Design, Synthesis, Molecular Dynamics Simulation and Functional

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Design, Synthesis, Molecular Dynamics Simulation and Functional Evaluation of a Novel Series of 26RFa Peptide Analogues Containing a Mono- or Polyalkyl Guanidino Arginine Derivative Karima Alim, Benjamin Lefranc, Jana Sopková-de Oliveira Santos, Christophe Dubessy, Marie Picot, Jean A Boutin, Hubert Vaudry, Nicolas Chartrel, David Vaudry, Julien Chuquet, and Jérôme Leprince J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01332 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Journal of Medicinal Chemistry

jm-2018-013325 3rd revised version

Design, Synthesis, Molecular Dynamics Simulation and Functional Evaluation of a Novel Series of 26RFa Peptide Analogues Containing a Mono- or Polyalkyl Guanidino Arginine Derivative

Karima Alim,1 Benjamin Lefranc,1,2 Jana Sopkova-de Oliveira Santos,3 Christophe Dubessy,1,2 Marie Picot,1 Jean A. Boutin,4 Hubert Vaudry,1,2 Nicolas Chartrel,1 David Vaudry,1,2 Julien Chuquet,1 and Jérôme Leprince1,2,* 1INSERM

U1239, Laboratory of Neuronal and Neuroendocrine Differentiation and

Communication, Normandy University, 76000 Rouen, France 2Cell

imaging platform of Normandy, Normandy University, 76000 Rouen, France

3CERMN, 4Institut

Normandy University, 14000 Caen, France

de Recherches Internationales Servier, 50 rue Carnot, 92150 Suresnes, France

ABSTRACT 26RFa, the endogenous QRFPR ligand, is implicated in several physiological and pathological conditions such as the regulation of glucose homeostasis and bone mineralization; hence QRFPR ligands display therapeutic potential. At the molecular level, functional interaction occurs between residues Arg25 of 26RFa and Gln125 of QRFPR. We have designed 26RFa(20-26) analogues incorporating arginine derivatives modified by alkylated substituents. We found that the Arg25 side-chain length was necessary to retain the activity of 26RFa(20-26) and that Nmonoalkylation of arginine was accommodated by the QRFPR active site. In particular, [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) appeared to be 25-fold more potent than 26RFa(20-26) and displayed a position in a QRFPR homology model slightly different to that of the unmodified heptapeptide. Other peptides were less potent than 26RFa(20-26), exhibited partial agonistic activity or were totally inactive in accordance to different ligand-bound structures. In

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vivo, [(Me)ωArg25]26RFa(20-26) exerted a delayed 26RFa-like hypoglycemic effect. Finally, Nmethyl substituted arginine-containing peptides represent lead compounds for further development of QRFPR agonists.

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INTRODUCTION In vertebrates, the term RFamide-related peptide (RFRP) designates a family of biologically active peptides that possess the Arg-Phe-NH2 (RFamide) motif at their C-terminal extremity. Since the discovery of 26RFa in 2003 no novel RFRP has supplemented this family of regulatory peptides.1-3 Indeed, data mining in the mammalian genome databases using the RFGK or RFGR motif as queries failed, suggesting that the RFRP family is complete and composed, in human, by neuropeptide AF/neuropeptide FF (NPAF/NPFF), prolactin-releasing peptide (PrRP), RFRP1(GnIH)/RFRP-3, metastin/kisspeptin and 26RFa/QRFP peptides.4 The cDNA encoding the 26RFa precursor has been cloned in mammals including human,1,2 bovine,2 mouse2,3 and rat,1,2 as well as in birds5,6 and fish.7,8 Analysis of the human 26RFa precursor indicates that prepro26RFa may generate several additional peptides notably an N-terminally extended form, 43RFa, now known as QRFP9 and a truncated form, 26RFa(20-26), that is strongly conserved across vertebrate species, although it has never been chemically isolated so far. In the human hypothalamus and spinal cord, processing of the precursor generates both 26RFa and QRFP,10 while in the rat and chicken brain, the mature forms are the 43-residue long QRFP11 and the 26-residue long 26RFa5 peptides, respectively. 26RFa and QRFP are the cognate ligands of the former human orphan receptor GPR103, also designed SP9155 or AQ272,3 and now renamed QRFPR.9,12 QRFPR is similarly activated by both 26RFa and QRFP, leading to a dose-dependent increase in cAMP formation in cultured rat anterior pituitary cells1,2 and an increase in intracellular calcium concentration ([Ca2+]i) in CHO-G16 cells transfected by the human receptor.2,13,14 In the central nervous system (CNS), 26RFa/QRFP mRNA is expressed in discrete hypothalamic nuclei in rat,1-3 mouse11 and human.10 The prepropeptide gene is also expressed in human endocrine glands particularly in the pituitary and prostate.3 In human, the highest expression of QRFPR is observed in the cerebral cortex, hypothalamus and vestibular nuclei3 whereas, in peripheral organs, the receptor gene is highly expressed in the retina, pituitary, heart, kidney, testis and bone.2,15,16 Tissue distribution of 26RFa and QRFPR is consistent with the involvement of this peptide system in several physiological and pathophysiological processes17,18 such as regulation of energy homeostasis18,19 and bone mineralization.16 26RFa also stimulates the hypothalamo-pituitary gonadal axis,2,7,20-23 increases locomotor activity24 3 ACS Paragon Plus Environment


and modulates analgesia25-27 and glucose-evoked insulin secretion19,28,29 suggesting that QRFPR ligands should be amenable to drug development.9 Although less potent than 26RFa in activating QRFPR,13 the C-terminal heptapeptide 26RFa(2026)

(GGFSFRF-NH2) mimics the orexigenic and gonadotropic effects of 26RFa.20,24 Structure-

activity relationship studies reveal that replacement of the Ser23 residue by a norvaline leads to an analogue, [Nva23]26RFa(20-26) (LV-2073), that is 3 times more potent than the native heptapeptide.13 Further studies have led to the design of [Cmpi21, aza-β3-Hht23]26RFa(21-26) (LV2172), which is more potent than 26RFa(20-26) in mobilizing [Ca2+]i and more stable in serum.14 This pseudopeptide also exerts a long-lasting orexigenic effect in mice.14 Similarly, the Nterminal degradation-sensitive Gly-Gly peptide bond has been replaced by a fluoro-olefin moiety that exhibits isosteric and isoelectronic properties similar to those of the native amide bond.30 The resulting [Ψ20,21(CF=CH)]26RFa(20-26) compound (LV-2098) displays a five-fold longer half-life in human serum and an activity similar to that of the parent 26RFa(20-26) peptide.30 Structure of G-protein-coupled receptors (GPCRs) and mechanisms controlling ligand/receptor binding are required for rational drug design. Using the X-ray structure of the β2-adrenergic receptor as a template, we have recently built a 3D molecular homology model of human QRFPR in which the bioactive peptide 26RFa(19-26) has been docked.31 A strong intermolecular interaction has been predicted between the guanidino group of the Arg25 residue of 26RFa and the Gln125 residue side-chain of QRFPR that was subsequently validated by QRFPR site-directed mutagenesis as a pivotal interaction exploitable to the development of QRFPR antagonists.31 In fact, asymmetric dimethylation of the side-chain of arginine led to a first QRFPR peptide antagonist, [(Me,Me)ω,ωArg25]26RFa(20-26) (LV-2185), able to significantly reduce the 26RFa-evoked [Ca2+]i increase in CHO cells transfected by the human QRFPR.31 The arginine residue of the RFamide motif plays a critical role in the biological activities of RFRPs (for review, see4). For instance, the positively charged arginine of FMRF-NH2 is an essential residue for regulating access to the modulatory site of acid-sensing ionic channels.32 In mammals, substitution of the arginine moiety by an alanine dramatically reduces the agonistic activity of 26RFa(20-26) and kisspeptin-10.13,33 To go further in the design of potent and low molecular weight peptide ligands of QRFPR, we have designed and synthesized a new series of 4 ACS Paragon Plus Environment

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Arg25-modified 26RFa(20-26) analogues and evaluated the impact of these modifications on the activation of QRFPR by assessing the Ca2+-mobilizing activity of the compounds in stably transfected human QRFPR CHO cells. 26RFa(20-26) and three analogues distinguished by their pharmacological profiles have been selected for molecular dynamics (MD) simulation studies in the human QRFPR homology model, and the most promising compound has been evaluated in vivo in a glucose homeostasis paradigm.



RESULTS AND DISCUSSION We have previously demonstrated that the guanidino function of the arginine 25 residue of 26RFa(19-26) establishes a strong interaction with the lateral carbonyl group of glutamine 125 of QRFPR that contributes to the receptor activation process.31 Indeed, impairing this ligand/receptor contact by gem-dimethylation of the Nω-guanidino group of Arg25 in 26RFa(2026) yields to an antagonist of QRFPR.

31 In order to develop more potent QRFPR ligands, we have

designed a series of mono- or polyalkyl-guanidino arginine derivatives of 26RFa(20-26) as well as of their homo- and norarginine counterparts (5a-14c, Table 1). All peptides were synthesized by standard Fmoc-based solid phase methodology as previously described.34 Since Fmoc derivatives of arginine, norarginine (Nar), homoarginine (Har), Nωmethyl-arginine, Nω,Nω-dimethyl-arginine, and Nω,Nω’-dimethyl-arginine are commercially available, they were directly introduced in the synthetic route yielding 26RFa(20-26) (4b, LV2021), [Nar25]26RFa(20-26) (4a, LV-2290), [Har25]26RFa(20-26) (4c, LV-2291), [(Me)ωArg25]26RFa(2026) (5b, LV-2186), [(Me)

ωArg25]26RFa (16, LV-2294), [(Me,Me)ω,ωArg25]26RFa (20-26) (6b, LV-2185),

and [(Me,Me)ω,ω’Arg25]26RFa(20-26) (7b, LV-2199), respectively (Supplemental Fig. S1-S3, S5, S8, S11, S34). Other analogues were obtained by the efficient strategy developed by Hamzé et al.35 as described in Scheme 1, which gives access to Nω-mono, Nω,Nω-di, Nω,Nω’-di or Nω,Nω,Nω’trialkylated Nar, Arg, or Har containing peptides by reaction of resin-bound S-methylnorisothiocitrulline, -isothiocitrulline or -homoisothiocitrulline moieties or their Nω-methyl counterparts with a series of primary or secondary amines (Supplemental Fig. S4, S6-S7, S9S10, S12-S33). Briefly, three precursors, protected with a Boc group at their N-terminal extremity and containing, in position 25, a 2,4-diaminobutyric acid (Dab, 1a), an ornithine (Orn, 1b) or a lysine (1c) residue, orthogonaly protected by an allyloxycarbonyl (Alloc) moiety, were first assembled on solid support (Scheme 1). After selective deprotection of the free lateral amines, reaction of 1 with Fmoc-isothiocyanate (Fmoc-NCS) followed by Fmoc removal, led to the corresponding grafted northio- (2a), thio- (2b) and homothiocitrulline (2c) derivatives, while treatment with methyl-isothiocyanate led to Nω-methyl-northio (2a’), Nω-methyl-thio (2b') and Nω-methyl-homothiocitrulline (2c') containing peptides, respectively (Scheme 1). As


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previously described, after piperidine-assisted Fmoc deprotection, resin beads were found to be negative to ninhydrin reaction indicating that carbamate formation did not occur from a nucleophile attack of the side-chain amino group on the carbonyl moiety of Fmoc-NCS.35,36 In contrast to Fmoc-NCS, three successive additions of methyl-isothiocyanate were needed to obtain a total conversion of the free amines into Nω-methyl-thiourea moieties, as determined by a ninhydrin-based colorimetric assay. This can be ascribed to better reactivity of the isothiocyanate due to the presence of the carbamate function that increases its electrophilic character. After S-methylation of the thiourea group by methyl iodide as activation, polymerbound protected peptides 3 were separated in several portions to react with different primary and secondary amines to afford, after resin cleavage, side-chain deprotection and purification, 27 highly pure analogues of 26RFa(20-26) with 6-39% yield (Table 1, Supplemental Fig. S8-S34). As previously reported, guanidinylation with isobutylamine was less efficient than that obtained with all other primary or secondary amines used even with higher quantity (2x eq.) and increased time (20 h).35 Nω,Nω-diisopropyl-substituted compounds were not obtained by this method probably in reason of the steric hindrance of the isopropyl groups. The synthetic route is illustrated by the preparation of [(Me,Me)ω,ωHar25]26RFa(20-26) (6c, LV-2269) from homothiocitrulline containing compound (2c) (Scheme 1; Fig. 1). A small portion of 2c was cleaved by TFA/TIS/H2O (95:2.5:2.5) and analyzed by reversed-phase HPLC (RP-HPLC) and MALDI-ToF mass spectrometry. The expected analogue was obtained in 83% yield and 73% purity (Fig. 1A). [Lys25]26RFa(20-26) was not detected in the chromatogram showing that carbamate formation did not occur. Resin-bound peptide 2c was then methylated (CH3I, DMF, 3 x 1h) affording the S-methylated supported intermediates 3c which, after cleavage of a small portion, led to the expected peptide in 86% yield and 62.4% purity (Fig. 1B). No undesired extra-methylation and no trace of starting material were detected by MALDI-ToF mass spectrometry. Guanidinylation of 3c was achieved by reaction of 2 M dimethylamine solution in dry DMSO at 80°C for 16 h under an Ar atmosphere. Cleavage of the resulting polymersupported peptide provided [(Me,Me)ω,ωHar25]26RFa(20-26) (6c, LV-2269) in 50% crude yield and 55.5% purity (Fig. 1C). No precursor trace was detected indicating that guanidinylation was



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complete. Similarly, we did not detect homocitrulline containing peptide resulting from remaining traces of H2O in the reaction solvent. The pharmacological profile of these analogues was first assessed by testing their ability to increase [Ca2+]i in human QRFPR-transfected CHO cells (Table 2). As previously reported, 26RFa(20-26) (4b, LV-2021) was almost 40 fold less potent than 26RFa to activate QRFPR (Table 2).13 Homologation of the arginine side-chain (4c, LV-2291) did not significantly alter this potency, while methylene shortening led to the inactive [Nar25]26RFa(20-26) (4a, LV-2290) compound probably due to the lack of interactions with the Gln125 and/or Glu132 residues of QRFPR.31 As a matter of fact, all the peptides containing a mono-, a di-, or a tri-substituted guanidino Nar25 moiety were totally devoid of agonistic activity (Table 1, n = 2; Table 2). Similarly,

all

the

Har25

derivative-containing

peptides

were

inactive,

except

[(iBu)ωHar25]26RFa(20-26) (13c, LV-2289) that retained a weak potency to mobilize intracellular calcium in human QRFPR-transfected CHO cells (Table 1, n = 4; Table 2). In contrast, [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) was 25-fold more potent than the heptapeptide 26RFa(20-26) (4b, LV-2021) and only 1.5 times less potent than the full-length peptide 26RFa (Fig. 2A; Table 2). Replacement of the Nω-methyl group by a Nω-ethyl (9b, LV-2271), a Nω-propyl (12b, LV-2274) or a Nω-isobutyl (13b, LV-2288) substituent decreased the Ca2+-mobilizing activity of the analogues with potencies similar to that of the control peptide 26RFa(20-26) (4b, LV-2021), whereas Nω,Nω, Nω,Nω’ and Nω,Nω,Nω’ di- and trialkyl arginine-containing peptides (6b, LV-2185; 7b, LV-2199 and 8b, LV-2279) were unable to activate QRFPR (Fig. 2B,C; Table 1, n = 3; Table 2). It is noteworthy that Nω-methylation of the Arg25 moiety provoked the same beneficial effect in full-length 26RFa as in its C-terminal heptapeptide (Table 2). Indeed, [(Me)ωArg25]26RFa (16, LV-2294) was 6-fold more potent than 26RFa. Taken together, these data indicate that the Arg residue in position 25 has an optimal side-chain length for conferring to the analogue an agonistic activity towards QRFPR and that its Nω-methylation is accommodated by the active site. Furthermore, it has been reported that the C-terminal PheArg-Phe-NH2 part of 26RFa(19-26) is anchored into the QRFPR binding pocket by, at least, two interactions originated in the Nω and Nω’ atoms, individually involved in binding and activation process.31 Since mono-alkylation of the Nω atom, which does not affect the positive charge of 8 ACS Paragon Plus Environment

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the guanidino group, enhanced the potency of 26RFa(20-26) (Nω-methylation), we can speculate either the occurrence of a new favorable interaction inside the cavity or the beneficial modification of the electrostatic characteristics of the initial interaction. Similar observations have been reported for kisspeptin-10.37 In particular, the Nω-methyl-arginine containing analogue and its Nω-ethyl counterpart are, respectively, twice more than and as potent as kisspeptin-10 to stimulate [Ca2+]i in a functional assay of human KiSS1R.37 This similarity highlights common recognition and activation processes between all the RFRPs and their cognate receptors as previously suggested.31 In support of this hypothesis, human QRFPR shares 44-57% sequence similarity with the human NPFF1, NPFF2, GPR10 and KiSS1R in a LALIGN alignment.38 Compounds devoid of agonistic activity were subsequently evaluated for their ability to antagonize the 26RFa-evoked [Ca2+]i response. As a first step, a one-dose assay was used consisting in a 30-minute incubation of QRFPR-transfected cells with a 10-5 M analogue solution prior to an effective 10-7 M 26RFa application. As shown in figure 3, the nonpeptide antagonist 15 from AstraZeneca39 (Fig. 4) totally blocked the agonistic effect of 26RFa on calcium mobilization (Fig. 3). At the same dose, the arginine-modified containing compounds did not significantly reduce the 26RFa-induced calcium increase except [(Me,Me,Me)ω,ω,ω’Arg25]26RFa(20-26) (8b, LV-2279) which antagonized 47% of the agonistic response (Fig. 3A,B). At a concentration of 10-5 M, [(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185) did not significantly reduce the 26RFa-evoked response (Fig. 3). However, a concentrationresponse curve revealed that this compound inhibited the 26RFa-evoked [Ca2+]i increase dosedependently (Fig. 5A). The antagonistic effect of [(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185) plateaued at 10-4 M (Fig. 5B) with an IC50 of 6.0 µM and a maximal efficacy of 80% (Table 2). The [(Me,Me,Me)ω,ω,ω’Arg25]26RFa(20-26) (8b, LV-2279) peptide analogue and the pyrrolo[2,3c]pyridine derivative (15) displayed IC50 of 8.2 and 0.6 µM, and efficacy of 87.5 and 100%, respectively (Fig. 5B,C and Table 2). Furthermore, compound 15 concurrently shifted the 26RFa dose-response curve to the right in a concentration-dependent manner whereas LV-2279 (8b) only affected the efficacy of the calcium increase, indicating that the non-peptide molecule acts as a competitive QRFPR antagonist while the arginine-modified containing peptide [(Me,Me,Me)ω,ω,ω’Arg25]26RFa(20-26) (8b, LV-2279) behaves as a partial agonist, in very much 9 ACS Paragon Plus Environment


the same as [(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185) (Fig. 6). Indeed, at supra-micromolar concentrations both peptide compounds, LV-2279 (8b) and LV-2185 (6b), exhibited a residual agonistic effect (data not shown). Thus, it can be assumed that during the incubation of QRFPRtransfected cells with 10-5 M concentrations of the analogues, partial receptor internalization occurred leading to minimization of the 26RFa effect. We have previously reported that the RFamide motif of 26RFa(19-26) dives inside the binding cavity of the human QRFPR model and that the arginine moiety establishes two main intermolecular atomic interactions with the Gln125 and Glu132 residues of QRFPR.31 In order to correlate the increase, the decrease and the switch from agonistic to antagonistic activity with modifications of ligand/receptor interactions, we have positioned [(Me)ωArg25]26RFa(20-26) (5b, LV-2186), [(Me,Me)ω,ω’Arg25]26RFa(20-26) (7b, LV-2199) and [(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185) inside the QRFPR homology model, respectively. The generated complexes have been subsequently submitted to MD simulations to optimize ligand position into the QRFPR binding site. In comparison to the docking position of 26RFa(19-26),31 26RFa(20-26) (4b, LV-2021) accommodated slightly differently in the binding pocket. In particular, the Arg25 side-chain stretched more in 26RFa(20-26) (4b, LV-2021) than in 26RFa(19-26). Indeed, the  and ’ protons of Arg25 of 26RFa(20-26) (4b, LV-2021) settled two hydrogen bonds with the side-chains of the Ser325 and Asp91 moieties, two residues imbedded in the middle of TM7 and TM2, respectively (Fig. 7A), whereas they establish contact with Gln125 and Glu132 side-chains present in the top of TM3 in 26RFa(19-26).31 However, Gln125 and Glu132 were still involved in the interaction network of 26RFa(20-26) (4b, LV-2021) by establishing hydrogen bonds with the -carbonyl oxygen and the N proton of the Arg25 residue, respectively (Fig. 7A). Similarly, the N proton of Phe24 moiety was paired with the Gln318 side-chain. Interestingly, we found that the carbonyl oxygen of the Ser23 residue and the C-terminal amide proton were positioned in a way that suggests their engagement in an intramolecular hydrogen bond stabilizing the backbone conformation in a -turn-like structure (Fig. 7A). These data are consonant with the first structural study of 26RFa(20-26) (4b, LV-2021) conformation by NMR demonstrating that its secondary structure encompasses turn motifs.30 Moreover, a parallel-displaced stacking arrangement was observed between the two  systems of the Phe22 and Phe24 residues of 10 ACS Paragon Plus Environment

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26RFa(20-26) with a centroid-to-centroid distance of 4.4 Å (4b, LV-2021). The methylated sidechain of [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) was almost placed in the same position maintaining similar or different interactions with the Gln125 and Glu132 residues of TM3, the strictly conserved Asp91 (Asp2.50) moiety of class-A GPCR TM2 and the Ser325 residue of TM7 (Fig. 7B). Similarly, the contact between the backbone and the Gln318 residue as well as the intramolecular hydrogen bond stabilizing the -turn-like structure were retained suggesting that all these interactions constituted the hallmark of agonist docking. The main difference between 26RFa(20-26) (4b, LV-2021) and [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) laid in the fact that both aromatic rings of Phe24 and Phe26 flanking the Arg25 residue were oriented in opposite directions, suggesting that the methylated arginine side-chain of 5b (LV-2186) moved TM7 away from the helix bundle and enlarged the binding groove for accommodating the Phe24 side-chain (Fig. 7B). Therefore, the aryl moiety of Phe24 faced towards that of Phe322 and the measured distance between the m-proton of Phe322 and the centroid of the aromatic ring of Phe24 (3.4 Å) was consistent with a T-shape, edge-to-face, -stacking arrangement (Fig. 7B). This novel interaction correlated well with the fact that [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) was more potent than 26RFa(20-26) (4b, LV-2021) to activate QRFPR. Unexpectedly however, the bulky gem-dimethylated guanidine function of [(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185) did not modify the binding groove (Fig. 7C). MD simulations demonstrated that [(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185) was very flexible and showed no preference for turn conformations as observed in both agonist compounds. Nevertheless, all the contacts between agonists (4b, LV-2021 and 5b, LV-2186) and QRPFR were preserved except the hydrogen

bonds

involving

the

Ser325

and

Asp91

residues

(Fig.

7C).

Since

[(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185) reduced 26RFa-induced [Ca2+]i increase, these interactions are thought to be, at least in part, necessary for triggering the intracellular signaling cascade. Finally, [(Me,Me)ω,ω’Arg25]26RFa(20-26) (7b, LV-2199) was found to form a single contact with surrounding atoms at an intermolecular distance consistent with a hydrogen bond which complied with a very weak agonistic activity of this dimethylated compound (Fig. 7D). Taken as a whole, our MD simulation studies reveal that the pharmacological properties of 5b (LV-2186), 7b (LV-2199) and 6b (LV-2185) match with 11 ACS Paragon Plus Environment


different modeling positions and a different set of interactions inside the QRFPR model. Our results also confirm the role of the Gln125 and Glu132 residues of the QRFPR TM3 helix in the activation and/or binding processes, and identify Ser325 and/or Asp91 as a putative switch for triggering the intracellular signaling pathway. We next sought to compare in vivo the effect of the active truncated analog LV-2186 (5b) to that of 26RFa on insulin-induced hypoglycemia. To this aim, both peptides were intraperitoneally (i.p.) injected in 6-h fasted mice. As previously reported,19 i.p. administration of 26RFa (500 µg/kg) significantly potentiated insulin-evoked hypoglycemia between 30 and 90 min after insulin (0.75 U/kg i.p.) challenge (Fig. 8). In contrast, [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) at the same molar dose (148 µg/kg i.p.) did not modify plasma glucose level during the insulin tolerance test (data not shown). However, at a 5-fold higher dose (741 µg/kg i.p.), [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) significantly enhanced hypoglycemia 90 min after its induction by insulin load (Fig. 8). This result, obtained in vivo, is consistent with a lesser potency of 5b (LV-2186) to activate QRFPR in vitro, in comparison to 26RFa (Table 2). In addition, its delayed effect may be attributed to physicochemical alterations that could lead to repercussions on its bioavailability, as discussed below. Replacement of arginine residues by guanidino-alkylated derivatives within bioactive peptide sequences was previously found to improve stability of Kiss1R agonists towards digestion by trypsin-like proteases,37 to emulate bradykinin B2 receptor affinity of the Hoe 140 antagonist,40 and to enhance potency and duration of activity of GnRH antagonists.41,42 The selectivity of peptide-based thrombin inhibitors is also modulated by this kind of modification.43 Similarly, specific peptide inhibitors targeting the substrate arginine-binding site of protein arginine Nmethyltransferases and containing a single Nω-ethyl-arginine residue have been designed.44,45 Incorporation of Nω-alkyl-arginine into peptides was also shown to increase lipophilicity37,46 and to modify the basicity of the N-alkyl-guanidino group.45,46 Generally, the alkyl substituent reduces available hydrogen bonding to the guanidino group while retaining a positive charge at physiological pH, and consequently modifies the peptide-receptor interactions of the alkylated ligand.


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CONCLUSIONS In conclusion, we have successfully synthesized 31 mono- or polyalkyl guanidino arginine derivative-containing 26RFa analogues by an efficient solid-phase methodology. In vitro evaluation of their pharmacological profiles in a calcium-mobilizing assay revealed that (i) a side-chain length corresponding to that of arginine is required to the analogue for displaying agonistic activity, (ii) Nω-alkylation of Arg25 is rather well tolerated and even leads to the most potent QRFPR agonist based on 26RFa(20-26) disclosed so far in the case of Nω-methylation and, (iii) other modifications yield partial agonists and inactive compounds. Molecular modeling of three prototypic analogues confirmed the involvement of the Gln125 and Glu132 residues in activation and/or binding to QRFPR, and identified Asp91 and Ser325 as putative QRFPR activation triggers. Our data also suggest that steric, electronic, logD and/or pKa differences between arginine derivatives can account for the pharmacological behavior of these peptides both in vitro and in vivo. Finally, incorporation of Nω-methyl-arginine at position 25 of 26RFa(2026)

may be exploited in the development of new QRFPR peptide ligands with higher agonistic

activity.


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Experimental Procedures Fmoc-amino acid residues, Boc-glycine, O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) and Rink amide 4-methylbenzhydrylamine (MBHA) resin were purchased from Christof Senn Laboratories (Dielsdorf, Switzerland), Novabiochem (Darmstadt, Germany) or IRIS Biotech (Marktredwitz, Germany). N,N-Diisopropylethylamine (DIEA), piperidine, acetic anhydride, trifluoroacetic acid (TFA), triisopropylsilane (TIS), tertbutylmethylether (TBME), tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), sodium diethyldithiocarbamate (DTC), dimethylsulfoxide (DMSO) and probenecid were supplied by Sigma-Aldrich

(Saint-Quentin-Fallavier,

France).

N-methylpyrrolidone

(NMP)

and

dimethylformamide (DMF) were from Biosolve (Dieuze, France), and dichloromethane (DCM), acetonitrile, phenylsilane (PhSiH3), iodomethane, Fmoc-isothiocyanate, methyl-isothiocyanate and all the amines were from Thermo Fisher Scientific (Courtaboeuf, France). α-Cyano-4hydroxycinnamic acid was from LaserBio Labs (Sophia-Antipolis, France). Hank’s balanced salt solution (HBSS) and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) were from Invitrogen (Courtaboeuf, France). Compound 25e was a generous gift from AstraZeneca. Peptide Synthesis All peptides were synthesized on a 433A Applied Biosystems automated peptide synthesizer (Villebon-Sur-Yvette, France) using the standard manufacturer’s procedures (0.25 mmol scale) on a Rink amide MBHA resin as previously described.34 All Fmoc-amino acids and Boc-glycine (1 mmol, 4 eq.) were coupled by in situ activation with HBTU (0.9 mmol, 3.6 eq.) and DIEA (2 mmol, 8 eq.) in NMP. Reactive side-chains were protected as follows: Ser, tert-butyl (tBu) ether; Arg, Har, pentamethyldihydrobenzofuran (Pbf) sulfonylamide; Nar, di-tertbutyloxycarbonyl (Boc)2 dicarbamate; Dab, Orn, Lys, allyloxycarbonyl (Alloc) carbamate. After each coupling reaction, N-acylation of unreacted amino functions was performed by addition of a large excess of acetic anhydride (0.5 M) and DIEA. Synthesis of Substituted Arginine Analogue Containing Peptides



After completion of the chain assembly, deprotection of the allyloxy carbamate was performed manually by Pd (0) under Ar as previously described.35,47,48 Briefly, PhSiH3 (24 eq.) dissolved in 6.5 mL of DCM was transferred to a sealed tube containing 1.2 g of Boc-peptidyl(Alloc)-resin (1 eq.) using an Ar flushed gas-tight syringe. A solution of Pd(PPh3)4 (0.3 eq.) in 19.5 mL of DCM was added and gently agitated for 1 hour at room temperature. The resin was then washed sequentially with DTC (0.02 M in DMF), DMF and DCM. A 0.2 M solution of either Fmocisothiocyanate or methyl-isothiocyanate (5 eq.) in DCM was added to the resin, stirred for 2 hours at room temperature, washed with DMF and DCM, and subsequently treated with 20% piperidine in DMF for Fmoc removal. The resin was then treated with a 0.2 M solution of iodomethane (5 eq.) in DMF, stirred for 1 hour at room temperature and washed with DMF. The iodomethane addition step was repeated twice. The resulting resin was split into 10 equal parts and transferred into hermetically closed tubes; a 2 M solution of amine (10 eq.) in dry DMSO was added under inert atmosphere. The mixtures were heated for 16 hours at 80°C to obtain, after DMF and DCM washes, mono-, di- or tri-alkylated norarginine-, arginine- or homoarginine-containing peptide bound resins. Peptide Cleavage and Purification All peptides were deprotected and cleaved from the resin by adding 10 mL of an ice-cold mixture of TFA/TIS/H2O (9.5:0.25:0.25, v/v/v) and stirring 3 hours at room temperature.49 After filtration, crude products were precipitated by addition of cold TBME and collected by centrifugation after 3 washes with TBME. Crude peptides were purified by RP-HPLC on a Vydac 218TP1022 C18 column (2.2 x 25 cm; Grace, Columbia, SC) using a linear gradient (10-50% or 20-50% over 50 min) of acetonitrile (0.1% TFA) at a flow rate of 10 mL/min. Absorbance was simultaneously monitored at 215 and 280 nm using a UV detector. Analytical RP-HPLC analysis, performed on a Vydac 218TP54 C18 column (0.46 x 25 cm; Grace), revealed that the purity of all peptides was higher than 97% (Table 1; Figure S1-S33). The authenticity of each peptide was verified by MALDI-TOF-MS on a voyager DE-PRO (AppleraFrance, Villebon-sur-Yvette, France) or on an UltrafleXtreme (Bruker Daltonik, Bremen, Germany) in the reflector mode with αcyano-4-hydroxycinnamic acid as a matrix.


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Cell Culture Stably transfected human QRFPR CHO cells were obtained as previously described.13,14 The cells were maintained in F-12 nutrient mixture (Ham-F12) medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and penicillin-streptomycin. Expression of Gα16 cells was maintained using selection antibiotic hygromycin B (200 µg/mL) and that of QRFPR using geneticin G418 (500 µg/mL) (Life Technologies, Villebon-Sur-Yvette, France) in a humidified 5% CO2 atmosphere at 37°C. Calcium Mobilization Assays Changes in intracellular Ca2+ concentrations induced by 26RFa analogues in CHO-Gα16-hQRFPRtransfected cells were measured on a benchtop scanning fluorometer flexstation III (Molecular Devices, Sunnyvale, CA) as previously described.13,14,33,50 Briefly, 96-well assay black plates with clear bottom (Corning international, Avon, France) were seeded at a density of 40,000 cells/well 24 h prior to assay. For profiling agonistic experiments, cells were loaded with 2 µM Fluo-4 acetoxymethyl ester (AM) (Invitrogen) during 1 h in the presence of 0.01% pluronic acid, washed thrice, and incubated 30 min with standard HBSS containing 2.5 mM probenecid and 5 mM HEPES. Compounds to be tested were added at final concentrations ranging from 10-11 to 10-5 M in HBSS, and the fluorescence intensity was measured during 3 min. To evaluate antagonistic potency of the test compounds, cells were incubated with each compound over 15 min after Fluo-4 AM loading. Then, during fluorescence recording, a pulse of 26RFa was administered at a final concentration of 10-7 M. After subtraction of the mean fluorescence background, the baseline was normalized to 100%. Fluorescence peak values were determined for each concentration of compound. Molecular Dynamics Simulations In order to investigate the stability of 26RFa(20-26) derivative/QRFPR complexes, MD simulations were carried out. Our previously reported computational homology model of QRFPR in complex with 26RFa(19-26) was used as a starting point.31 Appropriate deletion at position 19 and modifications of the Arg25 side-chain were introduced with BIOVIA Discovery Studio



(v.4.5). All MD simulations were initiated using CHARMM version c40b2 and CHARMM NAMD 2.12 with the all-atom CHARMM 36 force field.51-53 Each complex was inserted into preequilibrated membrane bilayer composed of POPC, cholesterol and POPG (60:20:20), in the presence of 0.15 M KCl and solvated by TIP3P water molecules to mimic physiological conditions using CHARMM-GUI solvator.54,55 Periodic boundary conditions were applied to the systems using the IMAGE algorithm. Van der Waals interactions were truncated using a force switching function between 10 and 12 Å and the particle mesh Ewald (PME) was used to calculate long-range electrostatic interactions.56 Finally, the SHAKE algorithm was applied to restrain all bonds involving hydrogen atoms and vacuum dielectric constant was used during all calculations.57 Equilibration of the systems was carried out in several steps using the diminishing constraints as proposed within CHARMM-GUI. In order to investigate the evolution of 26RFa derivative/QRFPR interactions, the systems ran freely for 10 ns under NPT conditions. Langevin dynamics with a damping coefficient of 1 ps-1 was used to maintain the system temperature and Nosé-Hover Langevin piston method to control the pressure at 1 atm. The generated trajectories were taken for subsequent analysis. Insulin Tolerance Test Ten to 14-week-old male C57Bl/6J mice (Janvier laboratory, Le Genest-Saint-Isle, France), weighing 22–25 g, were housed 5 per cage with free access to standard diet (U.A.R., Villemoisson-sur-Orge, France) and tap water. Animals were kept in a ventilated room at a temperature of 22 ± 1°C under a 12-h light/12-h dark cycle (light on between 7:00 AM and 7:00 PM). All the experiments were carried out between 9:00 AM and 6:00 PM in an adjacent testing room. Animal manipulations were performed according to the European Community Council Directive of November 24th 1986 (86:609:EEC), and were conducted by authorized investigators. Before the test, mice were fasted for 6 h with free access to water and then injected i.p. with 0.75 U/kg body weight of human insulin (Eli Lilly, Neuilly-sur-Seine, France) containing or not (vehicle) either 26RFa (500 μg/kg), or LV-2186 at a dose equimolar to that of 26RFa (148 µg/kg) or at a 5-fold excess (741 µg/kg). Blood plasma glucose concentrations were measured from tail vein samplings 0, 15, 30, 60 and 90 minutes after i.p. injection using an


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AccuChek Performa glucometer (Roche Diagnostic, Saint-Egreve, France). Statistical Analysis Calcium experiments were performed in triplicate, and data, expressed as mean ± SEM of at least three distinct experiments, were analyzed with the Prism 6.0 software (Graphpad Software, San Diego, CA). EC50 and the IC50 values were determined from concentrationresponse curves using a sigmoidal dose-response fit with variable slope from at least three independent determinations. Differences between 26RFa(20-26) and analogue activities were analyzed by the Mann-Whitney test. In vivo data, obtained on at least eight animals per group, were expressed as mean ± SEM, and were analyzed by two-way ANOVA followed by Tuckey honestly significant difference for post-ANOVA comparisons.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:. Molecular formula, MALDI-TOF MS spectrum and RP-HPLC analysis of the synthesized compounds (4a–14c, 16). MD simulations.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Benjamin Lefranc: 0000-0002-0367-2414 Jana Sopkova-de Oliveira Santos: 0000-0002-4829-8120 Christophe Dubessy: 0000-0001-9252-0959 Jean A. Boutin: 0000-0003-0068-7204 Nicolas Chartrel: 0000-0002-3469-3736 19 ACS Paragon Plus Environment


Jérôme Leprince: 0000-0002-7814-9927 Author Contributions All authors contributed in the conception and design of peptide analogues and experiments, and analyzed data. B.L synthesized material used in experiments. K.A, J.S.O.S, C.D and M.P conducted experiments. K.A and J.L wrote the manuscript. J.A.B, H.V and N.C contributed to critical revisions. Notes The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

ACKNOWLEDGMENTS This work was supported by INSERM, the University of Normandy Rouen, the European Regional Development Fund (ERDF, PeReNE and DO IT) and the Region Normandy. The authors thank the Centre Régional Informatique et d’Applications Numériques de Normandie (CRIANN) and the ERDF for the molecular modeling softwares. K.A was the recipient of a fellowship from the Region Normandy.

ABBREVIATIONS USED Alloc, allyloxycarbonyl; [Ca2+]i, intracellular calcium concentration; CNS, central nervous system; Dab, 2,4-diamino-butyric acid; GPCR, G protein-coupled receptor; Har, homoarginine; MD, molecular dynamics; Nar, norarginine; NPAF, neuropeptide AF; NPFF, neuropeptide FF; Orn, ornithine; PrRP, prolactin-releasing peptide; RFRP, RFamide-related peptide. KEY WORDS GPR103, QRFP receptor, peptide drug design, arginine derivatives, alkyl guanidino arginine, Ca2+ mobilization, molecular dynamics, insulin tolerance test.


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Lakowski, T. M.; ’t Hart, P.; Ahern, C. A.; Martin, N. I.; Frankel, A. Nη-substituted arginyl peptide inhibitors of protein arginine N-methyltransferases. ACS Chem. Biol. 2010, 5, 1053–1063.

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Thomas, D.; Koopmans, T.; Lakowski, T. M.; Kreinin, H.; Vhuiyan, M. I.; Sedlock, S. A.; Bui, J. M.; Martin, N. I.; Frankel, A. Protein arginine N-methyltransferase substrate preferences for different Nη-substituted arginyl peptides. ChemBioChem 2014, 15, 1607– 1613.


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Kennedy, K. J.; Lundquist, J. T.; Simandan, T. L.; Kokko, K. P.; Beeson, C. C.; Dix, T. A. Design rationale, synthesis, and characterization of non-natural analogs of the cationic amino acids arginine and lysine. J. Pept. Res. 2000, 55, 348–358.

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Thieriet, N.; Alsina, J.; Giralt, E.; Guibé, F.; Albericio, F. Use of Alloc-amino acids in solidphase peptide synthesis. Tandem deprotection-coupling reactions using neutral conditions. Tetrahedron Lett. 1997, 38, 7275–7278.

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Leprince, J.; Oulyadi, H.; Vaudry, D.; Masmoudi, O.; Gandolfo, P.; Patte, C.; Costentin, J.; Fauchère, J.-L.; Davoust, D.; Vaudry, H.; Tonon, M.-C. Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN. Design of a potent endozepine antagonist. Eur. J. Biochem. 2001, 268, 6045–6057.

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Chatenet, D.; Dubessy, C.; Boularan, C.; Scalbert, E.; Pfeiffer, B.; Renard, P.; Lihrmann, I.; Pacaud, P.; Tonon, M.-C.; Vaudry, H.; Leprince, J. Structure-activity relationships of a novel series of urotensin II analogues: identification of a urotensin II antagonist. J. Med. Chem. 2006, 49, 7234–7238.

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Dubessy, C.; Cartier, D.; Lectez, B.; Bucharles, C.; Chartrel, N.; Montero-Hadjadje, M.; Bizet, P.; Chatenet, D.; Tostivint, H.; Scalbert, E.; Leprince, J.; Vaudry, H.; Jégou, S.; Lihrmann, I. Characterization of urotensin II, distribution of urotensin II, urotensinrelated peptide and UT receptor mRNAs in mouse: evidence of urotensin II as the neuromuscular junction. J. Neurochem. 2008, 107, 361–374.

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Brooks, B. R.; Brooks III, C. L.; MacKerell Jr. A. D.; Nilsson, L.; Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; Caflisch, A.; Caves, L.; Cui, Q.; Dinner, A. R.; Feig, M.; Fischer, S.; Gao, J.; Hodoscek, M.; Im, W.; Kuczera, K.; Lazaridis, T.; Ma, J.; Ovchinnikov, V.; Paci, E.; Pastor, R. W.; Post, C. B.; Pu, J. Z.; Schaefer, M.; Tidor, B.; Venable, R. M., Woodcock, H. L.; Wu, X.; Yang, W.; York, D. M.; Karplus, M. CHARMM: The biomolecular simulation program. J. Comput. Chem. 2009, 30, 1545–1614.

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Table 1: Chemical Data for Synthesized Compounds. H N

H2N

O N H

O

H N O

O N H OH

O n( ) R1

Side-chain n( ) HN n( ) HN

n( ) HN

n( )

NH

N

n( )

NH N H

NH N

NH

NH N

NH

HN

n( ) HN

n( ) HN

n( ) HN

n( ) HN

N

NH N

NH N H

NH N H

NH N

N R2

O R3

Formula

MH+ calc.b

MH+ obs.c

Purity (%)

na

4a 4b 4c

LV-2290 LV-2021 LV-2291

C39H51N11O8 802.39 C40H53N11O8 816.41 C41H55N11O8 830.42

802.39 99.9 816.43 99.9 830.41 99.9

H

H

H

2 3 4

H

H

Me

2 3 4

5a 5b 5c

LV-2263 LV-2186 LV-2264

C40H53N11O8 816.41 C41H55N11O8 830.42 C42H57N11O8 844.44

816.52 99.9 830.42 99.9 844.46 99.9

Me

2 3 4

6a 6b 6c

LV-2268 LV-2185 LV-2269

C41H55N11O8 830.42 C42H57N11O8 844.44 C43H59N11O8 858.45

830.42 99.9 844.46 99.9 858.43 99.9

7a 7b 7c

LV-2276 LV-2199 LV-2277

C41H55N11O8 830.42 C42H57N11O8 844.44 C43H59N11O8 858.45

830.44 99.9 844.46 99.9 858.46 99.9

H

Me

Me

H

Me

2 3 4

Me

Me

Me

2 3 4

8a 8b 8c

LV-2278 LV-2279 LV-2280

C42H57N11O8 844.44 C43H59N11O8 858.45 C44H61N11O8 872.47

844.48 99.9 858.50 99.9 879.49 99.9

H

H

Et

2 3 4

9a 9b 9c

LV-2270 LV-2271 LV-2272

C41H55N11O8 830.42 C42H57N11O8 844.44 C43H59N11O8 858.45

830.43 99.9 844.55 99.9 858.47 99.9

10a LV-2265 10b LV-2266 10c LV-2267

C43H59N11O8 858.45 C44H61N11O8 872.47 C45H63N11O8 886.49

858.35 99.9 872.48 99.6 886.45 99.9

N H

NH

Compound

N

NH2

N H NH

R3

N H

HN

n( )

R2

NH2

N

n( )

R1

O

H N

H

Et

Et

2 3 4

H

Me

Et

2 3 4

11a LV-2281 11b LV-2282 11c LV-2283

C42H57N11O8 844.44 C43H59N11O8 858.45 C44H61N11O8 872.47

844.42 99.9 858.52 97.3 872.48 99.9

H

H

Pr

2 3 4

12a LV-2273 12b LV-2274 12c LV-2275

C42H57N11O8 844.44 C43H59N11O8 858.45 C44H61N11O8 872.47

844.60 99.9 858.48 99.9 872.54 98.5

H

H

iBu

2 3 4

13a LV-2287 13b LV-2288 13c LV 2289

C43H59N11O8 858.45 C44H61N11O8 872.47 C45H63N11O8 886.49

858.46 99.9 872.50 97.4 886.50 99.9

2 3 4

14a LV-2284 14b LV-2285 14c LV-2286

C42H55N11O8 842.42 C43H57N11O8 856.44 C44H59N11O8 870.45

842.52 99.9 856.47 99.9 870.47 99.9

H

Aze



an

= 2, norarginine derivative-containing peptides; n = 3, arginine derivative-containing peptides; n = 4, homoarginine derivative-containing peptides. bCalculated molecular masses. cObserved molecular masses measured by MALDI-ToF mass spectrometry.


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Table 2: Biological Data for Synthesized Compounds. Agonist Compound 26RFa

Antagonist

EC50 (nM)a

IC50 (nM)

Max effect (%)b

46 ± 30

nd

-

4a 4b 4c

LV-2290 2626 LV-2021 26 LV-2291

> 105 1640 ± 259 1910 ± 310

> 105 nd nd

-

5a 5b 5c

LV-2263 LV-2186 LV-2264

> 105 66.4 ± 16.2*** > 105

> 105 nd > 105

-

6a 6b 6c

LV-2268 LV-2185 LV-2269

> 105 > 105 > 105

> 105 6060 ± 922 > 105

80.0 -

7a 7b 7c

LV-2276 LV-2199 LV-2277

> 105 > 105 > 105

> 105 > 105 > 105

-

8a 8b 8c

LV-2278 LV-2279 LV-2280

> 105 > 105 > 105

> 105 8250 ± 569 > 105

87.5 -

9a 9b 9c

LV-2270 LV-2271 LV-2272

> 105 1370 ± 543 > 105

> 105 nd > 105

-

10a 10b 10c

LV-2265 LV-2266 LV-2267

> 105 > 105 > 105

> 105 > 105 > 105

-

11a 11b 11c

LV-2281 LV-2282 LV-2283

> 105 > 105 > 105

> 105 > 105 > 105

-

12a 12b 12c

LV-2273 LV-2274 LV-2275

> 105 1400 ± 308 > 105

> 105 nd > 105

-

13a 13b 13c

LV-2287 LV-2288 LV 2289

> 105 1500 ± 424 2747 ± 1460

> 105 nd nd

-

14a 14b 14c

LV-2284 LV-2285 LV-2286

> 105 > 105 > 105

> 105 > 105 > 105

-

15

-

nd

579 ± 161

100 31

ACS Paragon Plus Environment


16

LV-2294

6.9 ± 3.0

-

-

aData

are mean ± SEM of at least three distinct experiments performed in triplicate. bThe maximal effect, at a concentration of 10-4 M, is expressed as a percentage of the 26RFa-induced (10-7 M) mean calcium response inhibition. (***) P < 0.001 vs control 26RFa(20-26 ) as assessed by Mann and Whitney test. nd, not determined.


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Legend to figures Scheme 1: (color) General Synthetic Scheme for the Preparation of Substituted Norarginine- (n = 2), Arginine- (n = 3) and Homoarginine-containing Peptides (n = 4). The residue involved in the synthetic route is indicated in red. For clarity, N- and C-terminal parts have been removed on the second line. Figure 1: (no color) RP-HPLC Analysis of Crude Peptide Intermediates and [(Me,Me)ω,ωHar25]26RFa(20-26) (6c, LV2269)

Analogue.

Crude

homothiocitrulline-containing

peptide

2c

(A),

S-

methylhomoisothiocitrulline-containing peptide 3c (B), and [(Me,Me)ω,ωHar25]26RFa(20-26) (6c, LV-2269; C) were analyzed after resin cleavage and N-terminal and side-chain deprotection of resin grafted peptides. Figure 2: (no color) Effect of Graded Concentrations of Arg25-Modified 26RFa(20-26) Analogues on Basal [Ca2+]i Mobilization in CHO-Gα16-hQRFPR-transfected Cells. Representative dose-response curves of 26RFa(20-26) (4b, LV-2021; closed squares; A,B,C) and its analogues (closed circles) [(Me)ωArg25]26RFa(20-26) (5b, LV-2186; A), [(Et)ωArg25]26RFa(20-26) (9b, LV-2271; B), and [(Pr)ωArg25]26RFa(20-26) (12b, LV-2274; C). Data are mean ± SEM of triplicate. The EC50 calculated from dose-response curves were 66.4 ± 16.2 nM for 5b (LV-2186), 1370 ± 543 nM for 9b (LV2271) and 1400 ± 308 nM for 12b (LV-2274). Figure 3: (no color) Effect of Arg25-Modified 26RFa(20-26) Analogues on 26RFa-evoked [Ca2+]i Mobilization in CHOGα16-hQRFPR-transfected Cells. (A) Normalized response of 26RFa (10-7 M) on calciummobilization in the absence (HBSS control) or presence of 10-5 M of AstraZeneca antagonist 15 or Arg25-modified analogues. (B) Percentage of inhibition induced by HBSS, AstraZeneca antagonist 15 or Arg25-modified analogues on 26RFa-evoked [Ca2+]i increase. Data are mean ± SEM at least three independent determinations. (*) P < 0.05, (***) P < 0.001 vs. control (HBSS).



Page 34 of 45

Figure 4: (no color) Chemical Structure of the Pyrrolo[2,3-c]pyridine 15 Disclosed by AstraZeneca. (5-Bromo-1methyl-1H-pyrrolo[2,3-c]pyridin-2-yl){6-[(dimethylamino)methyl]-(+)4-methyl-3,4dihydroisoquinolin-2(1H)-yl}methanone. Figure 5: (no color) Effect of Graded Concentrations of Arg25-Modified 26RFa(20-26) Analogues on 26RFa-evoked [Ca2+]i Mobilization in CHO-Gα16-hQRFPR-transfected Cells. Representative dose-inhibition curves of Arg25-modified analogues [(Me,Me)ω,ωArg25]26RFa(20-26) (6b, LV-2185; A), [(Me,Me,Me)ω,ω,ω’Arg25]26RFa(20-26) (8b, LV-2279; B) and AstraZeneca antagonist 15 (C) on (10-7 M) 26RFa-evoked response CHO-Gα16-hQRFPR-transfected cells. Data are mean ± SEM of triplicate. The IC50 value calculated from the data was 6060 ± 922 nM for [(Me,Me)ω,ωArg25]26RFa(20-26)

(6b,

LV-2185),

8250

±

569

nM

for

[(Me,Me,Me)ω,ω,ω’Arg25]26RFa(20-26) (8b, LV-2279) and 579 ± 161 nM for 15. Figure 6: (no color) Effect of Arg25-Modified 26RFa(20-26) Analogues on 26RFa-evoked [Ca2+]i Mobilization in CHOGα16-hQRFPR-transfected Cells. Representative dose-response curves showing the calciummobilizing effect of 26RFa in the absence (closed squares; A,B) or presence of 10-5 M (closed circles) of AstraZeneca antagonist 15 (A) or [(Me,Me,Me)ω,ω,ω’Arg25]26RFa(20-26) (8b, LV-2279; B). Data are mean ± SEM of triplicate. Figure 7: (color) Representative Poses from MD Simulations of 26RFa(20-26) and Three Prototypic Analogues in the QRFPR Homology Model. Human 2-adrenoceptor (accession number: 2RH1) was used as a template.31 26RFa analogue backbones are displayed by sticks while TM2, TM3 and TM7 are represented by blue, green and orange ribbons, respectively. Hydrogen bonds and  stacking interactions are displayed in dashed and solid lines, respectively. The interatomic or atom-tocentroid distances (Å) are indicated. The Asp91 moiety of TM2, the Gln125 and Glu132 residues of TM3 and the Gln318, Phe322 and Ser325 moieties of TM7 are labeled and depicted by sticks. 34 ACS Paragon Plus Environment

Page 35 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A) 26RFa(20-26) (4b, LV-2021), orange sticks. (B) [(Me)ωArg25]26RFa(20-26) (5b, LV-2186), blue sticks.

(C)

[(Me,Me)ω,ωArg25]26RFa(20-26)

(6b,

LV-2185),

pink

sticks.

(D)

[(Me,Me)ω,ω’Arg25]26RFa(20-26) (7b, LV-2199), cyan sticks. Structures were examined using PyMOL (v2.1.1, Schrödinger, New York, NY). Figure 8: (no color) Effect of [(Me)ωArg25]26RFa(20-26) (5b, LV-2186) and 26RFa on Plasma Glucose Level in Mice

during an Insulin Tolerance Test. Representative time-course effects of phosphate buffer (n = 8, closed squares), 26RFa (n = 8, 500 µg/kg i.p, open squares) and [(Me)ωArg25]26RFa(20-26) (5b, LV-2186; n = 12, 741 µg/kg i.p., open circles) on insulin (0.75 U/kg i.p.)-induced hypoglycemia between 15 and 90 min after the concomitant insulin load. Data represent mean ± SEM of at least eight animals per group. (*) p

Design, Synthesis, Molecular Dynamics Simulation and Functional

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