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. 2021 Jul 16;26(14):4312. doi: 10.3390/molecules26144312

Point-Substitution of Phenylalanine Residues of 26RFa Neuropeptide: A Structure-Activity Relationship Study

Benjamin Lefranc 1,2,†, Karima Alim 1,†, Cindy Neveu 1,†, Olivier Le Marec 1,†, Christophe Dubessy 1,2, Jean A Boutin 3,4, Julien Chuquet 1, David Vaudry 1,2, Gaëtan Prévost 1, Marie Picot 1, Hubert Vaudry 1,2, Nicolas Chartrel 1, Jérôme Leprince 1,2,*
Editor: Katalin Prokai-Tatrai
PMCID: PMC8307317  PMID: 34299587

Abstract

26RFa is a neuropeptide that activates the rhodopsin-like G protein-coupled receptor QRFPR/GPR103. This peptidergic system is involved in the regulation of a wide array of physiological processes including feeding behavior and glucose homeostasis. Herein, the pharmacological profile of a homogenous library of QRFPR-targeting peptide derivatives was investigated in vitro on human QRFPR-transfected cells with the aim to provide possible insights into the structural determinants of the Phe residues to govern receptor activation. Our work advocates to include in next generations of 26RFa(20–26)-based QRFPR agonists effective substitutions for each Phe unit, i.e., replacement of the Phe22 residue by a constrained 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid moiety, and substitution of both Phe24 and Phe26 by their para-chloro counterpart. Taken as a whole, this study emphasizes that optimized modifications in the C-terminal part of 26RFa are mandatory to design selective and potent peptide agonists for human QRFPR.

Keywords: GPR103, QRFP, RFamide, intracellular calcium concentration, peptide analog

1. Introduction

The neuropeptides 26RFa (QRFP26) and its N-terminal extended form 43RFa (QRFP, Figure 1) are the endogenous ligands of the Gi/s/q coupled QRFP receptor (QRFPR), formerly known as the orphan receptor GPR103 [1,2,3]. Since the isolation of 26RFa from a European green frog Rana esculenta brain extract [4], prepro-26RFa cDNA has been characterized in numerous genomes of diverse vertebrates from fish to mammals [5]. However, very few mature peptides have been identified so far, leaving the 26RFa/QRFP precursor post-translational processing uncertain. Indeed, in avians, 26RFa orthologs have been isolated and sequenced from the Japanese quail [6] and zebra finch [7] while, in rodents, only the 43RFa counterpart has been characterized from rat [2,8]. In humans, both 26RFa- and 43RFa-immunoreactive forms have been detected in hypothalamic and spinal cord extracts [9]. Interestingly, the presence of a tribasic cleavage site (Arg-Lys-Arg/Lys) within the 26RFa sequence suggests that the C-terminal heptapeptide GGFSFRF-NH2, named 26RFa(20–26), could also be produced from the precursor. However, to date, this fragment, whose sequence is highly conserved in tetrapods, has not been identified as a mature product of the 26RFa/QRFP gene transcription.

Figure 1.

Figure 1

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Primary structures of human 26RFa and 43RFa/QRFP. <E denotes pyroglutamic acid.

In rats, the 26RFa/QRFP gene is primarily expressed in hypothalamic neurons which project in various brain areas [1,2,8,10]. Consistent with the widespread distribution of 26RFa/QRFP-containing fibers, 26RFa and/or QRFP are involved in the regulation of multiple physiological activities [5]. In particular, it is now firmly established that 26RFa and QRFP stimulate food intake in rodents [1,8,10,11,12,13,14,15,16]. There is also evidence that 26RFa and/or QRFP regulate several other neuroendocrine and cognitive functions including reproduction [17,18], anxiety [19], memory [20], cardiovascular activity [8] and nociceptive transmission [21,22,23], some of which depend, at least in part, on off-target interaction with QRFPR-related receptors [24,25]. In addition, recent data reveal that the 26RFa/QRFPR system controls glucose homeostasis at the periphery by increasing insulin sensitivity and inhibiting hepatic glucose production [26,27,28,29]. Thus, QRFPR positions as an attractive target for the development of innovative drugs. Indeed, there is now clinical evidence for possible indications of QRFPR ligands for the treatment of metabolic disorders, obesity and diabetes [5].

As a molecular signature, the RF-amide C-terminal extremity of 26RFa/QRFP and other RF-amide-related peptides (i.e., NPAF/NPFF, PrRP, RFRP-1(GnIH)/RFRP-3 and kisspeptin in humans [30]) represents the chemical determinant for bioactivity. Notably, deamidation or local side-chain substitution of the RF-amide moiety is generally associated with full or partial loss of activity and/or affinity of these neuropeptides [2,31,32,33]. The N-terminal part of RF-amide-related peptides diverges in their sequence and length, and conditions the whole or partial affinity and selectivity for one or several receptors, respectively [34,35]. For instance, 26RFa also binds with nanomolar affinity to both NPFF1 and NPFF2 receptors [24]. However, 26RFa can be downsized from the N-terminal extremity with a gradual decrease in its ability to mobilize intracellular calcium concentration ([Ca2+]i) in CHO cells transfected by the human QRFPR (hQRFPR) [36]. Since 26RFa(20–26) (LV-2021) mimics the orexigenic and gonadotropic effects of full-length 26RFa [12,17], it appears that this C-terminal heptapeptide can serve as a molecular scaffold for designing low molecular weight, potent and stable hQRFPR ligands. As a matter of fact, 26RFa(20–26) has already been successfully used to design nanomolar active agonists such as [Nva23]26RFa(20–26) (LV-2073) [36], [Cmpi21, aza-β3-Hht23]26RFa(21–26) (LV-2172) [37], and [(Me)ωArg25]26RFa(20–26) (LV-2186) [38], as well as proteolytic resistant pseudopeptides (Figure 2) [39]. According to our previous structure-activity relationship studies [for review, 5], these compounds exhibit modifications at positions occupied by residues classified as permissive to substitution or engaged in peptide bonds susceptible to degradation. In contrast, the residues of the -Phe24-Arg25-Phe26-NH2 triad are very sensitive to substitutions and only tolerate subtle amendments for improving bioactivity like the ω-methylation of arginine 25 [38]. To date, few replacements have been reported in the phenylalanine positions of 26RFa(20–26) [5]. As previously suggested for PrRP and kisspeptin [40], our work emphasizes that optimized modifications in the C-terminal part of 26RFa are mandatory to design selective and potent peptidergic ligands for hQRFPR.

Figure 2.

Figure 2

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Chemical structures of peptidic and pseudopeptidic human QRFPR ligands. (A) [Nva23]26RFa(20–26) (LV-2073); (B) [Cmpi21, aza-β3-Hht23]26RFa(21–26) (LV-2172); (C) [(Me)ωArg25]26RFa(20–26) (LV-2186); (D) [ψ(CF=CH)21,22]26RFa(20–26); (E) [ψ(CF=CH)20,21]26RFa(20–26).

Thus, the aim of the present study was to investigate molecular diversity at the unexplored Phe positions to go deeper into the structure-activity relationships of 26RFa for the design of potent hQRFPR agonists.

2. Results and Discussion

2.1. Impact of Modifications of Each Phenylalanine Residue

It is well-accepted that the (hetero)aromatic units of peptides and proteins are master residues for DNA recognition [41], folding [42] and receptor-ligand interaction [43]. In particular, His, Phe, Trp and Tyr contribute to three types of non-covalent interactions including π-hydrogen bonds, electrostatic cation-π interactions and van der Waals π-π interactions [44] for governing molecular recognition of a ligand into the binding site of the receptor and transduction processes.

Replacement of all three phenylalanine residues of 26RFa(20–26) (LV-2021, 1) with an alanine moiety identifies Phe24 and Phe26 as key residues for QRFPR activation, unlike Phe22 which is rather permissive to this substitution and thus weakly participates in the activity of the peptide [36]. Accordingly, Phe24 and Phe26 side chains strongly interact with hydrophobic regions at the bottom of the binding pocket of the hQRFPR homology model [45]. In order to optimize positions 22, 24 and 26 of LV-2021, we have successively replaced each phenylalanine of the heptapeptide 1 with different commercially available aromatic or aliphatic building blocks (Table 1, compounds 2–48) and evaluated their ability to mobilize [Ca2+]i in cultured Gα16-hQRFPR-transfected CHO cells (Table 2), as previously described [36]. Substitution with the isosteric residue 3-(2-thienyl)-alanine (Thi) yielded compounds LV-2050, LV-2051 and LV-2052 (2–4) that were less active than the parent peptide whatever the position concerned (Table 2). Similarly, incorporation of more hindered residues such as 3-(2-naphtyl)-alanine (2Nal; LV-2065, LV-2213, LV-2191, 5–7) and tryptophane (LV-2210, LV-2204, LV-2187, 8–10) altered the agonistic activity of the compounds, suggesting that there was little space available at the bottom of the orthosteric binding site as previously reported [45]. However, the para position of the Phe26 residue tolerated a bulky group or a substituent with different electronic effects. Indeed, [ptBuPhe26]26RFa(20–26) (LV-2238, 13), [Pcp26]26RFa(20–26) (LV-2193, 16) and [pNO2Phe26]26RFa(20–26) (LV-2194, 19) were significantly more potent than 26RFa(20–26) (LV-2021, 1) to increase [Ca2+]i in vitro (Table 2, Figure 3A–C). The same substitutions at positions Phe22 and Phe24 were either neutral (15, 17) or deleterious to activity (11, 12, 14, 18). Finally, methylene shortening of the phenylalanine side chain led to the inactive phenyl-glycine (Phg)-containing [Phg22]26RFa(20–26) (LV-2053, 20), [Phg24]26RFa(20–26) (LV-2054, 21; LV-2055, 22) and [Phg26]26RFa(20–26) (LV-2056, 23; LV-2057, 24) diastereoisomers, probably due to the lack of interactions with hydrophobic residues in the core of the hQRFPR binding pocket.

Table 1.

Chemical data for 26RFa(20–26) analogs substituted in positions 22, 24 and 26.

Peptide Structure of Residue 22, 24, 26 Code HPLC MS
Rt (min) a Purity (%) Calcd b Obsd c
1 26RFa(20–26) LV-2021 18.0 99.9 815.41 816.53
2 [Thi22]26RFa(20–26) graphic file with name molecules-26-04312-i001.jpg LV-2050 18.6 99.9 821.36 822.33
3 [Thi24]26RFa(20–26) LV-2051 18.6 99.9 821.36 822.37
4 [Thi26]26RFa(20–26) LV-2052 18.6 99.9 821.36 822.38
5 [2Nal22]26RFa(20–26) graphic file with name molecules-26-04312-i002.jpg LV-2065 20.4 99.9 865.42 866.45
6 [2Nal24]26RFa(20–26) LV-2213 20.8 99.9 865.42 866.46
7 [2Nal26]26RFa(20–26) LV-2191 20.6 99.9 865.42 866.57
8 [Trp22]26RFa(20–26) graphic file with name molecules-26-04312-i003.jpg LV-2210 18.9 99.9 854.42 855.27
9 [Trp24]26RFa(20–26) LV-2204 18.6 99.9 854.42 855.43
10 [Trp26]26RFa(20–26) LV-2187 19.3 99.9 854.42 855.48
11 [ptBuPhe22]26RFa(20–26) graphic file with name molecules-26-04312-i004.jpg LV-2068 22.1 99.9 871.47 872.49
12 [ptBuPhe24]26RFa(20–26) LV-2235 22.3 99.9 871.47 872.45
13 [ptBuPhe26]26RFa(20–26) LV-2238 22.4 99.9 871.47 872.55
14 [Pcp22]26RFa(20–26) graphic file with name molecules-26-04312-i005.jpg LV-2069 19.9 99.9 849.37 850.30
15 [Pcp24]26RFa(20–26) LV-2232 20.5 99.9 849.37 850.41
16 [Pcp26]26RFa(20–26) LV-2193 20.0 99.9 849.37 850.42
17 [pNO2Phe22]26RFa(20–26) graphic file with name molecules-26-04312-i006.jpg LV-2214 19.9 99.9 860.39 861.53
18 [pNO2Phe24]26RFa(20–26) LV-2236 19.0 99.9 860.39 861.46
19 [pNO2Phe26]26RFa(20–26) LV-2194 18.9 99.9 860.39 861.56
20 [Phg22]26RFa(20–26) dia 2 graphic file with name molecules-26-04312-i007.jpg LV-2053 24.2 97.8 801.39 802.40
21 [Phg24]26RFa(20–26) dia 1 LV-2054 21.1 99.9 801.39 802.40
22 [Phg24]26RFa(20–26) dia 2 LV-2055 22.2 98.3 801.39 802.40
23 [Phg26]26RFa(20–26) dia 1 LV-2056 21.0 99.9 801.39 802.70
24 [Phg26]26RFa(20–26) dia 2 LV-2057 21.6 97.3 801.39 802.62
25 [NMePhe22]26RFa(20–26) graphic file with name molecules-26-04312-i008.jpg LV-2066 18.9 99.9 829.42 830.53
26 [NMePhe24]26RFa(20–26) LV-2233 19.2 98.9 829.42 830.48
27 [NMePhe26]26RFa(20–26) LV-2242 18.8 99.9 829.42 830.48
28 [Tic22]26RFa(20–26) graphic file with name molecules-26-04312-i009.jpg LV-2211 20.3 99.9 827.41 828.42
29 [Tic24]26RFa(20–26) LV-2205 17.7 99.9 827.41 828.46
30 [Tic26]26RFa(20–26) LV-2189 18.5 99.9 827.41 828.53
31 [NPhe22]26RFa(20–26) graphic file with name molecules-26-04312-i010.jpg LV-2058 21.1 99.9 815.41 816.23
32 [NPhe24]26RFa(20–26) LV-2060 17.9 99.9 815.41 816.56
33 [NPhe26]26RFa(20–26) LV-2061 18.6 99.9 815.41 816.40
34 [AHPPA22]26RFa(20–26) graphic file with name molecules-26-04312-i011.jpg LV-2062 17.8 99.9 859.43 860.30
35 [AHPPA24]26RFa(20–26) LV-2063 17.7 99.9 859.43 860.40
36 [AHPPA26]26RFa(20–26) LV-2064 18.1 99.9 859.43 860.55
37 [DTrp22]26RFa(20–26) graphic file with name molecules-26-04312-i012.jpg LV-2237 18.2 99.9 854.42 855.56
38 [DTrp24]26RFa(20–26) LV-2070 17.7 99.9 854.42 855.53
39 [DTrp26]26RFa(20–26) LV-2188 18.2 99.9 854.42 855.39
40 [DTic22]26RFa(20–26) graphic file with name molecules-26-04312-i013.jpg LV-2212 18.4 99.9 827.41 828.49
41 [DTic24]26RFa(20–26) LV-2215 18.2 99.9 827.41 828.36
42 [DTic26]26RFa(20–26) LV-2190 18.3 99.9 827.41 828.41
43 [Oic22]26RFa(20–26) graphic file with name molecules-26-04312-i014.jpg LV-2067 18.6 99.9 819.44 820.56
44 [Oic24]26RFa(20–26) LV-2234 18.3 99.9 819.44 820.36
45 [Oic26]26RFa(20–26) LV-2241 18.1 99.9 819.44 820.45
46 [Cys22,24]26RFa(20–26) graphic file with name molecules-26-04312-i015.jpg LV-2105 11.5 99.9 725.27 726.28
47 [Cys22,26]26RFa(20–26) LV-2107 6.7 99.9 725.27 726.37
48 [Cys24,26]26RFa(20–26) LV-2106 11.8 99.9 725.27 726.32
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a Retention time determined by analytical RP-HPLC. b Theorical monoisotopic molecular weight. c m/z [MH]+ value assessed by MALDI-TOF-MS. AHPPA, (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid; 2Nal, 3-(2-naphtyl)-alanine; NMePhe, N-methyl-phenylalanine; NPhe, N-benzyl-glycine; Oic, octahydroindole-2-carboxylic acid; Pcp, (4-chloro)-phenylalanine; Phg, phenylglycine; pNO2Phe, (4-nitro)-phenylalanine; ptBuPhe, (4-terbutyl)-phenylalanine; Thi, 3-(2-thienyl)-alanine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Diastereoisomers are numbered according to their elution order in RP-HPLC.

Table 2.

Biological data for 26RFa(20–26) analogs substituted in positions 22, 24 and 26.

Peptide Code EC50 IC50 Imax
(nM) (nM) (%) a
1 26RFa(20–26) LV-2021 1640 ± 259 -
2 [Thi22]26RFa(20–26) LV-2050 3160 ± 1110 -
3 [Thi24]26RFa(20–26) LV-2051 2530 ± 1020 -
4 [Thi26]26RFa(20–26) LV-2052 7227 ± 96 -
5 [2Nal22]26RFa(20–26) LV-2065 1150 ± 170 -
6 [2Nal24]26RFa(20–26) LV-2213 15,300 ± 5400 -
7 [2Nal26]26RFa(20–26) LV-2191 3790 ± 1800 -
8 [Trp22]26RFa(20–26) LV-2210 1980 ± 880 -
9 [Trp24]26RFa(20–26) LV-2204 2040 ± 1600 -
10 [Trp26]26RFa(20–26) LV-2187 5060 ± 1300 -
11 [ptBuPhe22]26RFa(20–26) LV-2068 >10,000 6220 ± 2500 45
12 [ptBuPhe24]26RFa(20–26) LV-2235 >10,000 ND
13 [ptBuPhe26]26RFa(20–26) LV-2238 1040 ± 25 ** -
14 [Pcp22]26RFa(20–26) LV-2069 5000 ± 4500 -
15 [Pcp24]26RFa(20–26) LV-2232 850 ± 240 NS -
16 [Pcp26]26RFa(20–26) LV-2193 457 ± 71 ** -
17 [pNO2Phe22]26RFa(20–26) LV-2214 2130 ± 1100 -
18 [pNO2Phe24]26RFa(20–26) LV-2236 >10,000 ND
19 [pNO2Phe26]26RFa(20–26) LV-2194 491 ± 33 ** -
20 [Phg22]26RFa(20–26) dia 2 LV-2053 5010 ± 3600 -
21 [Phg24]26RFa(20–26) dia 1 LV-2054 >10,000 ND
22 [Phg24]26RFa(20–26) dia 2 LV-2055 >10,000 ND
23 [Phg26]26RFa(20–26) dia 1 LV-2056 >10,000 ND
24 [Phg26]26RFa(20–26) dia 2 LV-2057 >10,000 ND
25 [NMePhe22]26RFa(20–26) LV-2066 15,100 ± 4200 -
26 [NMePhe24]26RFa(20–26) LV-2233 1360 ± 750 -
27 [NMePhe26]26RFa(20–26) LV-2242 >10,000 ND
28 [Tic22]26RFa(20–26) LV-2211 327 ± 170 * -
29 [Tic24]26RFa(20–26) LV-2205 >10,000 ND
30 [Tic26]26RFa(20–26) LV-2189 7700 ± 4400 -
31 [NPhe22]26RFa(20–26) LV-2058 578 ± 110 * -
32 [NPhe24]26RFa(20–26) LV-2060 >10,000 ND
33 [NPhe26]26RFa(20–26) LV-2061 >10,000 ND
34 [AHPPA22]26RFa(20–26) LV-2062 2850 ± 380 -
35 [AHPPA24]26RFa(20–26) LV-2063 >10,000 ND
36 [AHPPA26]26RFa(20–26) LV-2064 >10,000 ND
37 [DTrp22]26RFa(20–26) LV-2237 6160 ± 2400 -
38 [DTrp24]26RFa(20–26) LV-2070 >10,000 ND
39 [DTrp26]26RFa(20–26) LV-2188 >10,000 >10,000 46
40 [DTic22]26RFa(20–26) LV-2212 4990 ± 2100 -
41 [DTic24]26RFa(20–26) LV-2215 6200 ± 2600 -
42 [DTic26]26RFa(20–26) LV-2190 >10,000 ND
43 [Oic22]26RFa(20–26) LV-2067 >10,000 ND
44 [Oic24]26RFa(20–26) LV-2234 >10,000 ND
45 [Oic26]26RFa(20–26) LV-2241 >10,000 ND
46 [Cys22,24]26RFa(20–26) LV-2105 >10,000 ND
47 [Cys22,26]26RFa(20–26) LV-2107 >10,000 ND
48 [Cys24,26]26RFa(20–26) LV-2106 >10,000 ND
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a Inhibition at a maximal concentration of 10−5 M. Data are mean ± SEM of at least three independent experiments performed in triplicate. AHPPA, (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid; 2Nal, 3-(2-naphtyl)-alanine; NMePhe, N-methyl-phenylalanine; NPhe, N-benzyl-glycine; Oic, octahydroindole-2-carboxylic acid; Pcp, (4-chloro)-phenylalanine; Phg, phenylglycine; pNO2Phe, (4-nitro)-phenylalanine; ptBuPhe, (4-terbutyl)-phenylalanine; Thi, 3-(2-thienyl)-alanine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Diastereoisomers are numbered according to their elution order in RP-HPLC. ND, not detectable. NS, not significant. * p < 0.05, ** p < 0.01 vs. control 26RFa(20–26) (LV-2021, 1) as assessed by Mann and Whitney test.

Figure 3.

Figure 3

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Effect of graded concentrations of Phe-modified 26RFa(20−26) analogs on basal [Ca2+]i mobilization in Gα16-hQRFPR-transfected CHO cells. Prototype dose–response curves of 26RFa(20−26) (LV-2021, 1, closed square, (A–E)) and its analogs (closed circles) [ptBuPhe26]26RFa(20–26) (LV-2238, 13, (A)), [Pcp26]26RFa(20–26) (LV-2193, 16, (B)), [pNO2Phe26]26RFa(20–26) (LV-2194, 19, (C)), [Tic22]26RFa(20–26) (LV-2211, 28, (D)) and [NPhe22]26RFa(20–26) (LV-2058, 31, (E)). Data are mean ± SEM of triplicate. The EC50 calculated from these representative dose–response curves were 1069 nM for 1 (LV-2021), 1076 nM for 13 (LV-2238), 416 nM for 16 (LV-2193), 489 nM for 19 (LV-2194), 305 nM for 28 (LV-2211) and 149 nM for 31 (LV-2058).

The heptapeptide 26RFa(20–26) does not encompass any secondary amide bonds within its backbone. Thus, nine analogs with N-methyl-phenylalanine (NMePhe), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) or N-benzyl-glycine (NPhe) were synthesized. These modifications were aimed at analyzing the significance of H-bond contributions of the amide NHs. Alkylation of these NHs without loss of agonistic activity would facilitate further analog design. N-methylation of Phe22 (LV-2066, 25) and Phe26 (LV-2242, 27) nullified the activity of the parent compound (EC50 > 10 µM, Table 2) revealing the critical role of the corresponding NHs in forming an H-bond with the hQRFPR pocket residues or another residue of the peptide, whereas N-methylation of Phe24 (LV-2233, 26) did not impair the whole activity of LV-2021 (1). The corresponding Tic-containing analogs (28–30) did not totally confirm these data since [Tic24]26RFa(20–26) (LV-2205, 29) and [Tic26]26RFa(20–26) (LV-2189, 30) were devoid of effect while [Tic22]26RFa(20–26) (LV-2211, 28) was 5-fold more potent than 26RFa(20–26) (LV-2021, 1) (Figure 3D). It is thus also plausible that the structural constraint induced by the Tic22 residue may keep the side chain in a favorable conformation for receptor activation, which was less accessible by free rotation in the native Phe side chain. The NPhe-peptoid analogs (31–33) behaved like the Tic-containing counterparts, and [NPhe22]26RFa(20–26) (LV-2058, 31) was almost as potent as LV-2211 (Table 2, Figure 3E). At this stage, conclusions are compromised since the local conformational rigidity to peptide backbone via pipecolinic acid bridge [46] and the shift of side chain functionality from the α-carbon to the NH offering flexibility to peptoid led to similar results. However, we can speculate that the Phe22 residue, which does not dive deeply into the binding pocket, accommodated rather well with backbone modifications at the odds to Phe24 and Phe26 which are strongly embedded in the orthosteric binding site [45]. An increase in H-bond capacity and flexibility by the introduction of (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), a marine Cyanobacterium symploca peptide naturally occurring γ-amino acid [47] (34–36) had no positive effect on the potency of the parent heptapeptide (Table 2).

We have previously reported that stereoinversion to D-Phe in [DPhe22]26RFa(20–26), [DPhe24]26RFa(20–26) and [DPhe26]26RFa(20–26) results in a complete loss of the [Ca2+]i response [36]. Similarly, successive D-Trp and D-Tic incorporation within the 26RFa(20–26) sequence in the same positions generated weak (LV-2237, 37; LV-2212, 40; LV-2215, 41) or inactive (LV-2070, 38; LV-2188, 39; LV-2190, 42) agonists (Table 2), confirming that not only side chain functionality but also its correct orientation play a critical role in the activity of the peptide.

Systematic replacement of the three Phe units with the constrained aliphatic residue octahydroindole-2-carboxylic acid (Oic) yielded LV-2067 (43), LV-2234 (44) and LV-2241 (45) that were totally devoid of Ca2+-mobilizing activity (Table 2). The Oic moiety might induce a turn, like the prolyl residue [48], that would destabilize at this point the correct folding of the peptide backbone in the hQRFPR binding pocket. Furthermore, these results also confirm the importance of aromaticity in positions 22, 24 and 26 of 26RFa(20–26) in the receptor activation process.

In methanol, 26RFa adopts a well-defined conformation consisting of a N-terminal amphipathic α-helical structure (Pro4–Arg17), preceding a C-terminal disordered region [49]. Interestingly, it has been shown that two Phe residues in an i, i+4 arrangement (Phe22 and Phe26 of 26RFa and 26RFa(20–26)) enthalpically stabilize an α-helix [50]. To assess such a conformation of the C-terminal extremity of the peptide inside the hQRFPR binding pocket during the recognition process, we prepared cyclic disulfide-bridged 26RFa(20–26) analogs (Table 1, 46–48) as already reported for the pentapeptide Met-enkephalin [51]. The activity profiles of [Cys22,26]26RFa(20–26) (LV-2107, 47), [Cys22,24]26RFa(20–26) (LV-2105, 46) and [Cys24,26]26RFa(20–26) (LV-2106, 48) did not confirm that the Phe residues of 26RFa(20–26) (LV-2021, 1) participate in the bioactive conformation of the peptide or that some mandatory features are absent of these analogs (Table 2).

Compounds inactive as agonists were evaluated as possible antagonists of 26RFa-evoked calcium increase in cultured Gα16-hQRFPR-transfected CHO cells. None of the members of this series were able to reverse the stimulatory effect of 10−7 M 26RFa on [Ca2+]i, except LV-2068 (11) and LV-2188 (39) which antagonized 45–46% of the agonistic response with, respectively, modest and very low IC50 (Table 2, Figure 4).

Figure 4.

Figure 4

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Effect of graded concentrations of Phe-modified 26RFa(20−26) analogs on 26RFa-evoked [Ca2+]i mobilization in Gα16-hQRFPR-transfected CHO cells. Prototype dose-inhibition curves of [ptBuPhe22]26RFa(20–26) (LV-2068, 11, closed circles) and [DTrp26]26RFa(20–26) (LV-2188, 39, closed squares) on 10−7 M 26RFa-evoked response. Data are mean ± SEM of triplicate. The IC50 value calculated from these representative dose–inhibition curves were 4495 nM for 11 (LV-2068) and 5841 nM for 39 (LV-2188).

To summarize, the three phenylalanine units exhibit differential contributions to the biological activity of 26RFa(20–26). The Phe22 residue appears to be the most permissive as it tolerated N-substitution like in [Tic22]26RFa(20–26) (LV-2211, 28) which was 5-folds more potent than the lead peptide. Conversely, most modifications of Phe24 and Phe26 did not improve the activity with the exception of para substituents of lesser size than that of a tertbutyl group. Indeed, [Pcp24]26RFa(20–26) (LV-2232, 15), [Pcp26]26RFa(20–26) (LV-2193, 16) and [pNO2Phe26]26RFa(20–26) (LV-2194, 19) were 2-, 3.5- and 3.3-fold more potent than 26RFa(20–26) (LV-2021, 1). Although the amide NH of Phe24 residue was probably not involved in H-bond, our results suggest that some flexibility of the peptide backbone is required at this point. Noteworthy, not only the H-bond capacity, but other features were also impacted by these modifications such as the side chain presentation geometry, amide cis/trans isomerization equilibrium, and/or β-sheet potential of the analog with a wide range of steric, electronic and hydrophobic characteristics.

2.2. Impact of Concomitant Modifications of Gly20, Gly21 and Phe22 Residues

The variety of conformations characterized in enkephalins [52] and other short peptide models containing contiguous Gly units [53] reveals an important heterogeneity in 3D structures of the Gly-Gly segment influenced by both neighboring residues and environment. In the homology model of hQRFPR, the Gly-Gly motif of 26RFa(19–26) seems to adopt an extended conformation [45] generally observed in β-strands that form part of β-sheets. We have previously investigated the local requirement of the N-terminal dipeptide extremity of 26RFa(20–26) and one of the most interesting results has been achieved by introducing a 4-carboxymethylpiperazine (Cmpi) unit in place of the native Gly-Gly pair which leads to the 5-fold more potent analog [Cmpi21]26RFa(21–26) (LV-2043) than the reference heptapeptide [37]. Herein, to probe the space around this peptidomimetic moiety, we have introduced 2-piperazino-2-aryl-acetic acid racemate derivatives (Figure 5) in place of the first three 26RFa(20–26) residues (Table 3) and evaluated the in vitro activity of each diastereoisomer (Table 4). In this series, analogs LV-2102 (52) and LV-2175 (55) containing 2-piperazino-2-[(4-fluoro)phenyl]acetic acid (Cmpi(4-FPhg)) and 2-piperazino-2-[(2-fluoro)phenyl]acetic acid (Cmpi(2-FPhg)) respectively, demonstrated similar [Ca2+]i-mobilizing activity in hQRFPR-transfected cells compared to the lead peptide, while other derivatives exhibited slightly (LV-2101, 51; LV-2103, 53; LV-2104 54; LV-2183, 63) or detrimentally lower potency (Table 4). Altogether, these results suggest that incorporation of an aryl substituent on the methylene of the Cmpi building block with the concomitant deletion of the Phe22 residues do neither improve the ability of [Cmpi21]26RFa(21–26) to activate hQRFPR nor reverse the 26RFa-evoked effect.

Figure 5.

Figure 5

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2-Piperazino-2-aryl-acetic acid derivatives used in place of the H-Gly-Gly-Phe- sequence and incorporated in 49–64. These peptidomimetic moieties were used as racemate.

Table 3.

Chemical data for 26RFa(22–26) analogs.

Peptide Derivative Code HPLC MS
Rt (min) a Purity (%) Calcd b Obsd c
1 26RFa(20–26) LV-2021 18.0 99.9 815.41 816.53
49 [Cmpi(Phg)22]26RFa(22–26) dia 1 LV-2099 12.8 99.9 756.41 757.46
50 [Cmpi(Phg)22]26RFa(22–26) dia 2 LV-2100 12.9 99.9 756.41 757.46
51 [Cmpi(4-FPhg)22]26RFa(22–26) dia 1 LV-2101 15.3 99.9 774.40 775.37
52 [Cmpi(4-FPhg)22]26RFa(22–26) dia 2 LV-2102 15.7 99.9 774.40 775.39
53 [Cmpi(3-FPhg)22]26RFa(22–26) dia 1 LV-2103 15.3 99.9 774.40 775.42
54 [Cmpi(3-FPhg)22]26RFa(22–26) dia 2 LV-2104 15.8 99.9 774.40 775.41
55 [Cmpi(2-FPhg)22]26RFa(22–26) dia 1 LV-2175 13.9 98.4 774.40 775.47
56 [Cmpi(2-FPhg)22]26RFa(22–26) dia 2 LV-2176 14.7 99.9 774.40 775.53
57 [Cmpi(4-TfmPhg)22]26RFa(22–26) dia 1 LV-2177 25.3 99.9 824.39 825.47
58 [Cmpi(4-TfmPhg)22]26RFa(22–26) dia 2 LV-2178 26.2 99.9 824.39 825.39
59 [Cmpi(4-MeOPhg)22]26RFa(22–26) dia 1 LV-2179 13.2 99.9 786.42 787.47
60 [Cmpi(4-MeOPhg)22]26RFa(22–26) dia 2 LV-2180 13.4 99.9 786.42 787.41
61 [Cmpi(3,4-diMeOPhg)22]26RFa(22–26) dia 1 LV-2181 10.9 99.9 816.43 817.37
62 [Cmpi(3,4-diMeOPhg)22]26RFa(22–26) dia 2 LV-2182 11.5 99.9 816.43 817.49
63 [Cmpi(3-Pyg)22]26RFa(22–26) dia 1 LV-2183 19.5 99.9 757.40 758.40
64 [Cmpi(3-Pyg)22]26RFa(22–26) dia 2 LV-2184 20.3 99.9 757.40 758.31
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a Retention time determined by analytical RP-HPLC. b Theorical monoisotopic molecular weight. c m/z [MH]+ value assessed by MALDI-TOF-MS. Cmpi(Phg), 2-piperazino-2-phenylacetic acid; Cmpi(2-FPhg), 2-piperazino-2-[(2-fluoro)phenyl]acetic acid; Cmpi(3-FPhg), 2-piperazino-2-[(3-fluoro)phenyl]acetic acid; Cmpi(4-FPhg), 2-piperazino-2-[(4-fluoro)phenyl]acetic acid; Cmpi(4-TfmPhg), 2-piperazino-2-[(4-trifluoromethyl)phenyl]acetic acid; Cmpi(4-MeOPhg), 2-piperazino-2-[(4-methoxy)phenyl]acetic acid; Cmpi(3,4-diMeOPhg), 2-piperazino-2-[(3,4-dimethoxy)phenyl]acetic acid; Cmpi(3-Pyg), 2-piperazino-2-(3-pyridyl)acetic acid. Diastereoisomers are numbered according to their elution order in RP-HPLC.

Table 4.

Biological data for 26RFa(22–26) analogs.

Peptide Derivative Code EC50 IC50
(nM) (nM)
1 26RFa(20–26) LV-2021 1640 ± 259 -
49 [Cmpi(Phg)22]26RFa(22–26) dia 1 LV-2099 >10,000 ND
50 [Cmpi(Phg)22]26RFa(22–26) dia 2 LV-2100 >10,000 ND
51 [Cmpi(4-FPhg)22]26RFa(22–26) dia 1 LV-2101 4050 ± 1200 -
52 [Cmpi(4-FPhg)22]26RFa(22–26) dia 2 LV-2102 1660 ± 788 -
53 [Cmpi(3-FPhg)22]26RFa(22–26) dia 1 LV-2103 3560 ± 440 -
54 [Cmpi(3-FPhg)22]26RFa(22–26) dia 2 LV-2104 2710 ± 1000 -
55 [Cmpi(2-FPhg)22]26RFa(22–26) dia 1 LV-2175 2480 ± 1113 -
56 [Cmpi(2-FPhg)22]26RFa(22–26) dia 2 LV-2176 >10,000 ND
57 [Cmpi(4-TfmPhg)22]26RFa(22–26) dia 1 LV-2177 >10,000 ND
58 [Cmpi(4-TfmPhg)22]26RFa(22–26) dia 2 LV-2178 >10,000 ND
59 [Cmpi(4-MeOPhg)22]26RFa(22–26) dia 1 LV-2179 >10,000 ND
60 [Cmpi(4-MeOPhg)22]26RFa(22–26) dia 2 LV-2180 >10,000 ND
61 [Cmpi(3,4-diMeOPhg)22]26RFa(22–26) dia 1 LV-2181 >10,000 ND
62 [Cmpi(3,4-diMeOPhg)22]26RFa(22–26) dia 2 LV-2182 >10,000 ND
63 [Cmpi(3-Pyg)22]26RFa(22–26) dia 1 LV-2183 4990 ± 2800 -
64 [Cmpi(3-Pyg)22]26RFa(22–26) dia 2 LV-2184 >10,000 ND
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Data are mean ± SEM of at least three independent experiments performed in triplicate. Cmpi(Phg), 2-piperazino-2-phenylacetic acid; Cmpi(2-FPhg), 2-piperazino-2-[(2-fluoro)phenyl]acetic acid; Cmpi(3-FPhg), 2-piperazino-2-[(3-fluoro)phenyl]acetic acid; Cmpi(4-FPhg), 2-piperazino-2-[(4-fluoro)phenyl]acetic acid; Cmpi(4-TfmPhg), 2-piperazino-2-[(4-trifluoromethyl)phenyl]acetic acid; Cmpi(4-MeOPhg), 2-piperazino-2-[(4-methoxy)phenyl]acetic acid; Cmpi(3,4-diMeOPhg), 2-piperazino-2-[(3,4-dimethoxy)phenyl]acetic acid; Cmpi(3-Pyg), 2-piperazino-2-(3-pyridyl)acetic acid. Diastereoisomers are numbered according to their elution order in RP-HPLC. ND, not detectable.

3. Materials and Methods

3.1. Reagents

All Fmoc-amino acid residues and building blocks and O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Christof Senn Laboratories (Dielsdorf, Switzerland) or PolyPeptide (Strasbourg, France). Rink amide 4-methylbenzhydrylamine (MBHA) resin was from Novabiochem (Darmstadt, Germany) and N-methylpyrrolidone (NMP), dimethylformamide (DMF) and dichloromethane (DCM) were from Biosolve (Dieuze, France). N,N-Diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), triisopropylsilane (TIS), tert-butylmethylether (TBME) were supplied from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Acetonitrile was from Fisher Scientific (Illkirch, France) and α-cyano-4-hydroxycinnamic acid (CHCA) matrix from LaserBioLabs (Valbonne, France).

3.2. Peptide Synthesis and Purification

All peptides and derivatives were synthesized by the solid phase methodology on Rink amide MBHA resin using a Liberty microwave-assisted automated peptide synthesizer (CEM, Saclay, France) and the standard manufacturer’s procedures at 0.1 mmol scale as previously described [54]. All Fmoc-amino acids and building blocks (0.5 mmol, 5 equiv) were coupled by in situ activation with HBTU (0.5 mmol, 5 equiv) and DIEA (1 mmol, 10 equiv). Peptides and derivatives were deprotected and cleaved from the resin by adding a TFA/TIS/H2O (99.5/0.25/0.25) mixture for 120 min at room temperature. After filtration, crude peptides were precipitated by addition of TBME, centrifuged and recovered by elimination of the supernatant (3 folds). Peptides and derivatives were purified by reversed-phase HPLC (RP-HPLC) on a 2.2 × 25 cm Vydac 218TP1022 C18 column (Grace, Epernon, France) using a linear gradient (10–40%, 10–50%, 10–60%, 20–40%, 20–50% or 20–60% over 45 min) of acetonitrile/TFA (99.9/0.1) at a flow rate of 10 mL/min. The purified peptides were then characterized by MALDI-TOF mass spectrometry on a UltrafleXtreme (Bruker, Strasbourg, France) using CHCA as a matrix. Analytical RP-HPLC, performed on a 0.46 × 25 cm Vydac 218TP54 C18 column (Grace), showed that the purity of all compounds was >97.3%.

3.3. Cell Culture

Stably transfected hQRFPR CHO cells were obtained as previously described [36,37,38]. 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 hQRFPR using geneticin G418 (500 µg/mL) (Life Technologies, Villebon-Sur-Yvette, France) in a humidified 5% CO2 atmosphere at 37 °C.

3.4. Calcium Mobilization Assays

Changes in intracellular Ca2+ concentrations induced by 26RFa(20–26) analogs in CHO-Gα16-hQRFPR-transfected cells were measured on a benchtop scanning fluorometer Flexstation III (Molecular Devices, Sunnyvale, CA, USA) as previously described [36,37,38,55,56]. Briefly, 96-well assay black plates with clear bottoms (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) for 1 h in the presence of 0.01% pluronic acid, washed thrice, and incubated for 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 the 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.

3.5. 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 8.0 software (Graphpad Software, San Diego, CA, USA). EC50 and the IC50 values were determined from concentration–response curves using a sigmoidal dose–response fit with variable slope from at least three independent determinations. Differences between 26RFa(20–26) and analog activities were analyzed by the Mann–Whitney test. p values < 0.05 were considered significant.

3.6. Nomenclature of Targets and Ligands

All targets and ligands used throughout this manuscript conform with the guidelines outlined by the International Union of Basic and Clinical Pharmacology and British Pharmacological Society (IUPHAR/BPS) Guide to Pharmacology [5,57].

4. Conclusions

The C-terminal extremity of 26RFa was previously identified as a pivotal region to modulate its signaling at hQRFPR [36]. In this work, the Phe22, Phe24 and Phe26 residues of 26RFa(20–26) were modified using series of point substitutions with natural, side chain-constrained, side chain-modified residues and peptidomimetic building blocks. Subtle chemical modifications in the sequence led to significant improvement in compound potency to activate hQRFPR. As such, a single modification of Phe22 with the steric restricted Tic residue decreased the EC50 value from 1640 ± 259 to 327 ± 170 nM, providing the most efficient modification at this sequence position. Anchored substituents in the para position of each Phe unit were the most versatile investigated modification. Thereby, the introduction of a para-chloro-phenylalanine in place of the native Phe24 moiety emerged as a favorable replacement. This finding follows the same trend observed in [Pcp26]26RFa(20–26), highlighting a limited room to accommodate aromatic residues around the Phe aryl group. Although combination of multiple point-effective modifications does not necessarily translate into an additive or synergic effect, we have explored each position of 26RFa(20–26) for complete optimization of its sequence. Future challenges will be to convert several of these point modifications to low molecular weight 26RFa analogs for metabolic disorder, obesity or diabetes therapies. We are confident that, by utilizing subtle amendments, we can design 26RFa(20–26)-based compounds with nanomolar potency, functional selectivity and in vivo bioavailability.

Abbreviations

AHPPA: (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid; [Ca2+]i, intracellular calcium concentration; Cmpi, 4-carboxymethylpiperazine; Cmpi(2-FPhg), 2-piperazino-2-[(2-fluoro)phenyl]acetic acid; Cmpi(4-FPhg), 2-piperazino-2-[(4-fluoro)phenyl]acetic acid; hQRFPR, human QRFP receptor; 2-Nal, 3-(2-naphtyl)-alanine; NMePhe, N-methyl-phenylalanine; NPhe, N-benzyl-glycine; Thi, 3-(2-thienyl)-alanine; Oic, octahydroindole-2-carboxylic acid; Phg, phenylglycine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

Author Contributions

B.L. contributed to compound syntheses, acquisition of data, data analysis/interpretation, drafting of the manuscript. K.A., C.N., O.L.M. and C.D. participated in acquisition of data and their analysis/interpretation. J.A.B., J.C., D.V., G.P. and M.P. provided valuable editorial comments and relevant bibliographic references. H.V. and N.C. contributed to critical revision of the manuscript. J.L. conceived and designed the experiments with B.L., K.A. and C.N., contributed to data analysis/interpretation, drafting of the manuscript and approval of the article. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by INSERM, the University of Normandy Rouen, the Region Normandy and the European Union. Europe gets involved in Normandy, with European Regional Development Fund (ERDF).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are generated during this study.

Conflicts of Interest

The authors declare no competing financial interest.

Sample Availability

Samples of the compounds 1–64 are available from the authors.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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