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Development of a single-chain peptide agonist of the relaxin-3 receptor using hydrocarbon stapling Keiko Hojo, Mohammed Akhter Hossain, Julien Tailhades, Fazel Shabanpoor, Lilian L.L. Wong, Emma E.K. Ong-Palsson, Hanna E. Kastman, Sherie Kieu-Y Ma, Andrew Lawrence Gundlach, K. Johan Rosengren, John D. Wade, and Ross A. D. Bathgate J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00265 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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Journal of Medicinal Chemistry

Development of a single-chain peptide agonist of the relaxin-3 receptor using hydrocarbon stapling. Keiko Hojo,†* M. Akhter Hossain,#ф* Julien Tailhades,# Fazel Shabanpoor,# ф Lilian L.L. Wong,# Emma E.K. Ong-Pålsson,# Hanna E. Kastman,# Sherie Ma,# Andrew L. Gundlach,#,£ K. Johan Rosengren,¥ John D. Wade,#ф* and Ross A.D. Bathgate#‡*. †

Faculty of Pharmaceutical Sciences and Cooperative Research Center of Life Sciences, Kobe Gakuin

University, Chuo-ku, Kobe, 650-8586, Japan. #

Florey Institute of Neuroscience and Mental Health and Florey Department of Neuroscience and Mental Health, University of Melbourne, Victoria 3052, Australia. ф

£

¥

‡

School of Chemistry, University of Melbourne, Victoria 3052, Australia.

Department of Anatomy and Neuroscience, University of Melbourne, Victoria 3052, Australia.

The University of Queensland, School of Biomedical Sciences, Brisbane, Queensland 4072, Australia

Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3052, Australia

KEYWORDS. H3 relaxin, human relaxin-3; H2 relaxin, human relaxin-2; RXFP; relaxin family peptide (receptor); GPCR, G protein-coupled receptor.

ABSTRACT

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G proteincoupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with onACS Paragon Plus Environment


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resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, αalkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain αhelical peptide agonist which will facilitate study of the function of relaxin-3.

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INTRODUCTION Relaxin-3 (also known as insulin-like peptide 7, INSL7) is a member of the insulin-relaxin family of peptides that was discovered in 2002.1 The human relaxin peptide sub-family consists of seven members, relaxin 1-3 and insulin like peptide (INSL) 3-6, that each have the same structural features as insulin, viz., the A- and B-chains are cross-braced by one intra-chain disulfide bond and two inter-chain disulfide bonds. Despite their structural similarity, they each possess different tissue expression profiles and distinct physiological roles (reviewed in; 2). Relaxin-3 is highly conserved among species from fish to humans1,3, 4 and is predominantly expressed in the brain with the most prominent expression in mammals in the nucleus incertus and other smaller populations in the brainstem.1, 5, 6 The endogenous receptor for relaxin-3 is the class A G protein-coupled receptor (GPCR) 135,7 which is classified by IUPHAR as RXFP3.8 Relaxin-3 neurons project to a number of brain regions where the peptide is localized in presynaptic vesicles of nerve terminals innervating RXFP3-positive neurons.9 Evidence has emerged that relaxin-3/RXFP3 signaling has a modulatory role in arousal, feeding, stress response and cognition and is thus a therapeutic target for psychiatric disorders, such as anxiety and depression.10 However in the brain, relaxin-3 is also able to bind to RXFP1, the cognate receptor for relaxin-2.2 This cross-reactivity complicates both in vitro and in vivo pharmacological studies and thus, the development of ligands that specifically activate RXFP3 but not RXFP1 is highly desirable for further exploration of the biological roles and therapeutic potential of the RXFP3 signaling system. The tertiary structure of human relaxin-3 (H3 relaxin) has been determined by solution NMR spectroscopy (Figure 1a).11 It has a core insulin-like structure that is similar to other relaxin family members.12, 13,14 The relaxin-3 A-chain forms two helical segments in an antiparallel position, and the middle segment of B-chain forms a central α-helix that is arranged perpendicular to the A-chain helices. The A-chain provides a scaffold for maintaining the helical structure within the B-chain. Previously a chimeric peptide consisting of INSL5 A-chain and relaxin-3 B-chain (R3/I5) was reported to be a ACS Paragon Plus Environment


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selective agonist for RXFP3 over RXFP1.15 Later studies demonstrated that a minimized analogue of relaxin-3 (Analogue 2, A2), which is a truncated variant of relaxin-3 lacking the intra-A-chain disulfide bond, is devoid of RXFP1 activity but retains full agonist activity at RXFP3 both in vitro and in vivo.16 Studies of H3 relaxin peptide mutants17 together with complementary studies on RXFP3 receptor mutants and modeling of the H3 relaxin/RXFP3 complex18, 19 have shown that the primary binding site is located within the surface of the helical domain in the B-chain (Figure 1b). The single B-chain alone has been shown to bind to RXFP3 albeit with considerably lower affinity and potency compared to native H3 relaxin.7 Although the α-helix is a predominant protein secondary structure that is an important recognition motif for protein-protein interaction, the single B-chain alone does not adopt an α-helical conformation.20 Consequently, the low affinity of the peptide was thought to be due to the lack of this secondary α-helical structure that, natively, is maintained by inter-molecular disulfide bridges with the A-chain.11 We have previously attempted to develop single B-chain analogues of H3 relaxin in which their native α-helix conformation is induced and maintained by the use of macrocyclization (“stapling”) techniques.16 Such techniques have been employed for numerous peptides to induce an α-helical conformation although the success of each stapling technique is highly sequence dependent (reviewed in; 21-23). More recently such techniques have been used to successfully develop potent stapled peptide agonists of peptide GPCRs.24-27 We previously utilized judiciously placed salt bridges, and single and bis-cystine bridges16 as potential means of stabilizing the bioactive conformations of the H3 relaxin Bchain. Unfortunately these approaches were not successful in that there was no improvement in the αhelical structure of the H3 relaxin B-chain analogs which we have clearly demonstrated is essential for RXFP3 binding affinity and activity.16


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Figure 1. (a) NMR solution structure of H3 relaxin. (b) Amino acid sequence of H3 relaxin highlighting residues involved in binding (blue) and activation (red). In the current studies, we instead examined the utility of hydrocarbon stapling, which has gained increasing popularity given the development of improved ring closure metathesis methods and the recognition of its effectiveness in helical induction.28-30 Additional stapling chemistries were also assessed for comparison. Notably, the successful production of an active simplified RXFP3-selective analogue will provide an important molecular probe for exploring relaxin-3/RXFP3 function in brain as well as a potential lead for the treatment of affective and cognitive disorders. RESULTS Design and synthesis of stapled peptides: A number of different chemical staples including, lactam, disulfide, thioether and “click chemistry” bonds have been successfully utilized for inducing helical structures in short peptides.23 Recently incorporation of a pair of stereochemically-constrained amino acid units, typically α-methyl, α-alkenyl amino acids, followed by their hydrocarbon ‘stapling’ has been shown to be effective.28, 30, 31 The staple is introduced to create approximately one (i and i+3 or i,i+4), ACS Paragon Plus Environment


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two (i,i+7), or three turns (i,i+11) of the helix, following ruthenium catalyzed ring-closing metathesis (RCM).21, 31 These types of cross links have the added advantage that in addition to locking in helical turns the α-methyl group puts steric constraints on the amino acid backbone that favors helical φ and ψ angles. Based on the positions of the key binding residues in the H3 relaxin B-chain from the NMR solution structure of H3 relaxin, we predicted that positions GluB13, AlaB17 and ThrB21 within the human relaxin-3 B-chain to be the best stapling points, as these residues are positioned on the opposite face of the helix relative to the RXFP3 interacting residues. Table 1. Stapled H3 relaxin peptides analogues synthesized and characterized in this study compared to the synthetic H3 relaxin B-chain. Peptide

Name

Sequence

H3 B-chain

H3 B1-27 (C10/22S)

RAAPYGVRLSGREFIRAVIFTSGGSRW

1

H3 Ac-B6-27 (13/17 HCa)

Ac-GVRLSGR-S5FIRS5-VIFTSGGSRW

2

H3 B6-27 (13/17 HC)

GVRLSGR-S5FIRS5-VIFTSGGSRW

3

H3 Ac-B8-27 (13/17 HC)

Ac-RLSGR-S5FIRS5-VIFTSGGSRW

4

H3 B8-27 (13/17 HC)

RLSGR-S5FIRS5-VIFTSGGSRW

5

H3 Ac-B10-27 (13/17 HC)

Ac-SGR-S5FIRS5-VIFTSGGSRW

6

H3 B10-27 (13/17 HC)

SGR-S5FIRS5-VIFTSGGSRW

7

H3 Ac-B8-27 (17/21 HC)

Ac-RLSGREFIR-S5VIFS5-SGGSRW

8

H3 B8-27 (17/21 HC)

RLSGREFIR-S5VIFS5-SGGSRW

9

H3 Ac-B8-27 (15/19 HC)

Ac-RLSGREF-S5RAVS5-FTSGGSRW

10

H3 Ac-B6-27 (13/17 SSb)

Ac-SGR-homoCFIRhomoC-VIFTSGGSRW

11

H3 Ac-B6-27 (13/17 Lc)

Ac-SGR-lactam[EFIRK]-VIFTSGGSRW

12

EK linear H3 Ac-B6-27

Ac-SGREFIRKVIFTSGGSRW

13

H3 Ac-B6-27 (C10/22S)

Ac-SGREFIRAVIFTSGGSRW

A2

Analog 2

RAAPYGVRLCGREFIRAVIFTCGGSRW CKWGASKSEISSLC

a

c

HC- Hydrocarbon staple (S5: (S)-2-(4-pentenyl) alanine); bSS- homocystine staple (homoC);

L- lactam staple. ACS Paragon Plus Environment

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Table 1 lists our designed stapled peptides. As a previous truncation study has indicated that the N-terminal part of the B-chain is largely dispensable for binding,16 variants with different degree of truncation were designed to find a minimal active structure. Peptides 1-6 that contain cross-links between positions GluB13 and AlaB17, and peptides 7 and 8 have the cross-link position approximately one helical turn towards the C-terminus between AlaB17and ThrB21. In peptide 9 the cross-link was introduced in place of IleB15 and IleB19. Although structure activity data has highlighted that IleB15 is a strong contributor to receptor binding and IleB19 is also proximal to the binding site, introduction of additional hydrophobicity in the form of the hydrocarbon linker at these sites could potentially create additional favorable contacts. For direct comparison with different stapling strategies we also designed the disulfide and lactam stapled peptides 10 and 11 that have an 8-atom macrocyclic bridge across the residue 13-17 helical turn but lack the α-methyl substitutions. Finally two linear forms of peptide 5, peptide 12 with an AlaB17Lys substitution to create a potential salt bridge with GluB13 and peptide 13 the native form of peptide 5. All peptides listed in Table 1 were readily synthesized by solid-phase peptide synthesis using Fmoc chemistry. RCM and lactam bond formation reactions were carried out on the solid support. Disulfide bond formation was by 2,2’-dipyridyl sulfide. After purification by preparative RP-HPLC, chemical characterization by analytical RP-HPLC and MS (Supplementary Table 1; Supplementary Figure 2) followed by quantification by amino acid analysis, the stapled peptides were used for bioassay. In vitro activity of 13/17 hydrocarbon stapled peptides: Peptides 1-6 were tested for their ability to bind and activate RXFP3. All of the peptides demonstrated markedly improved binding affinity compared to the H3 relaxin B-chain (Figure 2a, Table 2). This was accompanied by a significant increase in the ability of the peptides to inhibit forskolin induced cAMP responses in


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RXFP3 cells (Figure 2c, Table 2). The activities of all the peptide variants were equivalent including the shortest version, peptide 5, and were only slightly lower than native H3 relaxin. Although peptide 1 had slightly lower activity than the other peptides there was insufficient material to test it more than two times. Importantly the data clearly demonstrates there are no activity differences between acetylated or non-acetylated peptides. Peptides 2-6 were then tested for their ability to activate the related receptor RXFP4.

Figure 2. In vitro activity of 13/17 stapled peptides. a) Competition binding using Eu-labeled H3/I5 and c) cAMP inhibition activity of peptides in CHO-K1-RXFP3 cells. b) Competition binding using Eu-labeled INSL5 and d) cAMP inhibition activity of peptides in CHO-K1-RXFP4 cells.


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There was insufficient peptide 2 or peptide 4 to fully complete the binding studies and none of peptide 1 for RXFP4 testing. As shown in Figure 2 both H3 relaxin and H3 relaxin B-chain can bind to and activate RXFP4 with the H3 relaxin activity being slightly higher than the native peptide INSL5.

Figure 3. In vitro activity of peptides with alternative hydrocarbon stapling points. a) Competition binding using Eu-labeled H3/I5 and c) cAMP inhibition activity of peptides in CHO-K1-RXFP3 cells. b) Competition binding using Eu-labeled INSL5 and d) cAMP inhibition activity of peptides in CHO-K1-RXFP4 cells. Peptides 3, 4 and 6 all demonstrated significantly improved binding compared to the H3 relaxin B-chain with no significant differences in binding affinity (Figure 2b, Table 2). Importantly the increases in binding affinity were modest in comparison to their binding to


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RXFP3. These binding changes were similar to modest increases in activity seen in cAMP activity assays in CHO-K1-RXFP4 cells (Figure 2d, Table 2). Peptides 2-6 all demonstrated significant but modest improvements in activity compared to H3 relaxin B-chain. In vitro activity of peptides with alternative stapling points: We then tested the activity of peptides with alternative staple positions to ascertain if alternative hydrocarbon stapling would also be effective in increasing peptide activity. Peptides 7 and 8 with a 17/21 staple and peptide 9 with a 15/19 staple were compared with peptide 5 the shortest active peptide with the 13/17 staple. Interestingly peptides 7 and 8 bound to RXFP3 with a similar affinity to the H3 relaxin Bchain (Figure 3a, Table 2). Unfortunately there was not sufficient peptide 9 for testing of binding activity. Most importantly peptides 7 and 8 demonstrated very poor activity, whereas peptide 9 had no activity, in cAMP inhibition assays in RXFP3 cells (Figure 3c, Table 2). The peptides demonstrated distinct differences in binding and activity in RXPF4 cells. Peptides 7 and 8 did not demonstrate any binding in the competition assays but displayed weak cAMP inhibitory activity (Figure 3b,d, Table 2). However peptide 9 demonstrated similar affinity to the H3 relaxin B-chain but significantly higher potency than this peptide in cAMP inhibition assays (Figure 3b,d, Table 2). The activity of peptide 9 was still significantly lower than peptide 5 (Table 2). In vitro activity of non-hydrocarbon 13/17 stapled peptides and linear control peptides: We next tested the activity of peptides with alternative chemical 13/17 staples in parallel with linear non-stapled versions of peptide 5. Importantly, peptide 13 the linear version of peptide 5 and peptide 12 a version of peptide 13 with a potential salt bridge in the 13/17 position, demonstrated very poor binding affinity at RXFP3 (Figure 4a) and significantly lower activity than the H3 relaxin B-chain (Figure 4c, Table 2). Additionally they demonstrated poor binding and activity at


10

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RXFP4 demonstrated that in the absence of the 13/17 staple they have similar or lower activity than the H3 relaxin B-chain (Figure 4b,d, Table 2). We tested two alternative chemical staples in the 13/17 position, peptide 10 with a homo-cystine linkage and peptide 11 with a lactam linkage. Both peptides demonstrated similar binding affinities to the H3 relaxin B-chain and linear peptides at RXFP3 (Figure 4a, Table 2). However while the lactam showed similar activity to the H3 relaxin B-chain peptide the activity of the homo-cystine variant was equivalent to the linear peptides. Interestingly, while both peptides displayed minimal binding to RXFP4, they did show differences in activity (Figure 4b,d, Table 2). The data clearly demonstrates that the homocystine and lactam staples at 13/17 are not able to mimic the improved activity seen with the hydrocarbon staple. The homo-cystine variant showed similar activity to the linear peptides, but the lactam variant demonstrated significantly higher potency than the H3 relaxin B-chain in cAMP inhibition assays (Figure 4b,d, Table 2). The activity of the lactam peptide was still significantly lower than peptide 5 (Table 2).

Activity of peptides at the RXFP1 receptor: The peptides were also tested for their ability to activate the RXFP1 receptor. All of the peptides demonstrated either no activity at concentrations up to 10 µM or slight activity only at 10 µM, similar to the H2 relaxin B-chain peptide (Table 2; Supplementary Figure 1). As peptides 1-6 all showed similar activities on RXFP receptors the shortest variant peptide 5 was chosen for more detailed characterization.


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Figure 4. In vitro activity of peptides with alternative 13/17 staples. a) Competition binding using Eu-labeled H3/I5 and c) cAMP inhibition activity of peptides in CHO-K1-RXFP3 cells. b) Competition binding using Eu-labeled INSL5 and d) cAMP inhibition activity of peptides in CHO-K1-RXFP4 cells.

(ERK)1/2 phosphorylation: The ability of peptide 5 to activate ERK1/2 kinase phosphorylation in CHO-K1-RXFP3 cells was determined in comparison to H3 relaxin. Both peptide 5 and H3 relaxin induced ERK1/2 phosphorylation in a concentration dependent manner and there was no difference in the potency of peptide 5 in comparison to H3 relaxin [pEC50 values of 9.92 ± 0.10 (n = 3) and 9.83 ± 0.10 (n = 3), respectively (Figure 5a)].


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Table 2. Pooled binding affinity (pKi) and functional in vitro activity (pEC50) data for relaxin3 analogs RXFP3 Ligand

Eu-H3/I5 pKi

RXFP4 cAMP pEC50

Eu-INSL5 pKi

ф

RXFP1 cAMP pEC50

cAMP pEC50

INSL5

-

No activity

8.39 ± 0.14 (3)

8.67 ± 0.08 (5)

No activityϷ

H3 relaxin

7.73 ± 0.04 (3)#

9.08 ± 0.07 (5)#

8.64 ± 0.15 (3)#

8.94 ± 0.13 (4)#

9.36 ± 0.22 (4)

H3 B-chain

5.53 ± 0.09 (3)***

5.93 ± 0.02 (3)***

5.76 ± 0.38 (4)***

5.77 ± 0.15 (3) +,***

0.3Å. CD Spectroscopy: CD spectra were collected on a Jasco J-800 spectropolarimeter at 25℃ in 1 nm increments. Samples were prepared in 10 mM sodium phosphate buffer (pH 7.4) containing 6% CH3CN to give a total peptide concentration of 10.97 µM.


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ANCILLARY INFORMATION ASSOCIATED CONTENT Supporting Information. Supplementary Figure 1, 2 and supplementary Table 1 are available as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors* Ross Bathgate, e-mail: [email protected], phone: +61 3 90356735 Keiko Hojo, e-mail: [email protected], phone: +81 78 9741551 Akhter Hossain, e-mail: [email protected], phone: +81 3 83440414 John Wade, e-mail: [email protected], phone: +61 402 200980 ABBREVIATIONS USED: RXFP3; Relaxin family peptide 3, GPCR; G protein-coupled receptor, INSL; insulin like peptide, IUPHAR; International Union of Pharmacology, A2; Analogue 2, H3; human 3, H2; human 2, RCM; olefin metathesis, DCE; 1,2-dichloroethane, RP-HPLC; Reverse Phase - High performance liquid chromatography, TFA; trifluoroacetic acid, CHO-K1; Chinese hamster ovary cells, HEK; Human embryonic kidney fibroblast cells, Eu; Europium, ANOVA; Analysis of variance, ERK; extracellular signal-regulated kinase, icv; intracerebroventricular, R3/I5; relaxin3 B-chain/INSL5 A-chain peptide, NMR; Nuclear magnetic resonance, HSQC; Heteronuclear single quantum coherence, cAMP, Cyclic adenosine monophosphate


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ACKNOWLEDGMENT (This research was partly funded by NHMRC (Australia) project grants (508995) to JDW and RADB, and (1065481 and 1066369) to RADB, KJR and ALG. This research was also funded by The Naito Foundation (Japan) Subsidy for Female Researchers to KH. We are grateful to Tania Ferraro and Sharon Layfield for assistance with cell-based assays and to Feng Lin for amino acid analysis. We thank Prof Andrea Robinson and Dr Alessia Belgi (Monash University, Australia) for assistance with the RCM reactions. During these studies, MAH was the recipient of a Florey Foundation Fellowship. ALG and RADB are NHMRC Senior Research Fellows, and JDW is an NHMRC Principal Research Fellow. KJR is an Australian Research Council Future Fellow. Studies at the Florey were supported by the Victorian Government's Operational Infrastructure Support Program. REFERENCES 1.

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