Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue

Praveen Praveen, Julien Tailhades,* K. Johan Rosengren, Mengjie Liu, John D. Wade,

Note

Eﬀects of C‑Terminal B‑Chain Modiﬁcations in a Relaxin 3 Agonist

Analogue

Ross A. D. Bathgate,* and Mohammed Akhter Hossain *

Cite This: https://dx.doi.org/10.1021/acsmedchemlett.0c00456

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sı Supporting Information

ABSTRACT: The receptor for the neuropeptide relaxin 3, relaxin

family peptide 3 (RXFP3) receptor, is an attractive pharmaco-

logical target for the control of eating, addictive, and psychiatric

behaviors. Several structure−activity relationship studies on both

human relaxin 3 (containing 3 disulﬁde bonds) and its analogue

A2 (two disulﬁde bonds) suggest that the C-terminal carboxylic

acid of the tryptophan residue in the B-chain is important for

RXFP3 activity. In this study, we have added amide, alcohol,

carbamate, and ester functionalities to the C-terminus of A2 and

compared their structures and functions. As expected, the C-

terminal amide form of A2 showed lower binding aﬃnity for

RXFP3 while ester and alcohol substitutions also demonstrated

lower aﬃnity. However, while these analogues showed slightly

lower binding aﬃnity, there was no signiﬁcant diﬀerence in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic

acid, suggesting the binding pocket is able to accommodate additional atoms.

KEYWORDS: Insulin-like peptide, relaxin 3, H3 relaxin, GPCR, solid phase peptide synthesis, peptidomimetic

uman relaxin 3, H3 relaxin, is a member of the relaxin

relaxin-like activity, both in vitro and in vivo. Our SAR

studies on H3 relaxin and the related INSL5 peptide revealed

that a free C-terminus carboxylic acid (COOH) of the B-chain

−4

hormone family.

While H3 relaxin interacts with the

endogenous relaxin family peptide 3 (RXFP3) receptor, it can

also bind to and activate related peptide receptors, RXFP1 and

14,15

is favored for RXFP3 and RXFP4

receptor activity.

However, this is not the case for H2 relaxin as C-terminally

amidated H2 relaxin was found to be as potent as a free acid

peptide in terms of RXFP1 activity. Thus, the eﬀects of C-

terminal modiﬁcations are peptide and receptor-dependent.

The aim of this study was to investigate the eﬀects of C-

terminal modiﬁcations on the high yielding A2 analogue that is

RXFP4. Current literature suggests that RXFP3 is a potential

therapeutic target for psychiatric disorders such as anxiety and

depression. Central administration of RXFP3 agonists into

rats increases feeding behaviors and that of antagonists inhibits

food consumption and addictive behaviors. These data

suggest that RXFP3 is also a potential target for pharmaco-

more easily acquired and in much higher overall yield. We

designed and synthesized four novel A2 analogues. Some of the

modiﬁcations at the carboxylic acid level might improve the A2

peptide’s stability with regard to carboxypeptidases while

others were designed to reversibly hide the C-terminal to

explore the possibility of a prodrug strategy (Scheme 1).

To study the eﬀects of C-terminal modiﬁcations of the A2

peptide, we chemically synthesized four novel analogues (A2

B-alcohol, A2 B-ester, A2 B-carbamate, and A2 B-amide)

logical control of eating and addictive disorders.

H3 relaxin is structurally similar to insulin with two chains

(

A and B) and three disulﬁde bonds. H3 relaxin structure−

activity relationship (SAR) data suggest that the B-chain of H3

relaxin is the sole determinant of RXFP3 activity. Brieﬂy, R8,

R12, I15, R16, and F20 are involved in receptor binding and

R26 and W27 are involved in receptor activation (Figure 1).

A recombinant approach has been developed for eﬃcient

preparation of H3 relaxin by the Guo group. However, the

eﬃcient production of full-length H3 relaxin by chemical

means for SAR studies remains challenging because of its

complex three disulﬁde bonded structure. Therefore, we

simpliﬁed the structure by removing the disulﬁde bond from

the A-chain and truncating 10 residues from the N-terminus of

the A-chain. This truncation study resulted in a minimized

Received: August 18, 2020

Accepted: October 20, 2020

analogue, A2. This high yielding analogue exhibits H3

XXXX American Chemical Society

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

Note

Scheme 1. Solid Phase Peptide Synthesis and Regioselective

Disulﬁde Bridge Formation to Generate A2 Analogues

(

Yields 3-15%; Table S1)

Figure 1. Human relaxin-3 representation based on the NMR data

highlighting all the key residues. Purple, A-chain; blue, B-chain

highlighting the amino acids involved in RXFP3 binding; red, the C-

terminal dipeptide important in RXFP3 activation; yellow, disulﬁde

bridges.

together with the parent A2 B-carboxylic acid (A2 B-acid) as

control (Scheme 1). For this study, two orthogonal thiol-

protecting groups (Acm and Trt) were used for each A- and B-

chains. The C-terminus of the B-chain was modiﬁed with

diﬀerent functionality while the C-terminus of the A-chain for

all analogues was achieved with a carboxylic acid.

Brieﬂy, the A-chain was generated from the preloaded Wang

resin after solid phase peptide synthesis (SPPS). The sequence

contains two Cys residues with diﬀerent protecting groups:

Cys(Trt) and Cys (Acm group). The TFA cleavage with 2,2-

dipyridyl disulﬁde (DPDS) supplement generated the N-

terminal Cys(SPy) which is ready to be conjugated with the B-

chain which contains one Cys with free thiol and one Cys with

Acm group. B-Chains with C-terminal amide and carboxylic

acid were obtained from commercially available resin after

SPPS and TFA cleavage to keep a free cysteine. Other C-

terminally modiﬁed B-chains were obtained after preparing

Fmoc-L -Trp(Boc)ol and the resins R1−3 (Supporting

Information ). Brieﬂy, the B-chain with a C-terminus alcohol

was obtained through a methodology in which the L-

tryptophanol (side chain Boc protected) is loaded onto the

Synthetic conditions: (i) 20% piperidine in DMF, 20 min, RT; (ii)

Fmoc-AA-OH, HCTU, DIEA, DMF, 1 h, RT; (iii) TFA/DODT/

TIS/H O with 10 equiv of DSPS, 2 h, RT; (iii)′ TFA/DODT/TIS/

H O, 2h, RT; (iv) PBS/ACN (80:20) at pH 7.4, 2 h, RT; (v) addition

of A-chain in 6 M GnHCl (pH 8.5) to B-chain in 6 M GnHCl (pH

.5), 15 min, RT; (vi) I , AcOH, 1 h, RT.

-chlorotrityl resin by its amino function; the acylation of the

hydroxyl group with Fmoc-L-Arg(Pbf)-OH (R1) and the SPPS

aﬀorded after TFA cleavage the iso- or depsi- peptide. Then,

an intramolecular O-to-N acyl transfer allowed the formation

of the desired product. The use of acetonitrile/PBS mixture

disulﬁde formation was performed by thioloysis and iodolysis

leading to the successful preparation of A2 analogues. The

analysis and puriﬁcation of the peptides (A-chain, B-chain and

two-chain peptides) were carried out by using reversed-phase

high performance liquid chromatography (RP-HPLC). The

peptides were identiﬁed by matrix-assisted laser desorption

ionization time-of-ﬂight mass spectrometry (MALDI-TOF

MS). The molecular weights of the ﬁnal products were as

(

20:80) was proven to be eﬃcient in order to avoid the

precipitation of the ﬁnal B-chain alcohol as previously used

with hydrophobic sequences. The C-terminus with a serinyl

ester was obtained after acylation of a supported Acetyl-L-

Serine by Fmoc-L-Trp(Boc)-OH (R2). After SPPS and TFA

cleavage, the presence of the acyl group on the serine blocks

the intramolecular O-to-N acyl transfer which consequently

leads to a stable ester bond in this regard. Lastly, the C-

terminal carbamate was generated by loading on an

unprotected Rink amide resin the carbonate obtained by

reacting Fmoc-L-Trp(Boc)ol with 4-nitrophenyl chloroformate

follows: A2 B-acid (A2) m/z 4546.541 [M + H] , calcd

4536.312; A2 B-ester m/z 4668.35 [M + H] , calcd 4662.249;

A2 B-alcohol m/z 4540.4907 [M + H] , calcd 4543.249; A2 B-

carbamate: 4569.5468 [M + H] , calcd 4563.249; A2 B-amide:

m/z 4539.4366 [M + H]+, calcd 4535.312. The purity of these

peptides (≥98%) was determined by using the peak area

integration of RP-HPLC traces. The detailed characterization

(trace of analytical HPLC and MALDI MS, purity, and yield)

0,21

(

R3).

The SPPS of the separate, selectively S-protected A-

and B-chains was followed by RP-HPLC. Subsequent stepwise

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

Note

of these analogues is provided in the Supporting Information

RXFP3 couples to the inhibitory G proteins Gi/o, the

analogues were tested for their ability to inhibit forskolin-

stimulated cAMP activation in CHO-RXFP3 cells. Interest-

ingly, unlike H3 relaxin B-chain modiﬁed peptides, while

some analogues appeared to show lower potency the diﬀerence

to the activity of A2 B-acid was not statistically signiﬁcant

binding (Figure 2 ). The parent A2 analogue (A2 B-acid) with a

Figures S1 −S6; Table S1 ).

The analogues were tested in cell-based assays for RXFP3

(

Figure 2B, Table 1). The high potency of the ester peptide

A2 B-ester) can likely be explained in terms of the prodrug

principle. The reporter gene assay used to measure cAMP

activity requires a 6 h stimulation during which we

hypothesized that the CHO-K1 cells are liberating esterases

into the media that will generate the native A2 B-acid as a

metabolite from the A2 B-ester (Figure 2B). The conversion of

A2 B-ester was conﬁrmed by MALDI TOF MS spectrometric

analysis of cell media at the end of stimulation which showed

that a high proportion of the peptide (∼30%) was converted to

A2 B-acid (Supporting Information; Figure S6). This prodrug

strategy can have useful application for generating long-lasting

molecules in which the parent drug or at least one of its

metabolites is activating RXFP3. The slightly higher potency of

the A2 B-alcohol and A2 B-ester compared to their reduced

aﬃnity is also possibly due to slight diﬀerences in the stability

of the peptides over the 6 h activation time of the reporter

assays. The A2 B-carbamate analogue demonstrated similar

potency to A2 acid, in line with its similar aﬃnity for RXFP3.

Overall, it is interesting to note that in comparison with H3

relaxin where C-terminal amide substitution resulted in 10

times lower binding and activity at RXFP3 , the modiﬁcation

at the C-terminal of the B-chain is less detrimental for A2. We

therefore also tested the peptides activity at the related RXFP4

receptor. The results reﬂected the peptides activity at RXFP3

such that all the analogues, other than the B-alcohol, showed

similar activity at RXFP4 (Figure 2C, Table 1).

It is possible that the less restrained structure of the

truncated peptide, which lacks a large portion of the chain and

one disulﬁde bond, allows A2 to have a more adaptable

binding mode. Similar eﬀects are seen for linear and

Figure 2. Competition binding (A) and cAMP activity assays of A2

and its analogues in (B) CHO-RXFP3 cells and (C) CHO-RXFP4

cells. Europium-labeled H3 B1-22R was used as the labeled ligand in

the competition binding assays.

unstructured antagonists of RXFP3. RXFP3 mutagenesis

and modeling data suggest that the aromatic ring of TrpB27

is stacked against the aromatic ring of RXFP3 Trp138 while

the C-terminal acid is in a position to form a salt bridge across

23,24

the binding pocket to the extended side chain of Lys271.

free B-chain C-terminal carboxylic acid showed higher aﬃnity

compared A2 B-amide (Figure 2A, Table 1) consistent with

our previous observation that amidated B-chain analogues of

H3 relaxin exhibited a lower binding aﬃnity for RXFP3

compared with B-chain acid analogues. However, while A2 B-

alcohol and A2 B-ester also showed lower binding aﬃnity the

aﬃnity of A2 B-carbamate was not signiﬁcantly diﬀerent to A2

B-acid. The analogues were then tested for RXFP3 potency. As

The modiﬁcations introduced here removes the negative

charge of the C-terminus but retains hydrogen bond acceptors

that may still interact with Lys271. The dual oxygens and the

ﬂexibility around the C−O−C bonds in the carbamate version

could potentially provide two hydrogen bond acceptors in the

correct arrangement for coordinating the amine group, similar

to a free carboxylic acid.

Table 1. Pooled binding aﬃnity (pK , pEC ) Data

RXFP3 in vitro assays

RXFP4

analogues

Eu-H3 B1-22R pK (n)

cAMP pEC₅₀(n)

H3 relaxin

A2: acid

7.78 ± 0.06

7.34 ± 0.14 (3)

6.73 ± 0.03 (3)**

6.96 ± 0.15 (3)

6.48 ± 0.16 (3)***

6.72 ± 0.09 (4)**

9.0 ± 0.07

8.94 ± 0.13 (4)

8.16 ± 0.18 (4)

8.15 ± 0.17 (3)

7.71 ± 0.10 (3)

6.55 ± 0.12 (3)***

7.84 ± 0.21 (3)

8.21 ± 0.08 (3)

8.36 ± 0.16 (3)

8.21 ± 0.17 (3)

7.88 ± 0.10 (3)

7.80 ± 0.13 (3)

A2: B-ester

A2: B-carbamate

A2: B-alcohol

A2: B-amide

RXFP3 and RXFP4 in vitro assays were carried out on A2 and the 4 analogues. p < 0.001. p < 0.01 vs analogue 2.

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

University of Melbourne, Parkville, Victoria 3010, Australia;

Note

Finally, we carried out circular dichroism (CD) studies to

understand the eﬀect of C-terminal modiﬁcations on their

secondary structures. We thus analyzed the secondary structure

of two analogues with reduced binding aﬃnity, A2 B-ester and

A2 B-amide and calculated the mean residual ellipticity (MRE)

3052, Australia; Department of Biochemistry and Molecular

Biology, The University of Melbourne, Parkville, Victoria 3010,

Australia; Email: bathgate@unimelb.edu.au

Mohammed Akhter Hossain − The Florey Institute of

Neuroscience and Mental Health, The University of Melbourne,

Parkville, Victoria 3052, Australia; School of Chemistry, The

orcid.org/0000-0002-9961-0006; Email: akhter.hossain@

unimelb.edu.au

(Figure 3 ). The other analogues were not tested due to

Authors

Praveen Praveen − The Florey Institute of Neuroscience and

Mental Health, The University of Melbourne, Parkville, Victoria

052, Australia

K. Johan Rosengren − School of Biomedical Sciences, The

University of Queensland, Brisbane, Queensland 4072,

Australia; orcid.org/0000-0002-5007-8434

Mengjie Liu − The Florey Institute of Neuroscience and Mental

Health, The University of Melbourne, Parkville, Victoria 3052,

Australia

John D. Wade − The Florey Institute of Neuroscience and

Figure 3. Circular dichroism spectroscopy overlapping showing that

the overall secondary structure is maintained despite the C-terminal

B-chain modiﬁcation.

Mental Health, The University of Melbourne, Parkville, Victoria

052, Australia; School of Chemistry, The University of

Melbourne, Parkville, Victoria 3010, Australia; orcid.org/

000-0002-1352-6568

insuﬃcient peptides. The CD spectra showed a typical α-

helical pattern with double minima at 208 and 222 nm. There

was no signiﬁcant alteration in the overall secondary structure

of A2 B-ester or A2 B-amide compared to A2 B-acid.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acsmedchemlett.0c00456

Author Contributions

In conclusion, we prepared four novel A2 analogues bearing

C-terminal modiﬁcations. We found that modiﬁcations at the

C-terminus of the B-chain of A2, unlike that of parent H3

relaxin, is generally tolerable for RXFP3 cAMP potency. This

result is supported by the fact that modiﬁcations did not alter

the overall structure of these peptides. The serinyl ester was

found to be as potent as the starting A2 analogue. Such results

suggest that the A2-ester converted to A2-acid during the cell

assays leading to higher potency than expected from its aﬃnity

for the receptor. This property together with the likely

improved stability in serum of this modiﬁcation could

potentially be utilized for developing A2 analogues with

longer-lasting eﬀects for animal studies.

All authors have given approval to the ﬁnal version of the

manuscript. Conceived and designed the experiments: J.T. and

M.A.H.. Performed the experiments: P.P., M.L., and J.T.

Analyzed the data: R.A.D.B. and M.A.H. Contributed

reagents/materials: J.D.W., R.A.D.B., and M.A.H. Wrote the

paper: P.P., J.T., K.J.R., J.D.W., R.A.D.B., and M.A.H.

Funding

J.D.W. and R.A.D.B. are recipients of NHMRC Research

Fellowships (1117483 and 1135837, respectively). K.J.R. and

R.A.D.B. were supported by an NHMRC Project Grant

(

1165801). Studies at the Florey Institute were supported by

the Victorian Government’s Operational Infrastructure Sup-

port Program.

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00456.

Notes

■

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

We are grateful to Tania Ferraro and Sharon Layﬁeld for

assistance with in vitro assays.

■

Description of peptide synthesis chemicals and reagents,

amino acid and resin preparations, solid phase peptide

synthesis, preparation for H3 B-chain bearing a C-

terminal alcohol, regioselective disulﬁde bond folding,

peptide characterization, in vitro assays (binding and

cAMP) (PDF)

ABBREVIATIONS

■

GPCR, G-protein coupled receptor; RXFP, relaxin family

peptide receptor; NMR, nuclear magnetic resonance spectros-

copy; SAR, structure−activity relationship; cAMP, cyclic

adenosine monophosphate; CHO, Chinese hamster ovary;

CD, circular dichroism; DIEA, diisopropylethylamine; DMF,

N,N-dimethylformamide; HCTU, (2-(6-chloro-1H-benzotria-

zole-1-yl)-1,1,3,3-tetramethylaminium hexaﬂuorophosphate;

MALDI-TOF, matrix assisted laser desorption ionization

time-of-ﬂight spectrometry; ACN, acetonitrile; AcOH, acetic

acid; MRE, mean residual ellipticity; Trt, Trityl; tBu, tertiary

AUTHOR INFORMATION

Corresponding Authors

Julien Tailhades − The Monash Biomedicine Discovery Institute,

Monash University, Clayton, Victoria 3800, Australia; EMBL

Australia, Monash University, Clayton, Victoria 3800,

Australia; orcid.org/0000-0003-0391-2526;

Email: julien.tailhades@monash.edu

■

Ross A. D. Bathgate − The Florey Institute of Neuroscience and

Mental Health, The University of Melbourne, Parkville, Victoria

butyl; TFA, triﬂuoroacetic acid; TIS, triisopropylsilane; H O,

water; DODT, 3,6-dioxa-1,8-octanedithiol; GnHCL, guanidine

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

Subra, G.; Martinez, J.; Amblard, M. Synthesis of peptide alcohols on

Note

hydrochloride; PBS, phosphate buﬀered saline; SPPS, solid

phase peptide synthesis.

flexibility of human relaxin-2. ACS Chem. Biol. 2015, 10 (3), 891−

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17) Tailhades, J.; Gidel, M.-A.; Grossi, B.; Lec

(

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