IC
50
values using the Cheng-Prusoff
equation, and the statistical differences in pK
i
values were
calculated using one-way ANOVA followed by Bonfer-
roni’s multiple comparison test for multiple group
comparisons.
Functional cAMP assay
A cAMP reporter gene assay was used to assess the receptor
signalling of INSL3 and analogue 31 in HEK-293T cell line
co-transfected with RXFP2 (LGR8) and a pCRE-b-galac-
tosidase reporter plasmid. The assay was carried out as
described previously (Scott et al. 2006). Briefly, co-trans-
fected cells were incubated with increasing concentrations
of INSL3 and analogue 31 for 6 h after which the medium
was removed and cells frozen at -80°C overnight. The
amount of cAMP-driven b-galactosidase expression was
measured by lysing the cells. Each concentration point was
performed in triplicate and the data expressed as the
mean ±SEM of three independent experiments.
Results and discussion
INSL3 binds to its receptor principally using residues
confined to the B-chain. Deletion of the INSL3 A-chain
leads to the loss of B-chain a-helical structure and, there-
fore, loss of receptor binding affinity of the B-chain in
isolation. The a-helix is the most abundant secondary
structure which accounts for 30% of all protein residues
(Barlow and Thornton 1988). The helix plays a crucial role
in many protein-mediated biological processes such as
receptor binding (Beck-Sickinger and Jung 1995) and thus
is an attractive target for the design of mimetics. However,
peptides derived from these regions may not be biologi-
cally active when in isolation. This is usually due to the
loss of a-helicity where the peptides exhibit little or no
secondary structure as a result of loss of stabilizing inter-
actions within the parent protein. As part of our ongoing
effort to design short mimetics of INSL3 B-chain with
antagonistic action, we have utilized and evaluated various
possible ways of increasing the a-helicity within the
B-chain of INSL3. Towards this aim, we have designed
three series of analogues that include disulfide (primarily
helix to strand) constraints, ito i?4 lactam constraints,
and helix-inducing N-caps and residues (Table 1).
All analogues were synthesized on the solid-phase and
subjected to analysis by RP-HPLC and mass spectrometry.
The on-resin cyclization of lactam analogues was difficult.
In the first instance, using standard coupling procedures,
the reaction did not give the desired cyclic compound. A
second attempt using different coupling reagents, PyBOP
126 F. Shabanpoor et al.
123
and HOAt, gave the lactam but also resulted in the for-
mation of various major side products. Best results were
obtained using HBTU as coupling reagent and heating to
75°C using microwave power, in that the reaction went to
completion with minor side-products formation only and
an overall yield of 10–15%.
The introduction of a disulfide constraint between the
strand and the helix of the B-chain had little impact on the
level of helicity but improved the receptor binding affinity
in the analogue 3(pK
i
=6.41 ±0.11) compared to its
linear counterpart analogue 1(pK
i
=5.31 ±0.24). In
order to determine the mode of receptor interaction of this
analogue, the residues H
B12
,R
B16
,V
B19
,R
B20
and W
B27
,
which have been shown previously to be important for
binding of native INSL3 to RXFP2, were each mutated in
analogue 3. Alanine mutation of Arg
B16
significantly
reduced the receptor binding affinity of analogue 3(Fig. 2).
The replacement of His
B12
and Val
B19
with Ala also caused
40–50 times reduction in the binding affinity of analogue 3
whereas replacement of Arg
B20
caused only a slight drop in
receptor binding affinity. Finally, replacement of Trp
B27
in
this analogue with Ala led to almost complete loss of
receptor binding affinity. This trend of single replacement
of binding residues in the B-chain disulfide constrained
analogue and its loss of receptor binding affinity resembles
that of native INSL3 (Rosengren et al. 2006), suggesting
that, despite their lower affinity for RXFP2, the B-chain
mimetics bind to the receptor in the same fashion as native
INSL3.
The small improvement in receptor binding of the linear
B-chain with the disulfide constraint between the strand
and the a-helix was probably not as a result of induction of
further a-helical structure as there was little difference in
the level of a-helicity between the linear compound 1and
its cyclic counterpart analogue 3. It is likely that the
disulfide constraint between the strand and the helix of the
B-chain pulls the helix and the strand—very flexible in
native INSL3—closer together and this is possibly causing
a conformational change in the spatial orientation of the
Trp
B27
at the C-terminus of the B-chain which might be
better placed for interaction with the receptor. To further
investigate this possibility, three residues (G
B23,24
and
P
B25
) toward the C-terminus of the B-chain were replaced
with alanine. This change resulted in an analogue (11) with
binding affinity (pK
i
=5.74 ±0.2, n=3) similar to that
of linear B-chain (analogue 1). The GGP residues appear to
act as a hinge that provides flexibility for the B-chain C-
terminal region and this flexibility may be required for
proper interaction of Trp
B27
with the receptor. Since Trp
B27
is a crucial binding residue in the B-chain analogues,
we further investigated the role of both the individual
indole and benzene rings by replacing Trp
B27
in analogue 3
with b-(benzothien-3-yl)alanine (Bal, analogue 9) and
b-(naphtha-1-yl)alanine (1-Nal, analogue 10). Bal is an
analogue of Trp that has sulfur instead of nitrogen in the
indole ring and 1-Nal is another analogue in which the
indole ring is replaced with a benzene ring. The drop in
the level of receptor binding affinity of these two analogues
was similar which shows both rings of Trp
B27
are equally
important in receptor interaction.
Further constraining analogue 3by incorporating an ito
i?4 disulfide constraint along the helix at Phe
B14
and
Leu
B18
gave analogue 12 which exhibited low level of
helicity even in the presence of 20% TFE. This peptide had
a very low level of receptor affinity compared to analogue
3. The ito i?4 disulfide constraint along the helix would
cause analogue 12 to lose its flexibility around the helical
region and prevent the peptide from adopting an active
conformation for high affinity receptor binding. This was
investigated by inserting only the ito i?4 disulfide
constraint at Phe
B14
and Leu
B18
in analogue 13.This
analogue did not show any affinity for receptor binding,
which is likely due to the loss of flexibility and inability to
adopt a conformation with the key receptor binding resi-
dues in a correct orientation to the binding pocket on the
receptor.
The introduction of a lactam bridge in the B-chain was
the second approach that was investigated for the restora-
tion of helical structure in the isolated B-chain of INSL3.
Side chain–side chain lactam bridges have long been used
as a convenient and flexible method for introducing con-
formational constraints into a peptide structure. The points
to consider in the design of peptides with a lactam bridge
are the spacing of the two residues to be linked, the side-
chain orientation and the position of the bridge. A Glu–Lys
Fig. 2 Competition binding studies of native INSL3, disulfide
constrained analogue 3and its Ala-substituted analogues (Table 1).
Europium-labelled-INSL3 (0.3 nM) was used as a labelled ligand in
the competition binding assay in the presence of increasing concen-
tration of INSL3 and analogues. Singly substituting R
B12
(5) and
W
B27
(8) with Ala caused these peptides to almost completely lose
their receptor binding affinity. Replacing H
B12
(4) and V
19
(6) with
alanine also led to a major loss of receptor binding affinity. Mutation
of R
B20
had minor impact in the level of binding affinity
Effect of helix-promoting strategies on the biological activity 127
123
(E-K) lactam at the spacing of iand i?4 in the middle of
the peptide has been shown to be more effective at stabi-
lizing helical structure than two Glu-Lys lactams
positioned one at each end of the peptide (Houston et al.
1995). In the B-chain of INSL3, we introduced a lactam
bridge (i,i?4) in the mid-region of the B-chain a-helix
by mutating Phe
B14
and Leu
B18
to Glu, Asp, Lys or Dab.
Various orientations of the lactam bridge were evaluated in
the long form (analogues 14–16) (Fig. 3a) and truncated
B-chain of INSL3 (analogues 17–22).
Analogues 14 and 15 had higher a-helicity in PBS
(17 and 18%, respectively) compared to the linear analogue
1(10%); this small improvement in a-helicity was
accompanied by a small increase in receptor binding
affinity. In analogue 15, Lys
B18
was replaced with diam-
inobutyric acid (Dab) (analogue 16). This modification was
observed to not only reduce the level of a-helicity but also
cause a drop in receptor binding affinity (Table 1). This
may be due to the shorter side-chain of Dab which, on
cyclization with the Glu side-chain, results in an unfa-
vourable conformational change in the B-chain. The
introduction of a lactam bond in the truncated INSL3
B-chain analogues had little effect on either a-helicity or
receptor binding. Analogues 17 and 21with K-D and E-K
lactam orientation, respectively, were more effective in
inducing helical structure compared to D-K and K-E lac-
tams in analogues 18 and 20. Analogue 18 with D-K lactam
orientation did not have higher a-helicity compared to its
linear counterpart analogue 19. On the other hand, ana-
logue 21with E-K lactam orientation exhibited higher
a-helicity compared to its linear analogue 22. Both the
short and long forms of E-K orientated lactams (15 and 21)
had the highest helical content both in PBS and 20% TFE
compared to any other lactam orientation and this is in
accordance with previous literature reports (Houston et al.
1995). Despite some of the lactam constrained analogues
being able to induce a-helicity in the B-chain, this did not
result in high affinity binding. The introduction of a lactam
bridge in the mid-region of the B-chain backbone likely led
to the loss of B-chain flexibility and a more rigid structure,
which is no longer capable of adjusting binding residues to
align with the binding pocket on the receptor.
Analogues 23–25 show the effect of ‘‘capping’’ the
N-terminal of the truncated B-chain with acetyl and 2,4,5
benzenecarboxylate (Fig. 3B), the latter which has recently
been reported to increase the a-helicity of a model peptide
from 17 to 70% by providing carbonyl groups that act as
surrogate H-bond acceptors (Mimna et al. 2007). The
N-terminus capping of the B-chain a-helix was not effec-
tive in inducing a-helicity in PBS but in 20% TFE the level
of a-helicity increased significantly, which was likely a
reflection of the tendency of these peptides to adopt a
helical structure.
The a-helix-inducing effect of residues such as Ala and
Aib on the INSL3 B-chain helix was also investigated. The
use of a-helicogenic residues in enhancing the activity of
peptides has been demonstrated by substituting these at
specific positions in the helical N-terminal fragment of
parathyroid hormone PTH(1–11), which made the resulting
peptide 3,500 times more potent (Barazza et al. 2005). The
a-helix stabilization of alanine has been related to the
hydrophobic interaction, steric effects and solvation of
the polar groups of the a-helix backbone (Avbelj 2000;
Avbelj and Fele 1998; Bai and Englander 1994; Bai et al.
1993; Blaber et al. 1993,1994; Connelly et al. 1993).
Aib-based peptides have a remarkable tendency to form
a-helical conformations in solution due to the restricted
rotation about (N-Ca) and (Ca-C
1
) bonds, which is caused
by the presence of two methyl groups on the Caatom
(Ma et al. 2007; Marshall et al. 1990). Alanine and Aib
residues were incorporated along the a-helical segment of
INSL3 B-chain in place of valine in analogues 26–29.
None of these analogues had high helicity in PBS, although
the helicity increased upon addition of 20% TFE. These
peptides also showed very low receptor binding affinity due
to the lack of helical structure.
W27
L18 R20
R16
F14
V19
H12
O
OO
O
OH
OH
OH
N-term inus
C-terminus
BA
W27
R20
R16
V19
H12
K18
E14
E18
K14
K18
D14
D18
K14
1, 2, 4, 5-Benzene-tetraca rbox
y
lic d ia nh
y
dride
Fig. 3 a An INSL3 B-chain
analogue in which a lactam
constraint was incorporated in
the middle of the a-helix on the
side opposite to where the
binding residue is located.
F
14
and L
18
were replaced with
K, E, D or Dab in different
orientations. bTruncated INSL3
B-chain until G
B11
and the
N-terminus capped with either
acetic anhydride or 1,2,4,5-
benzene-tetracarboxylic
dianhydride
128 F. Shabanpoor et al.
123
The disulfide constrained analogue 30, where the
B-chain helix was stabilized using the C-terminal a-helical
region of the A-chain and analogue 31, which is a B-chain
dimer, exhibited 23 and 32% a-helicity in PBS, which was
higher than any other single B-chain analogues. The higher
level of a-helicity was accompanied by an increase in
receptor binding affinity of these two analogues (Fig. 4a).
To determine if 31 was able to activate the receptor and
induce intracellular cAMP production, the analogue was
tested in HEK-293T cells expressing RXFP2 (LGR8).
Native INSL3 induced cAMP production (pEC50 =
10.35 ±0.12) whereas analogue 31 was unable to activate
the receptor as intracellular cAMP accumulation was not
induced (Fig. 4b), which suggests that this peptide is an
antagonist of INSL3.
In summary, a systematic approach was taken to induce
a-helicity in the isolated INSL3 B-chain and study the
binding mode of interaction with the receptor. Isolated
INSL3 B-chain that was constrained with a disulfide bond
was found to bind to the receptor in the same way as native
INSL3. Attempting to induce an a-helical conformation by
constraining the B-chain of INSL3 did not fully compen-
sate for the stabilizing interactions of the missing A-chain
as constraining the B-chain by either a disulfide or lactam
bond did not increase a-helicity and receptor binding
affinity simultaneously. However, constraining the B-chain
with a short region of A-chain or dimerizing the B-chain
not only increased the a-helicity but also the receptor
binding affinity. On this basis, we have identified two novel
INSL3 antagonists which can be used as lead compounds
to be further minimized and optimized for development as
clinically useful INSL3 antagonists which may be used as
potential male and female contraceptives.
Acknowledgments We thank Tania Ferraro for help with binding
assays. This work was funded by National Health and Medical
Research Council of Australia Project grants #350245 and 509048 to
JDW, RADB and RAH.
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