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ScienceDirect
Synthetic relaxins
Mohammed Akhter Hossain1,2 and John D Wade1,2
The relaxin subfamily of peptides within the human insulin
superfamily consists of seven members including relaxin-2 and
relaxin-3. The former is a pleiotropic hormone that is a
vasodilator and cardiac stimulant in the cardiovascular system
and an antifibrotic agent whereas the latter is primarily a
neuropeptide involved in stress and metabolic control. Both
possess the unique three-disulfide heterodimeric peptide
structure of insulin. Consequently, the synthesis, both chemical
and biological, of relaxin-2 and relaxin-3 has long represented a
special challenge to further understanding their structural and
functional relationships. This review highlights past and recent
developments in the use of chemical and recombinant DNA
methods of synthesis of these peptides and current resulting
knowledge of their biology.
Addresses
1
Florey Institute of Neuroscience and Mental Health, Victoria 3010,
Australia
2
School of Chemistry, University of Melbourne, Victoria 3010, Australia
Corresponding author: Wade, John D (john.wade@florey.edu.au)

Current Opinion in Chemical Biology 2014, 22:4755

This review comes from a themed issue on Synthetic biomolecules
Edited by Paul F Alewood and Stephen BH Kent

http://dx.doi.org/10.1016/j.cbpa.2014.09.014
1367-5931/# 2014 Elsevier Ltd. All right reserved.

24-residue A-chain and 29-residue and 27-residue B-chain

respectively with modest sequence homology other than
the three cystine crosslinks (Figure 1).
Unlike insulin which acts via a tyrosine kinase receptor,
both relaxin-2 and relaxin-3 act via G protein-coupled
receptors known as RXFP1 and RXFP3 respectively
[1]. Relaxin-2 is produced in the corpus luteum and/
or placenta of all mammals and exerts multiple essential
actions on the reproductive tract to support pregnancy,
facilitate delivery, and prepare the mammary gland for
lactation. It has also been shown to be produced locally in
many tissues (Figure 2) and to have much wider biological
actions. Notably, atrial cardiocytes produce and secrete
relaxin-2, and its RXFP1 receptor was also identified in
the heart indicating that this organ is a target for this
peptide [3]. A plethora of studies have shown that relaxin2 is cardioprotective due to its antifibrotic, anti-hypertrophic, anti-inflammatory and vasodilatory actions [4]. It
is a potent compensatory mediator in human congestive
heart failure. Human relaxin-2 (also commonly called H2
relaxin) passed Phase III clinical trials in late 2012 for the
treatment of acute heart failure [5] and was granted
Breakthrough Therapy designation status in late
2013 by the US Food and Drug Administration. By
contrast, relaxin-3 and its RXFP3 receptor are primarily
expressed in neurons of the nucleus incertus and adjacent
brainstem areas of the brain and at much smaller levels in
the testes and prostate (Figure 2). It has potent anxiolytic
and anti-depressive actions and is now recognized to be
an important modulator of brain stress and mood systems
and may also have key roles in feeding and metabolism
[3,6].

Introduction

Synthesis of relaxin

The insulin family of proteins consists of 10 members in

the human with insulin being the ancestral gene. These
members include insulin, insulin-like growth factors
(IGF) I and II, and a now-classified relaxin subfamily
that comprises seven members: relaxin-1, relaxin-2 and
relaxin-3 and four other insulin-like peptides (INSL 3, 4,
5 and 6) [1]. Each member, except IGFs I and II,
possesses a two peptide chain structures crosslinked with
three disulfide bonds in the same disposition as for
insulin. The relaxin-1 gene (RLN1) is only present in
humans and other higher primates but the expression
product has not been detected in vivo. The human RLN2
gene corresponds to the sole RLN1 gene found in other
mammalian species and the expression product is known
as relaxin-2 or simply relaxin. The final relaxin member to
be identified, relaxin-3, is the ancestral peptide of the
relaxin subfamily [2]. Relaxin-2 and relaxin-3 consists of a

Via chemical synthesis of the two chains and random

chain combination

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The first attempt to chemically assemble H2 relaxin was

based upon the first reported successful chemical synthesis of bovine insulin in the mid-1960s. Earlier landmark studies had shown that native insulin could be Sreduced into its two separate chains which could then be
combined and re-oxidized in high pH buffer to generate
biologically active peptide [7]. Motivated by this finding,
bovine insulin A-chain and B-chain were separately produced by solution phase fragment synthesis and then
combined in solution to spontaneously oxidatively fold
to generate the native three cystine bonds [8]. Improved
yields of folding were obtained when the more readily
handled and purified S-sulfonated forms of the A-chain
and B-chain were used in the presence of reducing agent
[9]. These early results clearly showed that the primary
Current Opinion in Chemical Biology 2014, 22:4755

48 Synthetic biomolecules

Figure 1

A chain
H2 relaxin Z-L-Y-S-A-L-A-N-K-C-C-H-V-G- C-T-K-R-S-L-A-R-F-C
H3 relaxin D-V-L-A-G-L-S-S-S-C-C-K-W-G-C-S-K-S-E-I-S-S-L- C
H3 relaxin
R-A-A-P-Y-G-V-R-L-C-G-R-E-F- I-R-A-V-I-F-T-C-G-G-S-R-W
H2 relaxin D-S-W-M-E-E-V-I-K-L-C-G-R-E-L-V-R-A-Q-I-A-I-C-G-M-S-T-W-S
B chain
R12

W27

R16

I19

F20

H3 relaxin

R12

R16
I19
W27
A20

H2 relaxin

Current Opinion in Chemical Biology

Primary structures (upper) of human relaxin-2 (H2) and relaxin-3 (H3) and their tertiary structures (lower) as determined by X-ray crystallography and
solution NMR spectroscopy respectively. Z represents pyroglutamic acid.

structure of the peptide chains were the key determinants

for the correct alignment and subsequent cystine formation, itself remarkable given that the number of statistically possible disulfide heterodimers of the two insulin
chains is 12.
However, following preparation A-chain and B-chain of
H2 relaxin by Bocchemistry solid phase peptide synthesis
(SPPS), it was found that subsequent use of the mixed
S-sulfonate/thiol oxidative folding approach failed to
yield target protein molecule. This was principally due
to poor solubility of the B-chain whether in its S-reduced
or S-sulfonated form. This limitation was initially overcome by the use of more soluble C-terminal chain shortened (125) or lengthened (133) B-chain [10].
Subsequently, the native S-reduced B-chain (129) could
be dissolved in a complex buffer containing both organic
solvents and denaturing agents and, after addition of both
reduced A-chain and dithiothreitol in pH 12 aqueous
buffer, successful combination of the two chains of both
human relaxin-1 and H2 relaxin (which share an identical
B-chain sequence) was achieved [11] (Figure 3I). Overall
folding yields could be as high as 50% relative to the
starting B-chain. This approach was employed to produce
relaxin-2 from different species including rat and gorilla
[12,13]. Unexpectedly, no in vitro oxidative refolding
conditions were found under which successful combination of the two synthetic chains of human relaxin-3
could be achieved. Recourse to chemical regioselective
disulfide bond synthesis was required in order to obtain
this peptide for subsequent biological study (see below).
Although random chain combination has largely ceased to
be an approach for the production of synthetic relaxin-2, it
was used to determine folding pathways [14]. It is also a
Current Opinion in Chemical Biology 2014, 22:4755

potentially cheaper approach for the scale-up preparation

of native synthetic protein compared to the more laborious sequential selective disulfide bond formation
approach (see below). Recently, Barlos and colleagues
showed that oxidation to Met(O) of one of the two Met
residues within the H2 relaxin B-chain (Met25) significantly improved both its chemical synthesis and its subsequent solubility [15]. This latter aspect, in turn,
enabled enhanced oxidative folding with either
S-reduced or mixed isomers of disulfide bicyclic A-chain
to generate correctly folded Met(O)B25-relaxin that was
reduced to MetB25 with ammonium iodide in excellent
overall yield (48% relative to starting B-chain).
Via chemical synthesis of the two chains and
regioselective disulfide bond formation

An important advance in chemical peptide synthesis was

the development of novel orthogonal Cys thiol protecting groups that allowed selective removal of one set of
protecting groups under controlled conditions in the
presence of others. This enabled selective exposure of
free thiols for subsequent disulfide formation and permitted the development of alternative, effective strategies for the chemical synthesis of multi-cystine peptides
and proteins including, critically, insulin [16]. A significant advance in the solid phase synthesis of INSLs was
heralded by the successful synthesis of H2 relaxin via
regioselective disulfide bond formation, in which each of
the three disulfide bonds were formed sequentially by
stepwise removal of pairs of orthogonal S-protecting
groups followed by oxidation of the resulting liberated
thiol groups [17]. A complex eight-step strategy was
followed in which the A-chain was assembled by Fmoc
chemistry SPPS and contained three different S-protecting groups, each removable by a different chemical
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Synthetic relaxins Hossain and Wade 49

Figure 2

Heart

Kidney

Skin

lax
in

Uterus

H2

re

Liver

RXFP1
Brain
Kidney

Prostate/Testis

H3 relaxin

RXFP3
Current Opinion in Chemical Biology

Key sites of expression of relaxin-2 and relaxin-3 and their cognate receptors in RXFP1 and RXFP3 throughout the body.

mechanism: acetamidomethyl (Acm), trityl (Trt) and

p-methylbenzyl (MeBzl). After cleavage of the peptides
from the solid support by treatment with trifluoroacetic
acid, which also deprotected Cys(Trt), the two side chaindeprotected cysteines (A10,15) were oxidized with iodine
in 50% acetic acid and the resulting intramolecular disulfide bond-containing peptide then treated with HF to
remove the MeBzl group from the C-terminal Cys residue. Cys(A11) remained protected. The B-chain, prepared by Boc-SPPS contained a Cys(Acm) and a Cys
residue bearing a 3-nitro-2-pyridylthio (Npys) group;
the Npys was displaced in pH 4.5 buffer by the side
chain thiol of Cys(A24) within the A-chain intermediate
to produce the first interchain disulfide bond. The final
intermolecular disulfide bond was formed by iodolysis of
the pair of Cys(Acm) residues. Nin-Deformylation of the
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two Trp residues within the B-chain and subsequent

reduction of the Met(O) residues via treatment with base
and then ammonium iodide in aqueous trifluoroacetic
acid respectively led to synthetic H2 relaxin in very
low overall yield as an obvious consequence of the
numerous intermediate purification steps.
The successful chemical synthesis of the silkworm insulin-like peptide, bombyxin-IV, represented a major
advance in regioselective disulfide bond formation between the two synthetic chains [18]. The strategy was
simpler and employed only Fmoc chemistry solid phase
synthesis for the two chains that were selectively Sprotected with a combination of Trt, Acm and tert-butyl
groups (tBu). After A-chain intramolecular disulfide bond
formation by air-oxidation of the two free thiols, an
Current Opinion in Chemical Biology 2014, 22:4755

50 Synthetic biomolecules

Figure 3

(I) SPPS and folding by random oxidation

SH

SH

SH

A chain

SH

SH
SH
SH
D S W M E E V I K L C G R
E L V R A Q I A I C G M S T W S

B chain

SH

Acm

Z L Y S A L A N K C C H V G C T K R S L A R F C

Z L Y S A L A N K C C H V G C T K R S L A R F C

A chain

S-tBu

Acm
L

S
S A
S
F C
L A
A R
N K
C C H V G C T K R S L
S-tBu

Acm
Z

S
S
S A
F C
L A
A R
N K
C C H V G C T K R S L

Oxidative folding

S-tBu
SH

Acm

D S W M E E V I K L C G R
E L V R A Q I A I C G M S T W S

B chain
Z

(III) Recombinant DNA synthesis

S
S
S A
F C
L A
A R
N K
C C H V G C T K R S L
D
S
S
S
W
Acm
M
W
E E
S T
S
M
V I
K L
C G
I
C G R
E L V R A Q I A

C-peptide
Z

Acm
L

Y S

S
S
C
A L
R F
A N
L A
K C
S
C H V G C T K R S
S
S
S
W
S
M
W
E E
S T
S
M
V I
G
K L
C
C G R
A I
E L V R A Q I

Prorelaxin
Oxidative folding
SH

S
S
S A
F C
L A
A R
N K
C C H V G C T K R S L
S
S
S
S
W
S
M
W
E E
S T
S
V I
G M
K L
I C
C G R
A
E L V R A Q I

(II) SPPS and regioselective disulfide formation

Relaxin

C-peptide
SH

SH

Z L Y S A L A N K C C H V G C T K R S L A R F C
SH
SH
SH
D S W M E E V I K L C G R E L
V R A Q I A I C G M S T W S

Signal
Peptide

B chain

Preprorelaxin

C-

pe

pti

de

A
C
S
B
Plasmid
Current Opinion in Chemical Biology

Schemes for the synthesis of relaxin-2. (I) SPPS and random oxidative folding of the two chains, (II) SPPS and sequential disulfide bond formation
within the two chains, and (III) recombinant DNA synthesis of pro-peptide, oxidative folding and cleavage of the C-peptide.

important novel procedure was applied. Treatment of the

resulting peptide intermediate with 2,20 -dipyridyldisulfide in trifluoromethanesulfonic acid led to displacement
of the C-terminal tBu group and formation of Cys(pyridinylsulfenyl; Pys) derivative without affecting the
A-chain intramolecular disulfide bond. This Pys derivative reacted with the free thiol of Cys(B22) of the B-chain
Current Opinion in Chemical Biology 2014, 22:4755

to form the first intermolecular disulfide. The second and

final intermolecular disulfide was generated by iodolysis
of the pair of Cys(Acm) residues [18].
The strategy used for the synthesis of bombyxin-IV was
shown to work well for synthesis of H2 relaxin although
the overall yield was modest, being about 10% relative to
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Synthetic relaxins Hossain and Wade 51

the starting crude B-chain intermediate (Figure 3II). It

also allowed the preparation of relaxin-2 from other
species including horse and mouse [19,20], and led to
the first-ever acquisition of human relaxin-3 which was
previously shown to be refractive to production via the
random chain refolding approach [2,21]. Unlike with H2
relaxin, careful placement of the thiol protecting groups
was also required in order to minimize the detrimental
effect of the final iodine oxidation step used for disulfide
linking of the Cys(Acm) residues. The quantity of peptide thus obtained enabled its tertiary structure determination by 2-D NMR spectroscopy in which it was shown
to possess, not unexpectedly, the characteristic insulinlike fold [22] (Figure 1). The synthetic strategy has also
enabled the acquisition of rat relaxin-3 [23] and of other
members of the relaxin subfamily of peptides including
INSL3 and INSL5 together with their tertiary structure
determination [24,25].
Despite the effectiveness of this strategy, the multiple
steps required for not only the separate chain syntheses
but also the subsequent individual disulfide bond formation invariably lead to overall modest yields of target
peptide (generally around 10% relative to the limiting
chain). This may not be critical for the preparation
of analogs of INSLs for structural and functional study
but poses severe limitations for the acquisition of adequate material for pre-clinical studies. Consequently,
further improvements are desirable for the chemical
synthesis of such peptides, in particular, higher yields
by SPPS of the two chains, novel S-protection that
affords yet additional dimensions of orthogonality,
improved or alternative chemical disulfide formation
that avoids damage to other residues such as tryptophan
and tyrosine, and, finally, refined purification procedures. In this respect, the recent developments of
one pot regioselective disulfide formation of insulin
and of photolabile S-protecting groups show considerable promise [26,27,28].
Recombinant DNA synthesis of relaxin

Following the pioneering development of recombinant

DNA methods for the successful production of proteins, it
was not unexpected that the medically essential insulin
would be the first peptide to be subjected to this form of
synthesis. The first approach involved the separate
expression of each of the A-chain and B-chain followed
by their purification and random oxidative combination
[29]. This was followed by an improved process involving
the expression of pro-insulin which is now the current
method of choice for insulin production [30] as it entails
just one sequence of fermentation and purification steps.
Consequently, a similar method was also adapted developed for the expression of relaxin. As its C-peptide is
102 residues long compared to insulins 35 residues [3],
H2 relaxin was expressed as a single chain peptide but
containing a short mini-C-peptide of just 13 residues
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that was in part selected from the corresponding region

within IGF-1. Judiciously placed Asp and Arg residues at
the B-C and C-A intersections enabled selective postexpression in E. coli and oxidative folding cleavage of the
mini-C-peptide by sequential treatment with Asp N and
Arg C enzymes [31] (Figure 3III). Overall yields of
purified native two-chain H2 relaxin were significantly
higher than following random combination of the individual A-chain and B-chain. This approach was also
employed to prepare a number of Ala-mutated analogs
of H2 relaxin, and it remains the method of choice for
scale-up production of clinical grade material.
A similar approach was adopted for the production of
human relaxin-3 in mammalian cells although yields of
correctly folded product were initially low [32,33,34].
More recently, Guo and colleagues have refined this
expression process using E. coli [35]. A 12-residue
mini-C-peptide was employed which is flanked by an
Asp residue at the B-C junction which, together with the
natural Asp at the N-terminal of the A-chain, allows
eventual liberation of the mini-C-peptide by treatment
with a single enzyme, Asp-N that cleaves the peptide
bond at the N-terminal side of an Asp residue. The miniC-pro-relaxin-3 also contained an N-terminal hexa-His
tag for ease of post-expression purification. Following
cleavage from the Ni2+ column by Aeromonas aminopeptidase, in vitro refolding of the human relaxin-3 precursor
was significantly enhanced by direct disulfide reshuffling in redox buffer that avoids the handling of a fully
reduced and prone to aggregate peptide. On completion
of folding and correct disulfide bond formation, the miniC-peptide was enzymatically liberated to yield human
relaxin-3 in good overall yield (up to 3 mg/l of E. coli
culture broth cf less than 1 mg/l in earlier attempts).

Structurefunction relationship studies

A summary of structureactivity relationship is provided
in Figure 4. A comparison of the H2 relaxin primary
structure with relaxin-2 from different species reveals a
strong sequence similarity in the mid-region of the Bchain where two arginine residues are located on the same
surface of the a-helix. These residues, ArgB13 and
ArgB17, together with IleB20 were later found to be
essential for binding to and activating RXFP1 resulting
in a triangular contact region referred to as the
RXXXRXXI binding cassette [36,37]. The residues in
this cassette have been demonstrated to interact with the
large extracellular domain of RXFP1 [38]. An additional
interaction of the peptide with the transmembrane exoloops of the receptor is predicted to involve the A-chain of
the peptide, in particular residues ThrA16, LysA17, and
PheA23 [39]. TyrA3 and PheA23 were also found to be
critical for peptide folding [40]. These structureactivity
data indicate that the active site of H2 relaxin for interaction with RXFP1 probably consists of the mid-region of
the B-chain helix and part of the C-terminal region of the
Current Opinion in Chemical Biology 2014, 22:4755

52 Synthetic biomolecules

Figure 4

H2 relaxin A-chain

H3 relaxin A-chain
N-terminus
truncation
caused progressive loss of
RXFP1 binding and activation

Ala mutation at 16 and 17 enhanced RXFP2 cAMP

stimulation
Ala mutation at 22 reduced RXFP2 activity
Y3 and F23 are important for structural fold and for
RXFP2 activity
S19 and L20 are important of RXFP1 activity
4 residues from the N-terminus can be truncated
without losing RXFP1 activity

10 residues from the Nterminus can be truncated

without losing RXFP3 binding and activation

A chain
H2 relaxin

Z-L-Y-S-A-L-A-N-K-C-C-H-V-G-C-T-K-R-S-L-A-R-F-C
D-V-L-A-G-L-S-S-S-C-C-K-W-G-C-S-K-S-E-I-S-S-L-C H3 relaxin

R-A-A-P-Y-G-V-R-L-C-G-R-E-F-I-R-A-V-I-F-T-C-G-G-S-R-W
H2 relaxin D-S-W-M-E-E-V-I-K-L-C-G-R-E-L-V-R-A-Q-I-A-I-C-G-M-S-T-W-S

H3 relaxin

B chain
H2 relaxin B-chain
Mid-region, particularly R13, R17 and I20 are
important for RXFP1 acitivity
W29 is important for RXFP2 activity
6 residues from the N-terminus can be truncated
without losing RXFP1 activity
5 residues from the C-terminus can be truncated
without much losing RXFP1 activity

H3 relaxin B-chain
R8, R12, I15, R16 and F20 are
involved in RXFP3 binding
R26 and R27 are involved in
RXFP3 activation
9 residues from the N-terminus
of the B-chain can be truncated without losing RXFP3
activity

Domain minimization from SAR study

24
5
A-L-A-N-K-C-C-H-V-G-C-T-K-R-S-L-A-R-F-C

V-I-K-L-C-G-R-E-L-V-R-A-Q-I-A-I-C-G
7
24
mini-H2 relaxin

24
11
C-K-W-G-A-S-K-S-E-I-S-S-L-C

C-G-R-E-F-I-R-A-V-I-F-T-C-G-G-S-R-W
10
27
A6 (H3 analogue 6)
Current Opinion in Chemical Biology

Summary of the structureactivity relationships derived from synthetic H2 and H3 relaxin analogs. These two peptides share common structural motifs
including the binding cassette RXXXRXXI and three insulin-like disulfide bonds between the A-chain and B-chain and within the A-chain. The minimum
primary structures of the two peptides are also shown.

A-chain. Further sequential truncations were undertaken

at each of the N-termini of the A-chain and B-chain and
the C-terminus of the B-chain which were thought not to
be important for RXFP1 activity. More than 30 analogs
were evaluated and the results identified a minimized
Current Opinion in Chemical Biology 2014, 22:4755

analog, mini-H2 relaxin, that is one-third smaller than

the native hormone and retains good RXFP1 activity
[41,42] (Figure 4). The RXFP1-selective relaxin analog
H2:A(4-24)(F23A) has also been prepared bearing a postsynthesis click chemistry-conjugated fluorophore (Cy5.5)
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Synthetic relaxins Hossain and Wade 53

at the B-chain N-terminus, with conservation of the

disulfide bonds and biological activity. It was used to
probe brain RXFP1 signaling while tracking its distribution following central or peripheral administration [43].
Importantly, Silvertown and colleagues [44] showed that
a lentivirus engineered to produce pro-B-R13/17K H2
relaxin where ArgB13 and 17 were replaced by Lys
exhibited antagonist properties in vitro by competing
with relaxin-induced signaling. As the expressed form
was likely to have been a prohormone, a two-chain analog
containing the two point mutations was chemically
assembled and also shown to possess antagonist activity
and thus represents an important probe of biological
function [45].
Numerous human relaxin-3 analogs have also been prepared and the results of in vitro assays have shown that
truncation is tolerated from the N-termini of the A-chain
and B-chain by up to 10 and 9 residues respectively
without major loss of receptor binding or activation
[41,46]. The residues in the B-chain central a-helix that
are involved in RXFP3 binding have also been identified,
and it was shown that activation is mediated via two
critical C-terminal residues, ArgB26 and TrpB27 [47].
Replacement of the intra-A-chain disulfide bond with a
dicarba bond did not cause loss of biological activity and
also increased in vitro serum stability [48]. Furthermore,
replacement of the A-chain with that from INSL5 (R3/I5)
confers selectivity to RXFP3 and RXFP4 by substantially
reducing affinity for the relaxin receptor, RXFP1 [47,49].
Remarkably, even greater selectivity for RXFP3/4 over
RXFP1 can be achieved by removal of the intra-molecular disulfide bond within the A-chain of relaxin-3 resulting
in the peptide Analog 2. This peptide is substantially
easier to synthesize than native relaxin-3 or the R3/I5
chimera. It has been further modified by truncation of the
A-chain to yield a still-smaller yet potent relaxin-3 agonist
known as Analog 6 (Figure 4) which can activate RXFP3
in vivo to increase feeding and significantly reduce
anxiety-like behavior in adult rats [46].

Conclusions
The development of efficient chemical and recombinant
DNA synthesis methods for the preparation of relaxin-2
and relaxin-3 have led to an extraordinary increase in our
knowledge of their functional roles. With H2 relaxin
poised to enter the clinic for the treatment of acute heart
failure, this understanding is more than ever critical for
the development of new, smaller analogs with improved
receptor specificity and in vivo stability. That relaxin-3 is
the ancestor to the relaxin family of peptides and a potent
neuropeptide places further demands on fully elucidating
its function in the most complex of all organs, the brain.
The current methods that enable the efficient preparation
of these complex peptides have been, and continue to be,
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critical for advancing our understanding of the biology

and therapeutic potential of these fascinating peptides.

Acknowledgements
We thank the NHMRC (Australia) for research financial support over many
years. JDW is an NHMRC Principal Research Fellow. Research at The
Florey Institute of Neuroscience and Mental Health is supported by the
Victorian Government Operational Infrastructure Support Program.

References and recommended reading

Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
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Bathgate RA, Halls ML, van der Westhuizen ET, Callander GE,
Kocan M, Summers RJ: Relaxin family peptides and their
receptors. Physiol Rev 2013, 93:405-480.
An extensive review of all aspects of the chemistry and biology of the
relaxins, their related peptides and receptors.

2.

Bathgate RA, Samuel CS, Burazin TC, Layfield S, Claasz AA,

Reytomas IG, Dawson NF, Zhao C, Bond C, Summers RJ et al.:
Human relaxin gene 3 (H3) and the equivalent mouse relaxin
(M3) gene. Novel members of the relaxin peptide family. J Biol
Chem 2002, 277:1148-1157.

3.

Shabanpoor F, Separovic F, Wade JD: The human insulin

superfamily of polypeptide hormones. Vitam Horm 2009,
80:1-31.

4.

Teichman SL, Unemori E, Teerlink JR, Cotter G, Metra M: Relaxin:

review of biology and potential role in treating heart failure.
Curr Heart Fail Rep 2010, 7:75-82.

5.


Teerlink JR, Cotter G, Davison BA, Felker GM, Filippatos G,

Greenberg BH, Ponikowski P, Unemori E, Voors AA, Adams KF Jr
et al.: Serelaxin, recombinant human relaxin-2, for treatment of
acute heart failure (RELAX-AHF): a randomised, placebocontrolled trial. Lancet 2013, 381:29-39.
Important description of the first successful Phase 3 clinical trial of H2
relaxin (serelaxin) for the treatment of heart failure.

6.

Smith CM, Walker AW, Hosken IT, Chua BE, Zhang C, Haidar M,
Gundlach AL: Relaxin-3/RXFP3 networks: an emerging target
for the treatment of depression and other neuropsychiatric
diseases? Front Pharmacol 2014, 5:46.

7.

Du YC, Zhang YS, Lu ZX, Tsou CL: Resynthesis of insulin from

its glycyl and phenylalanyl chains. Sci Sin 1961, 12:452-454.

8.

Kung YT, Du YC, Huang WT, Chen CC, Ke LT: Total synthesis of
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