Abstract
Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the ethical guidelines, outlined in our author and reviewer resource centre, still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. Accepted Manuscript
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the author guidelines. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the ethical guidelines, outlined in our author and reviewer resource centre, still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. Accepted Manuscript rsc.li/obc Organic & Biomolecular Chemistry www.rsc.org/obc ISSN 1477-0520 COMMUNICATION Takeharu Haino et al. Solvent-induced emission of organogels based on tris(phenylisoxazolyl) benzene Volume 14 Number 1 7 January 2016 Pages 1–372 Organic & Biomolecular Chemistry View Article Online View Journal This article can be cited before page numbers have been issued, to do this please use: A. Murza, X. Sainsily, J. Cote, L. Bruneau-Cossette, É. Besserer-Offroy, J. Longpre, R. Leduc, R. Dumaine, O. Lesur, M. Auger-Messier, P. Sarret and E. Marsault, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB02247B. Structure-activity relationship of novel macrocyclic biased apelin receptor agonists Alexandre Murza,a,c,‡ Xavier Sainsily,a,c,‡ Jérôme Côté,a,c Laurent Bruneau-Cossette,a,c Élie Besserer-Offroy,a,c Jean-Michel Longpré,a,c Richard Leduc,a,c Robert Dumaine,a Olivier Lesur,b,c Mannix Auger-Messier,b Philippe Sarreta,c,$ and Éric Marsaulta,c,$,* a Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke (QC), J1H 5N4, Canada. b Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke (QC), J1H 5N4, Canada. c Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke (QC), J1H 5N4, Canada. ‡ These authors contributed equally to this work. $ These authors contributed equally to directing this study. AUTHOR INFORMATION
Corresponding Author
* Prof. Éric Marsault Phone: +1 819.821.8000 ext 72433 Fax: +1 819.564.5400 Email: eric.marsault@usherbrooke.ca
Notes
The authors declare no competing financial interest. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.
Abstract
Apelin is the endogenous ligand for the G protein-coupled receptor APJ and exerts a key role in regulating cardiovascular functions. We report herein a novel series of macrocyclic analogues of apelin-13 in which the N- and C-terminal residues as well as the macrocycle composition were chemically modified to modulate structure-activity relationships on the APJ receptor. To this end, the binding affinity and the ability to engage G protein-dependent and G protein-independent signalling pathways of the resulting analogs were assessed. In this series, the position and the nature of the Cterminal aromatic residue is a determinant for APJ interaction and β-arrestin recruitment, as previously demonstrated for linear apelin-13 derivatives. We finally discovered compounds 1, 4, 11 and 15, four potent G protein-biased apelin receptor agonists exhibiting affinity in the nanomolar range for APJ. These macrocyclic compounds represent very useful pharmacological tools to explore the therapeutic potential of the apelinergic system. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.
Introduction
The apelin receptor (APJ, APLNR, or angiotensin receptor-like 1) is a member of the class A G-protein-coupled receptor (GPCR) superfamily and shares 31% sequence identity with the angiotensin II receptor type 1 (AT1R).1 APJ was deorphanized in 1998 when its endogenous ligand, apelin, was isolated from bovine stomach extracts.2 Apelin is derived from a 77-amino acid precursor, pre-proapelin, which is converted into several isoforms after proteolysis: apelin-36, -17, -13 (or its pyroglutamate analogue Pyr1-apelin13),2–4 the latter being the major form in circulation.5 The apelinergic system has been detected in many peripheral tissues, such as the heart, kidney and pancreas, as well as the central nervous system.3,6–8 Besides several potential therapeutic indications such as diabetes, obesity, gastrointestinal diseases, cancer and HIV infection,9,10 the most notable and best characterized role of the apelinergic system remains its functions in the cardiovascular system, where apelin effects vascular, inotropic effects and impacts fluid homeostasis.11–14 Indeed, intravenous administration of apelin-13 causes a lowering of mean arterial pressure (MAP) in rodents via a nitric oxide (NO)-dependent mechanism,2,3,15 an effect that was confirmed in healthy volunteers and heart failure patients.16,17 Activation of APJ triggers intracellular G-protein-dependent signalling pathways such as inhibition of cAMP production4,6 and phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), Akt and p70S6K.18–20 Upon agonist binding, APJ also recruits both β-arrestin1 and β-arrestin2, which leads to receptor internalization via different intracellular trafficking routes depending on the agonist.21 Remarkably, a second endogenous ligand of APJ named ELABELA (ELA, also known as toddler or apela), was O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. recently discovered.22,23 Similarly to apelin, ELA activates the Gαi/o-dependent signalling pathways, recruits β-arrestins and modulates the cardiovascular system.24,25 The conformation of apelin-17 in solution was elucidated by 2D NMR and circular dichroism by Langelaan et al, identifying no α-helical or β-sheet secondary structure.26 However, the Arg2-Pro3-Arg4-Leu5 moiety of apelin-13 was reported to adopt a more ordered structure. This hypothesis is further supported by molecular modeling studies27 and alanine scan that pinpoint Arg2, Arg4 and Leu5 as key residues involved in binding to APJ and in Gαi/o-dependent signalling. 6 The C-terminal Phe residue of apelin-13 also plays a critical role for APJ interaction and activation.6,28–31 The structure-activity relationship (SAR) of apelin is the subject of a growing body of literature and was reviewed recently.32 Macrocyclic analogs of the endogenous ligands of APJ are excellent tools to better understand the SAR of the apelin/APJ complex. Indeed, to interact with its target, the ligand has to adopt a binding conformation, which may increase entropic cost. If constrained in a binding conformation via macrocyclization, the requisite rearrangement to bind to the target is lower, which would be expected to increase affinity.33 Several groups have exploited this strategy to design macrocyclic analogues of apelin-13 (Figure 1). The endocyclic position of the main pharmacophore of apelin-13, Arg2-Pro3-Arg4Leu5, was constrained in a macrocycle based on modeling studies suggesting the existence of a β-turn in this part of the peptide.27 This led to the discovery of a cyclic APJ receptor antagonist with an affinity of 93 nM.34 Likewise, using molecular dynamics simulations of apelin/APJ interactions, Brame et al recently reported a macrocyclic analogue of apelin-13, MM07, where two Cys residues were cyclized via a disulfide O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. bridge around the aforementioned pharmacophore.35 This macrocycle, described as an agonist biased toward G-protein activation versus β-arrestins recruitment, caused a dosedependent increase in cardiac output following intravenous administration to rats and raised forearm blood flow in healthy volunteers. A patent (US 2013/0196899 A1) also reports a series of cyclic apelin-13 derivatives incorporating a variety of linkers such as disulfides, esters and amides (Figure 1). In order to further investigate the SAR of the apelin-13/APJ system and based on our previous exploration of the C-terminal part of the peptide, we designed and synthesized a novel series of macrocyclic analogues of apelin-13 bridged with alkene functionalities. Binding affinity was assessed using a conventional radioligand displacement assay, whereas activation of Gαi1 and GαoA subunits and recruitment of β-arrestins1 and 2 was measured using bioluminescence resonance energy transfer (BRET) assays, in order to define their signalling profiles. Finally, the impact on blood pressure of selected macrocycles was evaluated. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.
Results and Discussion
Design and synthesis of macrocyclic analogues of apelin-13 Macrocycles were synthesized on solid phase using the Fmoc strategy (Scheme 1) and as described previously (see also Methods section).30 At the end of peptide elongation, the alkene functions of the two allylglycines were cyclized via Ring Closing Metathesis (RCM) with Hoveyda-Grubbs second generation catalyst in 1,2-dichloroethane under microwave irradiation.36–38 This reaction, when used on solid phase, is associated with pseudo-high dilution that reduces undesirable dimer formation.39 To be noted, the ratio of (E) to (Z) isomers could not be characterized. Indeed, no separation was observed on UPLC-MS or preparative HPLC, and NMR of selected compounds proved uninformative (data not shown). Final resin cleavage with concomitant side chain deprotection was performed with a mixture of trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIPS) (Scheme 1). The crude product, obtained by precipitation in tert-butyl methyl ether (TBME), was purified by reverse-phase chromatography to >95% purity as determined by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). The design of the macrocyclic analogues of Pyr1-apelin-13 (subsequently called apelin13) was based on the structure-activity relationship (SAR) described in the literature. The N-terminal residues Arg2-Pro3-Arg4-Leu5-Ser6 are important for binding to APJ.6,30 Molecular modeling studies suggest that the Ser6 side chain could form a hydrogen bond with the Leu5 backbone carbonyl to stabilize a β-turn, a conformation that putatively favours interactions with the APJ receptor.27 The C-terminal Phe13 residue of apelin-13 is also important since it can modulate affinity for APJ, recruitment of β-arrestins, and is crucial for receptor internalization.6,28,30,31 Therefore, apelin-13 appears to possess two O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. distant epitopes that constitute the core scaffold in this series of macrocycles. The Ala scan performed by Medhurst et al. showed that His7Ala and Met11Ala mutations in apelin-13 have no impact on binding and signalling.6 Based on the above observations, His7 and Met11 were replaced by allylglycine (AllylGly) residues, pivotal for RCM, thereby positioning the macrocycle between the two important epitopes of apelin-13 as described in Table 1. Finally, Pro12 was initially removed and the N-terminal Pyr1 residue was deleted considering its lack of impact for APJ interaction.6 Gratifyingly, compound 1 possessed nM affinity, with a 20-fold loss compared to apelin-13 (Ki 7.1 nM vs 0.37 nM). This represents a good starting point, confirming that the macrocycle is well positioned and its conformation allows binding to APJ. The subsequent sections describe SAR investigation around this macrocyclic template. Modifications of the exocyclic C-terminal residue The C-terminal Phe13 residue of apelin-13 has been identified as a key modulator of binding affinity as well as cAMP and β-arrestin2 signalling.29–31 To better understand the role of this residue, analogues in which C-terminus Phe was replaced by O-benzylTyrosine (Tyr(OBn)) were synthesized, since this residue was found to markedly improve binding affinity in our previous works. This residue was also replaced by Ala or deleted, providing compounds 2-4 (Table 1). Interestingly, the absence of aromatic side chain does not impact binding for APJ (3, Ki 9.6 nM vs 1, Ki 7.1 nM), which contrasts with the Phe13Ala substitution in linear apelin-13 that led to a >10-fold decrease.6 Likewise, the lack of the C-terminal residue only mildly decreased affinity (4, Ki 16 nM) suggesting that this series of macrocycles possibly interacts with the C-terminal pocket of APJ in O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. slightly different ways compared to apelin-13. The Tyr(OBn) residue was then selected to replace Phe in position 13. Indeed, in apelin-13, this modification led to an analogue with an affinity in the pM range (20 pM), a 60-fold improvement vs the native peptide.30 The higher volume and π-stacking abilities of Tyr(OBn) seem to fit well in the C-terminal orthosteric pocket of APJ, which has been suggested to be wide and rich in aromatic residues.28,40 This observation remained true in this study since compound 2 exhibits a 4- fold increase in binding affinity compared to compound 1 (2, Ki 1.7 nM) (Table 1). Tyr(OBn) was therefore kept constant for the following compounds. Influence of Tyr(OBn) positioning and introduction of spacers We next examined whether the exocyclic position of the Tyr(OBn) residue in the macrocycle is optimal for APJ interaction. Thus, compounds 5 and 6 were synthesized. In these macrocycles, Tyr(OBn) is endocyclic and positioned between the Gly and Pro residues (5) or between the Lys and Gly residues (6), with a concomitant ring size increase from 17 to 20 atoms. As shown in Table 2, these changes led respectively to a 20-fold and 400-fold loss in binding affinity compared to 2 (5, Ki 29 nM; 6, Ki 662 nM vs 2, Ki 1.7 nM). Thus, distance between the two epitopes and ring conformational rearrangements induced by the Lys-Gly-Pro moiety are better tolerated by the orthosteric binding site of the APJ receptor. Overall, in this series, keeping the Tyr(OBn) residue in exocyclic position was the best option for further SAR analysis. Although several studies strongly suggested the Arg2-Pro3-Arg4-Leu5-Ser6 residues of apelin-13 to be necessary for APJ interaction,26,27 this N-terminal portion is not sufficient to account for peptide binding. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. To further probe the C-terminal end of these new macrocyclic analogues of apelin-13, different amino acids inducing variable conformations and having different size were introduced as spacers between the ring and the Tyr(OBn) residue (Table 3). Compounds 7 and 10 displayed Pro or Ala residue as spacer, presumably imposing different backbone conformations. Surprisingly, affinity for APJ was barely influenced by these modifications compared to the parent macrocycle 2 (7, Ki 3.4 nM; 10, Ki 1.7 nM versus 2, Ki 1.7 nM). Likewise, compounds 8 (Ki 0.49 nM) and 9 (Ki 7.1 nM), bearing respectively the more flexible β-Alanine (βAla) and γ-aminobutyric acid (γAbu) spacers, exhibited binding affinity for the APJ receptor in the same range as apelin-13 (Ki 0.37 nM) and 2 (Ki 1.7 nM). Altogether, these results suggest that several solutions can be considered to position the critical C-terminal aromatic amino acid in the binding pocket of the APJ receptor.29,30,41
Modifications of endocyclic positions
The size, rigidity and nature of endocyclic amino acids are crucial determinants for the interaction with APJ in this series. Accordingly, endocyclic positions were modified as described in Table 4. The endocyclic Lys-Gly-Pro moiety was replaced by 8- aminocaprylic acid and βAla-βAla residues with Tyr(OBn) and Pro-Tyr(OBn) amino acids at the C-terminal end (compounds 11-13), in order to understand the role of individual residues in binding with the APJ receptor. These modifications are expected to both remove potential pharmacophoric elements, but also to increase ring flexibility. Compound 11, in which the Lys-Gluy-Pro residues of 2 are replaced by an 8- aminocaprylic residue, exhibited a slightly decreased affinity compared to 2 (11, Ki 11 O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. nM vs 2, Ki 1.7 nM). This is surprising considering the importance of this modification. However, compounds 12 and 13 led to a substantial drop in binding affinity (12, Ki 790 nM, 13, Ki >10000 nM) compared to that of 2 and 11. The presence of a peptide bond within the βAla-βAla fragment is expected to introduce a local geometrical constraint42 compared to 8-aminocaprylic acid, which appears to be deleterious for interactions with APJ. To assess whether the cationic side chain of the endocyclic Lys is important for receptor binding, this residue was replaced by norleucine (Nle, compound 14, Table 4). Compared to 2, 14 elicited a 30-fold loss of binding affinity (14, Ki 53 nM), suggesting that the Lys residue is important for receptor binding. One can hypothesize that the amine function is engaged in a polar interaction with APJ or could be involved in an intramolecular interaction that contributes to stabilize an active conformation. Overall, these results demonstrated that the macrocycle is of primary importance for receptor interaction in this template, and plays a pivotal role for positioning the C-terminal residue in the binding pocket of APJ, a key pharmacophore to trigger signalling. Nevertheless, the way the macrocycle docks into the receptor remains to be determined.
N-terminal SAR study
The SAR study was completed by targeting the N-terminal peptidic moiety, Arg-ProArg-Leu-Ser. APJ site-directed mutagenesis, nuclear magnetic resonance (NMR) and circular dichroïsm (CD) studies suggested that the two Arg side chains of apelin-13 could be implicated in electrostatic interactions with residues Glu20 and Asp23 of APJ.43 Furthermore, it was also speculated that the guanidino functionalities would be separated from each other by approximately 9 Å.44 Gerbier et al recently reported, using molecular O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. modeling, that residues Glu174 and Asp284 of APJ interacted with the two Arg residues of apelin.40 We were thus interested to determine if the guanidino functionality of these two residues are essential to maintain affinity for APJ. Starting from reference compound 2, Arg residues were replaced by Lys or ornithine (Orn) (15 and 16, Table 5). These modifications led to a slight decrease in receptor binding compared to 2 (15, Ki 6.9 nM, 16, Ki 7.3 nM versus 2, Ki 1.7 nM). Considering that electrostatic abilities of protonated amines are lower than that of guanidino functionalities, these results are in accordance with previous works.44 The difference of chain length between Orn and Lys seems to be neutral on APJ interactions. Furthermore, N-terminal truncation of 2, leading to analogues 17 (deletion of N-terminal Arg-Pro residues) and 18 (deletion of N-terminal Arg-Pro-Arg-Leu-Ser residues), induced a marked reduction in receptor binding, showing respectively 150-fold and close to 1000-fold lower affinities compared to 2 (17, Ki 258 nM, 18, Ki 1642 nM). Despite lower affinity for APJ, it is worth noting that analogue 18 is the first one lacking the critical N-terminal Arg-Pro-Arg-Leu binding determinant yet still able to conserve appreciable binding to the APJ receptor.30 Indeed, compound 17 with only one Arg residue maintains submicromolar affinity. Surprisingly, the macrocyclic moiety with the C-terminal Tyr(OBn) residue, analogue 18, is still able to bind to APJ, in sharp contrast with previously described N-terminal truncated analogues of apelin-13 in which the deletion of Pyr1-Arg2-Pro3-Arg4-Leu5 completely abrogated binding for APJ.30 These results further reinforce our hypothesis that this macrocyclic series of analogues interacts with the orthosteric pocket of the APJ receptor via binding sites topologically distinct compared to apelin-13. Finally, to probe the importance of the N-terminal amine function, compounds 19-21 were synthesized (Table 5). The addition O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. of a pyroglutamic acid (Pyr) residue or acetylation of the primary amine elicited a minor drop in binding affinity compared to 2 (19, Ki 6.2 nM, 20, Ki 3.4 nM). However, the desamino-Arg analogue 21 exhibited a 65-fold decrease in receptor binding (21, Ki 113 nM). Altogether, these results suggest that the N-terminal amine can be substituted or alkylated in this series of analogues, which could be useful to prevent N-exopeptidase degradation, while retaining some level of APJ interaction. Assessment of G-protein activation and β-arrestins recruitment The signalling profile of this series of macrocyclic analogues was assessed, with an emphasis on Gαi1 and GαoA dissociation and recruitment of β-arrestin1 and 2 in HEK293 cells stably expressing the human APJ receptor using Bioluminescence Resonance Energy Transfer (BRET)-based biosensors.45,46 Following apelin-13 stimulation, doseresponse curves for Gαi1, GαoA, β-arrestin1 and β-arrestin2 exhibited half-maximal responses (EC50) respectively of 1.0, 2.3, 73 and 69 nM, as represented in Figure 2. To be noted, none of the macrocyclic analogues reported herein was as potent as apelin-13. Despite a binding affinity close to that of apelin-13, reference compound 2 was associated with a substantial loss in second messenger signalling compared to apelin-13 (Gαi1 35 nM; GαoA 27 nM; β-arr1 553 nM; β-arr2 893 nM) (Table 6). The endocyclic position of the Tyr(OBn) residue, analogue 5, induced an important loss in β-arrestin recruitment (5, EC50 >10000 nM). Similarly to binding studies, in this series of macrocycles, the exocyclic configuration of the aromatic C-terminal amino acid seems to be optimal to maintain signalling profile. Introduction of spacers between the ring and the C-terminal residue also had an influence on signalling. Indeed, a slight potency O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. improvement was observed for 8 (linker: βAla) versus 2. On the other hand, a 10-fold decrease in signalling was associated with analogue 9 (linker: γAbu) compared to 8 (Gαi1 130 nM; GαoA 103 nM; β-arr1 5602 nM; β-arr2 4656 nM). This spacer impaired both binding and signalling, suggesting that the distance from the ring and orientation of the C-terminal residue are critical for both G-protein-dependent and -independent pathways. Modifications of the ring, the C- and N-terminal moiety with 1, 4, 11, and 15 trigger Gαi1 and GαoA signalling (1, Gαi1 59 nM; GαoA 189 nM, 4, Gαi1 62 nM; GαoA 64 nM, 11, Gαi1 190 nM; GαoA 174 nM and 15, Gαi1 56 nM; GαoA 50 nM), with no recruitment of βarrestins (1, 4, 11, and 15, β-arr 1 & 2 >10000 nM) (Figure 2, Table 6). Furthermore, compounds 1, 4, 11, and 15 bind to APJ in the nM range (Table 4 and 5). Therefore, these macrocyclic analogues exhibit a functional selectivity toward G-protein activation over β-arrestins signalling.47 Compounds 1, 4, 11, and 15 are the first reported apelin analogues that possess such a strong bias toward G-protein activation while maintaining an affinity close to that of apelin-13. These molecules thus represent powerful pharmacological tools to better understand the link between signalling and physiological effects, which is the object of ongoing investigation. Effects on blood pressure following bolus administration Previous studies demonstrated that apelin-13 can lower mean arterial blood pressure (MAP) in normal and spontaneously hypertensive rats when administered in bolus,25,30,48 although this was not the case with continuous infusion.49 Additionally, the C-terminal amino acid of apelin has been shown to play a critical role in lowering the mean arterial pressure (MAP).30,31,48 We thus investigated the effects of this series of macrocycles on O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. the modulation of blood pressure, by focusing on the higher affinity compounds. Those selected derivatives are represented in bold in Table 6 and their dose-response curves for each signalling pathway depicted in Figure 2. Analogues 2, 7, 8, 15, 19, 20 and apelin-13 were administered intravenously at a dose of 19.6 nmol/kg (similar dose to previous experiments) to male Sprague-Dawley rats, and their arterial blood pressure monitored via continuous intra-carotid measurements. For each compound, the max ∆MAP corresponding to the maximal drop in MAP was compared to rats treated with saline (Figure 3). Apelin-13 exerted a maximal hypotensive effect reaching -35 mmHg and analogue 8 induced a similar max ∆MAP at the equimolar dose of 19.6 nmol/kg. Compounds 2 and 19 tended to induce a less intense max ∆MAP compared to apelin-13, yet this effect was statistically significant compared to control. Interestingly, analogues 15 and 20 provoked no hypotensive effect at all following i.v. bolus administration. The rational between signalling and in vivo vascular effects for the apelinergic system are not completely understood. Indeed, previous studies suggested that the drop in MAP elicited by apelin analogues depends on the ability of those ligands to induce APJ internalization and recruit β-arrestins.15,31,48 Analogues 15 and 20, exhibiting EC50 >5000 nM on the βarrestins pathways (Table 6), induced no statistically significant hypotensive effect, in agreement with this hypothesis. However, further in-depth studies are required to fully understand this mechanism.
Conclusion
In this study, we report the design and synthesis of a novel series of macrocyclic analogues of apelin-13. We conducted a SAR investigation of these molecules and O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. evaluated their abilities to bind to the APJ receptor and activate the Gαi1 and GαoA subunits as well as to recruit β-arrestins1 and 2. Altogether, our results demonstrate that, with this macrocyclic scaffold, the C-terminal residue is crucial to modulate binding and signalling, especially for β-arrestin recruitment. Indeed, the Tyr(OBn) amino acid, when replaced by a non-aromatic residue or when positioned within the ring, markedly influences the capacity of APJ to recruit β-arrestins. This is in agreement with current knowledge generated with linear C-terminal modified apelin-13 derivatives.30,31 This study also led to the discovery of several potent G protein-biased APJ receptor agonists, particularly compounds 1, 4, 11, and 15, which exhibit binding affinity profiles in the nanomolar range. Additionally, despite its modest affinity of 1642 nM, analogue 18 is worth mentioning since it is devoid of the critical Arg-Pro-Arg-Leu binding determinant, which to our knowledge is unprecedented. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.
Experimental
Procedures for solid phase synthesis
Materials
Wang resin (4-benzyloxybenzyl alcohol resin) was purchased from ChemImpex International (USA). [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU) and Fmoc-protected (L)-amino acids were purchased from Matrix Innovation (Canada). Hoveyda-Grubbs catalyst 2nd generation was purchased from Sigma-Aldrich (Canada). All other reagents and solvents were purchased from Sigma-Aldrich (Canada), Fisher Scientific (USA) or ACP (Canada), and were of the highest commercially available chemical purity. All reagents and starting materials were used as received. Peptide synthesis was performed in 12 mL polypropylene cartridge with 20 µm PE frit from Applied Separations (USA).
Peptide synthesis
In a typical procedure, Wang resin (0.3 mmol/g, 0.3 g) was treated with triphenylphosphine (3 equiv), diisopropylazodicarboxylate (DIAD, 3 equiv) and Fmocprotected amino acid (3 equiv) in tetrahydrofuran (THF, 5 mL). The mixture was shaken overnight on an orbital shaker at room temperature (RT), then the resin was sequentially washed for 3-min periods with DCM (2x 5 mL), 2-propanol (1x 5 mL), DCM (1x 5 mL), 2-propanol (1x 5 mL), DCM (2x 5 mL). A capping solution of DCM/Ac2O/DIPEA (20/5/1, 5 mL) was then added and the mixture shaken for 1 h at RT and washed with the above solvent sequence. After Fmoc deprotection with 20% piperidine/DMF (N,N- O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. dimethylformamide) (2x 10 min), the subsequent Fmoc-protected amino acid (5 equiv) was attached in the presence of HATU (5 equiv) and DIPEA (10 equiv) in DMF (5 mL). Coupling reaction proceeded for 30 min, unless the amino acid was being added to a Pro residue, in which case reaction lasted for 60 min. Piperidine (20% in DMF) was used to deprotect the Fmoc group at every step. The resin was washed after each coupling and deprotection step with the above sequence of solvents. At the end of elongation, the resin was transferred in a microwave reactor, dried under vacuum for 2 h, flushed with Ar during 30 min. Hoveyda-Grubbs catalyst 2nd generation (0.2 equiv) was added, then the tube was flushed again with Argon for 15 min. Freshly distilled 1,2-dichloroethane (3 mL) was finally added, then the reaction was performed in a microwave apparatus (Discover Activent 10/35mL from CEM, USA) during 1 h at 77°C. The resin was then sequentially washed with DCM (3x 5 mL), MeOH (3x 5 mL) then DCM (3x 5 mL). Final resin cleavage was performed using a mixture of TFA (trifluoroacetic acid)/H2O/TIPS (triisopropylsilane), 95/2.5/2.5, v/v (4 mL / 0.3 g of resin) for 4 h at RT. After filtration, the peptide was precipitated in tert-butyl methyl ether (TBME) at 0°C, the suspension was centrifuged, the supernatant removed and the crude product re-dissolved in aqueous AcOH 10%. Purification by reverse-phase HPLC yielded the desire products, isolated as white powders after lyophilization.
Peptide purification and characterization
Crude peptides were purified by reverse-phase chromatography using a preparative HPLC from Waters (Autosampler 2707, Quaternary gradient module 2535, UV detector 2489, fraction collector WFCIII) equipped with an ACE5 C18 column (250 x 21.2 mm, 5 O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. µm spherical particle size) and water + 0.1% TFA and acetonitrile as eluents. To determine purity, analytical UPLC chromatograms were recorded on a Waters Acquity H-Class equipped with an Acquity UPLC BEH C18 column (1.7 µm particles size, 2.1 x 50 mm) using the following gradient: water + 0.1% TFA and acetonitrile (0→0.2 min: 5% acetonitrile; 0.2→1.5 min: 5%→95%; 1.5→1.8 min: 95%; 1.8 → 2.0 min: 95% → 5%; 2.0 → 2.5 min: 5%). All analogues possessed UV purity >95% and were characterized by mass spectrometry (Electrospray infusion ESI-Q-Tof from Maxis). Binding affinity and signalling pathways
Materials
High glucose Dulbecco’s Modified Eagle Medium (DMEM), G418 and penicillin/ streptomycin were purchased from Invitrogen Life Technologies (Canada). Foetal bovine serum (FBS) was purchased from Wisent (Canada) and bovine serum albumin (BSA) from BioShop (Canada). White opaque 96-well half area plates were purchased from PerkinElmer (Canada). Polyethylenimine (branched PEI) was obtained from Polysciences (USA). Coelenterazine-400A (DeepBlueC) was purchased from Biosynth AG (Switzerland). BRET2 measurements were performed on a GeniosPro plate reader from Tecan (Austria). Apelin-13[Glp65, Nle75, Tyr77][125I] (specific activity 820 Ci/mmol) was prepared using IODO-GEN (1,3,4,6-tetrachloro-3a, 6a-diphenyl-glycoluril; Thermo Scientific Pierce, Canada) as described by Fraker and Speck.50 Briefly, 10 µL of a 1 mM peptide solution was incubated with 20 µg of IODO-GEN, 80 µL of 100 mM borate buffer (pH 8.5), and 1 mCi of Na-125I for 30 min at RT, and was then purified by reversed-phase HPLC on a C18 column. The specific radioactivity of the labeled peptide O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. was determined by self-displacement and saturation-binding analysis.
Radioligand binding
HEK293 cells expressing the YFP epitope-tagged human APJ were washed once with PBS and subjected to one freeze-thaw cycle. Broken cells were then gently scraped in resuspension buffer (1 mM EDTA and 10 mM Tris-HCl, pH 7.5), centrifuged at 3500 g for 15 min at 4°C and resuspended in binding buffer (50 mM Tris-HCl buffer, pH 7.5, containing 0.2% BSA). Competitive radioligand binding experiments were performed by incubating cell membranes (15 µg) with 0.2 nM Apelin-13[Glp65, Nle75, Tyr77][125I] (820 Ci/mmol) and increasing concentrations of various analogues (10-11 to 10-5 M) for 1 h at RT in a final volume of 200 µL. Bound radioactivity was separated from free ligand by filtration through GF/C glass fiber filter plates (Millipore, Billerica, MA) pre-soaked for 1 h in PEI 0.2% at 4°C and washed 3 times with 170 µL of ice-cold binding buffer. Receptor-bound radioactivity was counted in a γ-counter 1470 Wizard2 form PerkinElmer (80% counting efficiency). Nonspecific binding was measured in the presence of 10-5 M unlabeled apelin-13 and represented less than 5% of total binding. Ki values were determined from dose-response curves as the unlabeled ligand concentration inhibiting 50% of [125I]-apelin-13 specific binding and using the Cheng-Prusoff equation.51 All binding data were calculated and plotted using GraphPad Prism 6 (La Jolla, CA) and represent the mean ± SEM of three determinations.
BRET experiments
HEK293 cells, seeded in T175 flasks, were allowed to grow in high glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 2 mM glutamine, and O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. 20 mM HEPES at 37˚C in a humidified chamber at 5% CO2. All transfections were carried out with polyethylenimine.52 After 24 h, cells were transfected with the plasmids coding for hAPJ, Gαi1-RlucII, GFP10-Gγ1, Gβ1 (from www.cDNA.org) (for BRET based Gαi1 activation assay) or hAPJ, GαoA-RlucII, GFP10-Gγ1, Gβ1 (for BRET based GαoA activation assay) or hAPJ-GFP10 and RlucII-βarrestin1 or RlucII-βarrestin2 (for BRET based β-arrestin recruitment) using polyethylenimine.30,45,46 To perform the BRET assay, cells were transferred into white 96 well plates (BD Falcon) at a concentration of 50 000 cells/well 24 h after transfection and incubated at 37˚C overnight. Cells were then washed with PBS and 90 µL of HBSS was added in each well. Then, cells were stimulated with analogues at concentrations ranging from 10-5 M to 10-11 M for 5 min at 37˚C (Gαi1 and GαoA) or for 30 min at RT (β-arrestin1 and 2). After stimulation, 5 µM of coelanterazine 400A was added to each well and the plate was read using the BRET2 filter set of a GeniosPro plate reader (Tecan, Austria). The BRET2 ratio was determined as GFP10em/RlucIIem. Data were plotted and EC50 values were determined using GraphPad Prism 6. Each data point represents the mean ± SEM of at least three different experiments each done in triplicate.
Animals
Adult male Sprague Dawley rats (Charles River Laboratories, St-Constant, Quebec, Canada) were maintained on a 12 h light/12 h dark cycle with access to food and water ad libitum. The animal experimental procedures in this study were approved by the Animal Care Committee of Université de Sherbrooke and were in accordance with policies and directives of the Canadian Council on Animal Care. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.
Hypotensive effects
Rats were anaesthetized with a mixture of ketamine/xylaxine (87 mg/kg : 13 mg/kg, i.m.) and placed in supine position on a heating pad. Mean, systolic, and diastolic arterial blood pressure, as well as heart rate, were measured through a catheter (PE 50 filled with heparinized saline) inserted in the right carotid artery and connected to a Micro-Med transducer (model TDX-300, USA) linked to a blood pressure Micro-Med analyzer (model BPA-100c). Another catheter was inserted into the left jugular vein for bolus injections (1 mL/kg, 5-10 s) of vehicle (isotonic saline), or analogues at dose of 19.6 nmol/kg. Rats were given vehicle first, then only one dose of a single analogue prior to euthanasia. For relative potency evaluation, changes in blood pressure from baseline to maximal effect post-injection in individual animals were determined. Data represents mean ± SEM of at least six different experiments. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.
Acknowledgements
Financial support from Université de Sherbrooke, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, Merck Sharpe & Dohme (donation to the Faculty of Medicine and Health Sciences of Université de Sherbrooke) and the FRQS-funded Réseau Québécois de Recherche sur le Médicament (RQRM) is acknowledged. The Institut de Pharmacologie de Sherbrooke (IPS) and MITACS are also acknowledged for scholarship grants to A.M and X.S. M.A.-M. is the recipient of a Heart and Stroke Foundation of Canada (HSFC) New Investigator award. P.S. is the recipient of the Canada Research Chair in Neurophysiopharmacology of Chronic Pain. E.M. is a member of the FRQNT-funded Proteo Network. The authors would also like to thank Prof. Michel Bouvier (Institut de Recherche en Immunologie et Cancer, Montréal, Québec, Canada) for the use of human Gαi1, GαoA, and β-arrestin biosensors. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.
Bibliographic references & notes
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Lesur, Crit. Care Med., 2016, 1. 50 P. J. Fraker and J. C. Speck, Biochem. Biophys. Res. Commun., 1978, 80, 849–857. 51 Y. Cheng and W. H. Prusoff, Biochem. Pharmacol., 1973, 22, 3099–108. 52 C. Ehrhardt, M. Schmolke, A. Matzke, A. Knoblauch, C. Will, V. Wixler and S. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Ludwig, Signal Transduct., 2006, 6, 179–184. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Scheme 1. Synthesis of macrocyclic analogues of apelin-13 O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Table 1. Modifications of the C-terminal exocyclic residue Compounds R Ring size Binding Ki (nM) a Apelin-13 0.37 ± 0.04 1 Phe 17 7.1 ± 1.8 2 Tyr(OBn) 17 1.7 ± 0.25 3 Ala 17 9.6 ± 1.1 4 -OH 17 16 ± 3.1 a Binding constants (Ki) calculated from measured radioligand binding IC50 values using the Cheng-Prusoff equation, values represent the mean ± SEM of three determinations. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Table 2. Influence of the Tyr(OBn) position within the cycle Compounds R1 R2 Ring size Binding Ki (nM) a Apelin-13 -- -- 0.37 ± 0.04 2 Lys-Gly-Pro Tyr(OBn) 17 1.7 ± 0.25 5 Lys-Gly-Tyr(OBn)-Pro -OH 20 29 ± 2.5 6 Lys-Tyr(OBn)-Gly-Pro -OH 20 662 ± 128 a Binding constants (Ki) calculated from measured radioligand binding IC50 values using the Cheng-Prusoff equation, values represent the mean ± SEM of three determinations. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Table 3. Introduction of spacers between the ring and Tyr(OBn) Compounds R Ring size Binding Ki (nM) a Apelin-13 -- 0.37 ± 0.04 2 -- 17 1.7 ± 0.25 7 Pro 17 3.4 ± 0.68 8 βAla 17 0.49 ± 0.12 9 γAbu 17 7.1 ± 1.5 10 Ala 17 3.3 ± 1.6 a Binding constants (Ki) calculated from measured radioligand binding IC50 values using the Cheng-Prusoff equation, values represent the mean ± SEM of three determinations. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Table 4. Modifications of endocyclic positions Compounds R1 R2 Ring size Binding Ki (nM) a Apelin-13 -- -- 0.37 ± 0.04 2 Lys-Gly-Pro Tyr(OBn) 17 1.7 ± 0.25 11 8-aminocaprylic Pro-Tyr(OBn) 17 11 ± 3.4 12 βAla-βAla Tyr(OBn) 16 790 ± 107 13 βAla-βAla Pro-Tyr(OBn) 16 >10000 14 Nle-Gly-Pro Tyr(OBn) 17 53 ± 8.1 a Binding constants (Ki) calculated from measured radioligand binding IC50 values using the Cheng-Prusoff equation, values represent the mean ± SEM of three determinations. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Table 5. N-terminal SAR Compounds R Ring size Binding Ki (nM) a Apelin-13 -- 0.37 ± 0.04 2 Arg-Pro-Arg-Leu-Ser 17 1.7 ± 0.25 15 Lys-Pro-Lys-Leu-Ser 17 6.9 ± 0.77 16 Orn-Pro-Orn-Leu-Ser 17 7.3 ± 3.1 17 Arg-Leu-Ser 17 258 ± 47 18 -H 17 1642 ± 797 19 Pyr-Arg-Pro-Arg-Leu-Ser 17 6.2 ± 1.3 20 Ac-Arg-Pro-Arg-Leu-Ser 17 3.4 ± 1.1 21 Desamino-Arg-Pro-Arg-Leu-Ser 17 113 ± 28 a Binding constants (Ki) calculated from measured radioligand binding IC50 values using the Cheng-Prusoff equation, values represent the mean ± SEM of three determinations. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Table 6. Profiling on Gαi1 and GαoA activation as well as β-arrestin1 and 2 recruitment Gαi1 GαoA β-arrestin1 β-arrestin2 EC50 (nM) a Apelin-13 1,0 ± 0,2 2,3 ± 0,2 73 ± 4,8 69 ± 12 1 59 ± 14 189 ± 63 >10000 >10000 2 35 ± 11 27 ± 32 553 ± 27 893 ± 262 3 7,5 ± 3,2 22 ± 11 622 ± 30 657 ± 198 4 62 ± 18 64 ± 2,2 >10000 >10000 5 82 ± 12 156 ± 23 >10000 >10000 6 - - - - 7 22 ± 7,4 30 ± 8 836 ± 132 854 ± 193 8 24 ± 8,5 21 ± 7,1 457 ± 17 524 ± 126 9 130 ± 45 146 ± 76 5602 ± 863 4656 ± 706 10 62 ± 16 95 ± 23 1399 ± 413 2279 ± 606 11 190 ± 38 174± 47 >10000 >10000 12 - - - - 13 > 10000 > 10000 >10000 >10000 14 87± 25 110 ± 22 2512 ± 781 2646 ± 334 15 56 ± 8,8 50 ± 12 >10000 >10000 16 234 ± 38 332 ± 83 >10000 >10000 17 - - - - 18 - - - - 19 33 ± 6,0 35 ± 2,5 787 ± 156 1022 ± 240 20 167 ± 73 237 ± 88 5555 ± 2555 7042 ± 90 21 191 ± 65 180 ± 26 >10000 >10000 a Concentration that produces 50% dissociation of the Gαi1 and GαoA subunits and recruitment of β-arrestin1 and 2, values represent the mean ± SEM of three determinations. Analogues in bold were tested for their impact to lower the mean arterial pressure in rats. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Figure 1. Amino acid sequences of selected macrocyclic derivatives of apelin-13.18,27,34,35 O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8. Figure 3. Maximal reduction of mean arterial pressure (Max ∆MAP) induced by apelin13 and selected macrocyclic analogues (19.6 nmol/kg i.v.) in anesthetized rats. Each bar represents the average value ± SEM obtained with 5-16 animals. Statistical analyses were performed with a one-way ANOVA followed by a Dunnett's multiple comparisons test. * <0.05, ** p <0.005 and **** p <0.0001 vs Saline. O rg an ic & B io m ol ec ul ar C he m is tr y A cc ep te d M an us cr ip t Pu bl is he d on 2 9 N ov em be r 20 16 . D ow nl oa de d by U ni ve rs ity o f C al if or ni a - Sa n D ie go o n 07 /1 2/ 20 16 0 6: 43 :5 8.