Discovery of New Allosteric Modulators of the Urotensinergic System

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Discovery of new allosteric modulators of the urotensinergic system through substitution of the URP phenylalanine residue Etienne Billard, Terence Hébert, and David Chatenet J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00789 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 10, 2018

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Journal of Medicinal Chemistry

Discovery of new allosteric modulators of the urotensinergic system through substitution of the Urotensin II-related peptide (URP) phenylalanine residue.

Étienne Billard,† Terence E. Hébert,‡ David Chatenet†,*

†INRS

- Institut Armand-Frappier, Groupe de Recherche en Ingénierie des Peptides et en

Pharmacothérapie (GRIPP), Université du Québec, Ville de Laval, Québec H7V 1B7, Canada ‡Department

of Pharmacology and Therapeutics, McGill University, Montréal, Québec

H3A 1A3, Canada

Abstract : Urotensin II (UII) and urotensin II-related peptide (URP) are functionally selective, suggesting that these two hormones might play distinct physiological through different interactions with their cognate receptor UT. Hypothesizing that the Phe3 residue of URP, which is also present in hUII, is a key-element of its specific activation with UT, we evaluated the impact of its replacement by non-natural amino acids in URP. Each compound was evaluated for its ability to bind UT, induce rat aortic ring contraction and activate Gq, G12 and β-arrestin 1 signalling pathways. Such modifications impaired contractile efficacy, reflected by a reduced aptitude to activate G12 in URP but not in the

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truncated but equipotent UII4-11. Moreover, we have identified two structurally different UT modulators: [D-Phe(pI)3]URP and [Bip3]URP, which exert a probe-dependent action against UII and URP. These compounds should help us understand the specific roles of these hormones as well as guide further therapeutic development.


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Introduction Discovered in 1999,1-4 the urotensin II receptor (UT), which binds two peptidic hormones termed urotensin II (human UII, hUII, H-Glu-Thr-Pro-Asp-[Cys-Phe-Trp-LysTyr-Cys]-Val-OH, 1) and urotensin II-related peptide (URP, Ala-[Cys-Phe-Trp-Lys-TyrCys]-Val-OH, 2), is regarded as a crucial regulator of physiological and pathophysiological processes of the cardiovascular system.5 Notably, the urotensinergic system is involved in the pathogenesis and progression of various diseases including metabolic disorders, congestive heart failure, atherosclerosis and both systemic and pulmonary hypertension.5 UII and URP are cyclic peptides that only differ by their extracyclic N-terminal residues, their bioactive cyclic core (-Cys-Phe-Trp-Lys-Tyr-Cys-) being conserved throughout species.5 Accordingly, structure-activity relationships (SAR) studies have clearly shown that both the nature and orientation of these intracyclic residues were extremely important for UT binding and activation. They further showed that the N-terminal domain of UII was unimportant for UII function since UII4-11 (3) retained binding affinity and contractile effects similar to UII.6-10 However, using dissociation kinetic experiments and virtual docking, we and others recently demonstrated the existence of an initial interaction between this N-terminal domain, and more particularly the glutamic and aspartic acids of UII, and UT that could lead to specific topological changes in receptor conformation and therefore subtle changes in how it is activated.11, 12 Accordingly, several studies have underlined differences in the biological activities of UII and URP including transcription initiation,13 cell proliferation,14 polyphosphoinositide turnover,14 ANP release,15 and myocardial contractile activities.15 Furthermore, in spontaneously hypertensive rats, expression of



URP and UT, but not of UII, was up-regulated suggesting that UII and URP may have distinct pathophysiological roles, at least in hypertension.16 Also, in patients with acute heart failure, a significant raise in UII and URP plasma was observed but surprisingly, plasma URP concentration was almost 10 times higher than that of UII suggesting a distinct processing of their precursor in pathological conditions.17 Taken together, these observations suggest that UII and URP show distinct patterns of functional selectivity, and that altered UT signaling in response to UII or URP could therefore lead to distinct role for each peptide in the pathogenesis and progression of UT-associated diseases including atherosclerosis, pulmonary arterial hypertension, heart failure, and cancer. Over the last few years, we have specifically focused on SAR of URP in order to identify crucial differences in the molecular pharmacology or UII and URP. We identified, through the replacement of the intracyclic tryptophan residue of URP, two molecules that can reduce the contractile efficacy of UII without altering URP-associated contraction, therefore exerting a probe-dependent action through a non-competitive mechanism.12, 18 Termed urocontrin (4, [Bip4]URP, Bip = 4,4′-biphenylalanine, Table 1, Figure 1) and urocontrin A (5, [Pep4]URP, Pep = (phenylethynyl)phenylalanine, Table 1, Figure 1), these two derivatives represent the first allosteric modulators of the urotensinergic system. More recently, we demonstrated that the Tyr6 residue represents a complex switch in URP that integrates both receptor-binding and efficacy-associated determinants.19 Notably, this study helped us identify [Pep6]URP (6, Table 1, Figure 1) as a biased UT agonist that was able to reduce URP- but not UII-mediated vasoconstriction. While several studies, including ours, have investigated the role of intracyclic Trp, Tyr, but also Lys residues, limited data are available regarding the specific role played by the


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phenylalanine residue and its physicochemical characteristics in UT recognition and activation.9 Its replacement by an alanine, i.e. [Ala6]UII (7, Table 1), [Ala3]URP (8, Table 1) and [Ala6]UII4-11 (9, Table 1), or its D-enantiomer, i.e. [D-Phe6]UII (10, Table 1) and [D-Phe3]URP (11, Table 1), dramatically reduced the ability to induce aortic ring contraction.6,

7

Using proteinogenic and non-proteinogenic residues, including cyclic

amino acids, it was noted that the physicochemical characteristics of the native phenylalanine were probably involved in activation processes while its orientation was instead involved in UT recognition.20 One of the most interesting and intriguing findings or the SAR analysis was the propensity of [Cha6]UII4-11 (12) (Cha: cyclohexylalanine, Table 1, Figure 1) to act as an antagonist while its corresponding URP analog, i.e. [Cha3]URP (13, Table 1, Figure 1), behaved as a weak partial agonist.20 Hence, depending on the N-terminal residue adjacent to the cyclic core, i.e. Asp vs Ala, the residue at position 6/3 in UII and URP seems to be differentially involved in UT interaction and activation. To further understand the role of the physicochemical characteristics of this residue in UT binding and signaling, we substituted the Phe residue with para-hindered aromatics (Figure 2) in URP and to some extend in UII4-11 and tested their ability to bind UT, to elicit rat aortic contraction and to activate the Gq, G12, and β-arrestin 1 (βarr1) signaling pathways. Our results suggest that the phenylalanine residue, whereas excluded from the first urotensinergic system pharmacophores,21 has in fact a crucial involvement in the signaling texture of UT ligands, and is also an access point to allosteric modulators



Results and Discussion

Rational. Prior to this study, we demonstrated that the replacement of Trp4 and Tyr6 residues of URP with linear, rigid and sterically hindered non-natural amino acid obtained through Pd-catalysed reactions starting from L/D-Phe(pI) allowed us to characterise several allosteric modulators.12, 18Considering that the intracyclic Phe3 is also an important residue of the conserved intracyclic portion, we decided to employ the same strategy in order to assess structural requirement at this position.

Binding Affinity of URP and related analogues. For each peptide, a purity higher than 98% was observed by RP-HPLC analysis and their molecular weight, as monitored by MS analysis, was in accordance with theoretical values (Table 2). As shown in Table 3, replacement of the Phe residue of URP with various hydrophobic L-substituents generated analogs (14-16) that retained significant binding affinity toward UT (Figure 3). For instance, 16 (pIC50 = 7.57 ± 0.19), which contains a bulkier side chain at position 3 compared to URP, binds UT with a potency similar to that observed for URP (pIC50 = 7.71 ± 0.09). It therefore appears that no major steric barrier in para of this residue is involved in its interaction with the UT binding pocket. While aromaticity is crucial to retain high binding affinity at hUT,20 introduction of a biphenylalanine (15; pIC50 = 8.27 ± 0.18, *P ≤ 0.05) at position 3 in URP or para-iodo-phenylalanine in a similar position in URP (14; pIC50 = 8.25 ± 0.12, **P ≤ 0.01) and at position 6 in UII4-11 (20; pIC50 = 8.40 ± 0.22, *P ≤ 0.05) significantly


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increased the affinity of these analogs compared to 2, suggesting that augmentation of the hydrophobic character in para is favorable for UT binding. Conversely, introduction of a small polar hydroxyl group in para of the phenylalanine in UII, i.e. [Tyr6]UII, is known to produce a ten-fold decrease in affinity.22 These results are consistent with docking experiments, performed with the NMR structures of P5U (22, [Pen5]UII4-11, Table 1, Figure 1) and urantide (23, [Pen5,D-Trp7,Orn8]UII4-11, Table 1, Figure 1), which revealed a close interaction of the Phe residue with two hydrophobic residues Val184 and Met188 of UT.23 Hence, enhancing the hydrophobicity at the para position of the phenyl ring may improve these interactions and consequently UT binding. In contrast to their Lcounterpart, analogs 17, 18, and 19 bind UT with weak affinity (pIC50 < 6), which is in accordance with the very low binding of [D-Phe3]URP,6 and [D-Phe6]UII.22 The loss of binding affinity may be the result of several non-exclusive parameters that include 1) perturbation of peptide secondary structure making it unsuitable for proper binding and/or 2) modification of the spatial arrangement of the phenyl-substituted side chain that prevents its proper interaction with intra- or intermolecular amino acids. Finally, we noted that the presence of an aspartic acid adjacent to the disulfide bridge, as in 20 and 21, had no particular impact on their binding propensity compared to 14 and 17.

Vasocontractile effects of URP and related analogues. Each compound was then evaluated for its propensity to induce aortic ring contraction (Table 3). All L-substituted URP analogs (Figure 4A), i.e. 14 (pEC50 = 7.50 ± 0.23, **P ≤ 0.01), 15 (pEC50 = 7.54 ± 0.44, *P ≤ 0.05) and 16 (pEC50 = 7.49 ± 0.32, **P ≤ 0.01), were significantly less potent than their parent peptide (pEC50 = 8.16 ± 0.08) but also



behaved as partial agonists, their maximum efficacy ranging from 25 to 53% (Table 3). All D-substituted URP analogs exhibited a very low efficacy (between 10 and 20%) but had a similar potency compared to URP (Figure 4B). While an increase in steric hindrance at that position is not problematic for UT binding, this physicochemical characteristic drastically altered vasocontractile activity. It was quite surprising that 17, 18, and 19, which bind UT with low affinity, acted as potent partial agonists. Similar observations were previously reported and linked to interspecies variability since the human and rat UT isoform only share 75% sequence identity.1 For instance, it was demonstrated that [Phe9]hUII, which also acted as a partial agonist in a rat aortic ring contraction assay, binds hUT with a greater affinity than rUT but triggers better Ca2+ mobilization mediated by rUT compared to hUT.22 Also, [DPhe6]hUII, which binds hUT with a better affinity than rUT, is 4 time more potent to activate Ca2+ mobilization through rUT compared to hUT.22 Altogether, these results suggest that the binding site accommodating the phenylalanine residue of URP may differ in hUT and rUT (Figure 5). Accordingly, it was observed that the phenyl ring in UII was in a close relationship with Met184 and Ile188 of rUT while in hUT,1, 24 those residues are respectively replaced by a valine and a methionine.23 To further support our assumption, it was reported that the low binding affinity for rUT compared to hUT of palosuran (24, 1-[2-(4-benzyl-4-hydroxypiperidin-1-yl)ethyl]-3-(2-methylquinolin-4-yl)urea, Figure 1), a potent UT antagonist, was correlated with a different interaction of its quinoline ring with the residue 184.25 Notably, the side chain of Met184 in rUT has an unfavorable interaction with the quinoline ring of palosuran, which is not present in hUT.25 Hence, these different analogues, whose residues at position 3 lie within the same environment as


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the quinoline ring of palosuran, might have a better interaction with rUT compared to hUT. Interestingly, 20 (pEC50 = 7.98 ± 0.21, Emax = 112 ± 6) behaved as a full agonist while its URP counterpart 14 acted as a partial agonist. The presence of an aspartic acid adjacent to the disulfide bridge, while having no impact on its binding profile, completely restored contractile efficacy of 20 compared to its URP counterpart 14. Once again, and as previously highlighted,11, 12, 19 these results revealed the importance of the first residue adjacent to the disulphide bridge in the biological activity of UT ligands. Notably, Asp4 in hUII is thought to establish an ionic bond with Arg193 of the extracellular loop 2 (ECL2) of hUT.11 Interestingly, the strict conservation of an acidic residue in all UII isoforms5 suggests that a similar interaction could still take place in UT isoforms as well. Hence, this complementary interaction (compared to URP) may locally modify the positioning of ECL2 and TM4, which encompass the binding pocket of the phenylalanine residue, and facilitate UT activation (Figure 5). Finally, inversion of the configuration of the para-substituted moiety at position 3 of 20, i.e. 21, dramatically reduced its contractile activity.

Vasocontractile antagonistic action against UII and URP Considering that most of the peptides in this library retained a relatively low efficacy, we tested them as UT antagonists at a concentration of 10-6 M (with the exception of the full agonist 20 and the partial agonist 14, whose efficacy was greater than 50%) (Table 4). Strikingly, 15 and 16, which produced, at 10-6M, a small but nonnegligible contraction, prevent UII- and URP-mediated contraction while their Dsubstituted counterpart, 18 and 19, had no antagonist activity. The antagonist activity of



15 and 16 is consistent with their high UT binding affinity and relatively low contractile action. In order to gain a better insight into the antagonist activity of 15, which appears to bind UT with a higher affinity than 16, we evaluated the impact of various concentrations on UII- and URP-mediated contraction (Table 5, Figure 6A and 6B). Figure 6 shows that increasing the concentration of 15 induced a concentration-dependent decrease of hUII and URP contractile efficiency. Our results show that the observed decrease in potency noticed for URP only is saturable (Table 5), which is not compatible with classical competitive antagonism. Although their mechanism of action cannot be clearly assessed, the ability of 15 and 16 to modulate the efficiency and potency of contraction induced by UII and URP may represent an allosteric mode of action or slow dissociation kinetics as previously reported for the antagonist GSK1562590 (25, 4′-[(1R)-1-[[(6,7-dichloro-3oxo-2,3-dihydro-4H-1,4-benzoxazin-4-yl)acetyl](methyl)amino]-2-(1-pyrrolidinyl)ethyl]3-biphenylcarboxamide hydrochloride).26 At the molecular level, and as mentioned earlier, the phenylalanine residue in UII and URP interacts with TM4 and ECL2, two domains that undergo important conformational changes in most class A GPCRs upon ligand binding. Notably, and as reported for the angiotensin II type 1 receptors,27 after the initial interaction, the receptor will transition from an “open-state” to a “lid-state” in which the ligand is locked inside the binding pocket.28 Therefore, considering the proximity of the phenyl ring at position 3 with ECL2 residues, and the loss of activity observed with para-substituted analogs, the antagonist activity of 15 and 16 may be explained by the disruption/stabilization of crucial interaction within this region that lock these ligands in a partially active state and blocks receptor access to UII and URP.


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Interestingly, 17 was able to significantly reduce UII maximal effect (Emax = 52 ± 4) without affecting its potency or URP-mediated contraction, therefore acting as a probe-dependent antagonist (Table 4, Figure 7). This probe-dependent activity of 17, which was not shared with the other D-analogues, is not easily explainable. However, and as recently reported with the probe-dependent antagonist [Pep6]URP, the data suggest an allosteric mode of action which could involve UT oligomers.19 Interestingly, palosuran, whose aromatic moiety also interacts with ECL2, is able to abolish reductions in perfusion pressure (PP) induced by UII but not URP in isolated rat hearts,15 therefore also acting as a probe-dependent antagonist. These results suggest the importance of ECL2 in the control of UT activation but also revealed that modification at position 3 in URP could open new vistas for the design of potent antagonists and probe-dependent allosteric modulators.

Gq, G12 and βarr1 activation. Aortic contraction is a multi-causal event involving several intracellular effectors. Considering the low contractile efficacy of URP compared to UII4-11-substituted analogues, we hypothesized that the observed partial agonism could be the result of biased signalling. Using BRET-based biosensors and a HEK 293 cell line stably expressing hUT (Table 6), we assessed the ability of each analog to activate Gq and G12, two effectors involved in UT-mediated aortic ring contraction, as well as βarr1.29, 30 Bias between Gq and G12 were calculated and tabulated in Table 7. All analogs behaved as full agonists for the Gq pathway albeit with variable potency (Table 6, Figure 8). While all Lsubstituted analogs retained a similar or better affinity for UT than URP, it is interesting



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to observe that the only compound that behaved as a full agonist in the aortic ring contraction, i.e. 20, is the only one that can trigger Gq activation (pEC50 = 8.15 ± 0.13) with a similar potency than URP (pEC50 = 8.44 ± 0.12). All the others have markedly reduced potency such as 16 (pEC50 = 7.56 ± 0.10, ***P ≤ 0.001) or 18 (pEC50 = 5.40 ± 0.10, ***P ≤ 0.001). In accordance with their lower binding affinity, D-substituted analogues appeared even less potent than their L-related analogs (Table 6). In line with our previous observations, it can be observed that increasing the bulkiness in para of the phenyl ring at position 3 in a URP scaffold may reduce receptor flexibility and consequently negatively affect Gq activation. Regarding G12 activation, except for 18 (pEC50 = 6.18 ± 0.25) and 19 (pEC50 = 6.42 ± 0.24), most of the analogs were able to activate G12 (Figure 9, Table 6) with potencies similar to that of URP (pEC50 = 7.04 ± 0.11). However, only UII4-11-substituted analogs 20 (Emax = 94 ± 5) and 21 (Emax = 91 ± 4) retained maximal efficacy similar to URP (Emax = 100 ± 4), with all URP-substituted analogs exhibiting a significantly lower efficacy such as 16 (Emax = 78 ± 4, **P ≤ 0.01) and 17 (Emax = 68 ± 4, ***P ≤ 0.001). Our results therefore show that substitution in para of the phenyl ring in UII4-11 and URP resulted in a different pattern of recognition and activation for G12, underlining the importance of the N-terminal domain/residues when designing ligands targeting specific UT conformations. However, considering that 14, 15 and 16 retained similar potencies to URP, an increase in the size of the para substituent had no impact on G12 activation potency. Interestingly, ratios of transduction coefficients of these two pathways revealed that while L-analogues appear relatively unbiased (Table 7), all the D-substituted, and ఛ

ఛ

more specifically 17 (7.8, ∆∆log( ) = 0.89 ± 0.08, ***P ≤ 0.001) and 18 (50, ∆∆log( ) ௄஺ ௄஺


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= 1.70 ± 0.08, ***P ≤ 0.001), are strongly biased toward G12 activation compared to URP. Finally, evaluation of these different analogs on βarr1 (Figure 10, Table 6) revealed that they all acted as either full (14, 16, 17, 20, 21) or partial (15, 18, 19) agonists for β-arrestin 1 recruitment. Also, since only the para-iodo substituted analogs 14 (pEC50 = 6.74 ± 0.19) and 17 (pEC50 = 6.45 ± 0.27 ) retained a potency similar to that of URP, while 15, 16, 18 and 19 were all significantly less potent than URP (Table 6 and Figure 10), alteration of the steric parameter but not side chain orientation seems correlated with a decrease in βarr1 activation potency. For global comparisons, transduction coefficients and maximal effects of the different analogs for the 3 different pathways were plotted in a radar graph (Figure 11). Here, it can be seen that only 20 possesses an activation pattern similar to that of URP. Altogether, while L-analogs do not appear to induce a strong bias toward particular pathways, inversion of configuration of the residue at position 3 of URP strongly favors G12 and βarr1 activation over Gq (Table 7). For instance, bias analysis revealed that 17 and 21, whose activation pattern differed only in G12 maximal activation, appear unbiased between G12 and βarr1 (G12/βarr1 ratio = 1.1 and 1.2, respectively) but biased toward G12 (G12/Gq ratio = 7.8 and 4.3, respectively) and βarr1 (βarr1/Gq ratio = 8.6 and 5.3, respectively) over Gq (Table 7). Altogether, the reduced ability of URP-based analogues modified at position 3 to stimulate G12 activation may reflect an altered UT active conformation following their binding.



Conclusion The study highlights, for the first time, the crucial role of the Phe residue of URP in the activation of UT and suggests that modifications at this position could open up new avenue in the design of allosteric modulators for the urotensinergic system. This residue was absent from all previously described UT antagonist pharmacophores.21 In the present study, we notably demonstrate that [D-Phe(pI)3]URP (17) was able to significantly reduce maximal UII effects without affecting URP-mediated contraction, while [Bip3]URP (15) presents at sub-nanomolar concentration a probe-dependent saturable competitive effect on URP only. The probe-dependent activity in the aortic ring contraction assay suggests that these compounds act through lateral allostery, as recently hypothesized,31 through UT homodimers. Also, we demonstrated the importance of the N-terminal domain of UII in UT activation. For instance, while [Phe(pI)3]URP (14) behaved as a partial agonist in aortic contraction, its UII4-11 counterpart [Phe(pI)6]UII4-11 (20), which only differs by the presence of an aspartic acid at the N-terminus instead of an alanine, acted as a full agonist. Finally, this study and others, demonstrate the importance of evaluating UT antagonism against both endogenous ligands, i.e. UII and URP. We therefore believe that some UT ligands described in the literature have been mischaracterized since no investigation was performed against URP-mediated functions and that such missing information could shed some light on the failure of UT antagonists in preclinical studies.


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Experimental section Peptidic synthesis and biological assays have been conducted according to our previously pubished article19 and the detailed experimental section is provided in the supporting information.



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Ancillary Information Detailed experimental section provided.

Corresponding Author *E-mail: [email protected]

Conflicts of interest The authors declare no conflicts of interest.

Acknowledgments We thank the Canadian Institutes of Health Research (CIHR, MOP-142184) and the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-201504848) for funding.

Abbreviations ACN, acetonitrile; Bip, biphenylalanine; Boc, tert-butoxycarbonyle; BOP, (Benzotriazol1-yloxy)tris(dimethylamino) bioluminescence

resonance

phosphonium energy

transfer;

hexafluorophosphate; Cha,

cyclohexyl-alanine;

BRET, DCM,

dichloromethane; DIEA, N,N-diisopropylethylamine; DMEM, Dulbecco's Modified Eagle's medium; DMF, dimethylformamide; FBS, foetal bovine serum; Fmoc, Fluorenylmethyloxycarbamate; GFP10, green fluorescent protein 10; GPCR, G proteincoupled receptor; HEK 293 cells, Human Embryonic Kidney 293 cells; K3[Fe(CN)6],


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potassium ferricyanide; MALDI-TOF, Matrix-assisted laser desorption/ionization-Time of

flight;

NMR,

nuclear

magnetic

resonance;

PdCl2(PPh3)2,

Bis(triphenylphosphine)palladium (II) dichloride; Pep, (phenylethynyl)-phenylalanine; Phe(pI), para-iodo-phenylalanine; PP, perfusion pressure; RlucII, Renilla luciferase II; RP-HPLC, Reverse phase-high performance liquid chromatography; SAR, structureactivity relationship; UII, Urotensin II; URP, urotensin II-related peptide; UT, urotensin II receptors.

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Grouped Tables and Artwork

Table 1. Amino acid sequences of URP (2) and analogues 4-13, 22 and 23. n◦

Peptide

2

URP

H-Ala-[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH

4

Urocontrin

H-Ala-[Cys-Phe-Bip-Lys-Tyr-Cys]-Val-OH

5

Urocontrin A

H-Ala-[Cys-Phe-Pep-Lys-Tyr-Cys]-Val-OH

6

[Pep6]URP

H-Ala-[Cys-Phe-Trp-Lys-Pep-Cys]-Val-OH

7

[Ala6]UII

H-Glu-Thr-Pro-Asp-[Cys-Ala-Trp-Lys-Tyr-Cys]-Val-OH

8

[Ala3]URP

H-Ala-[Cys-Ala-Trp-Lys-Tyr-Cys]-Val-OH

9

[Ala6]UII4-11

H-Asp-[Cys-Ala-Trp-Lys-Tyr-Cys]-Val-OH

10

[D-Phe6]UII

H-Glu-Thr-Pro-Asp-[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH

11

[D-Phe3]URP


12

[Cha6]UII4-11

H-Asp-[Cys-Cha-Trp-Lys-Tyr-Cys]-Val-OH

13

[Cha3]URP

H-Ala-[Cys-Cha-Trp-Lys-Tyr-Cys]-Val-OH

22

P5U

H-Asp-[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH

Sequence

Urantide H-Asp-[Pen-Phe-Trp-Orn-Tyr-Cys]-Val-OH 23 Substituted amino acids are indicated in bold letters. D-Amino acids are indicated in bold italic letters. Non-natural amino-acids (Bip, Pep, Cha, Pen and Orn) are depicted in figure 1.


Journal of Medicinal Chemistry 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

Page 24 of 59

Table 2. Amino acid sequences and analytical data for URP and its related analogues. n◦

Peptide

Sequence

Calculateda

Observedb

Purityc

2

URP


1017.43

1017.56

99

14

[Phe(pI)3]URP

H-Ala-[Cys-Phe(pI)-Trp-Lys-Tyr-Cys]-Val-OH

1143.33

1143.52

98

15

[Bip3]URP

H-Ala-[Cys-Bip-Trp-Lys-Tyr-Cys]-Val-OH

1093.46

1093.67

98

16

[Pep3]URP

H-Ala-[Cys-Pep-Trp-Lys-Tyr-Cys]-Val-OH

1117.46

1117.67

99

17

[D-Phe(pI)3]URP

H-Ala-[Cys-Phe(pI)-Trp-Lys-Tyr-Cys]-Val-OH

1143.33

1143.55

98

18

[D-Bip3]URP

H-Ala-[Cys-Bip-Trp-Lys-Tyr-Cys]-Val-OH

1093.46

1093.90

99

19

[D-Pep3]URP

H-Ala-[Cys-Pep-Trp-Lys-Tyr-Cys]-Val-OH

1117.46

1117.65

99

20

[Phe(pI)6]UII4-11

H-Asp-[Cys-Phe(pI)-Trp-Lys-Tyr-Cys]-Val-OH

1187.32

1187.34

99

[D-Phe(pI)6]UII4-11 H-Asp-[Cys-Phe(pI)-Trp-Lys-Tyr-Cys]-Val-OH 1187.32 1187.44 98 21 a b Theorical monoisotopic molecular weight as calculated with ChemDraw Ultra 7.0.1. m/z value assessed by MALDI-TOF-MS. DAmino acids are indicated in bold italic letters. cPercentage purity determined by high-performance liquid chromatography using the eluents: A (H2O with 0.1% TFA) and B (100% CH3CN) with a gradient slope of 2% B/min, at a flow rate of 1 mL/min on a Vydac C18 column. Detection at 229 nm.


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Table 3. Binding affinity and contractile activity of URP and related analogs. Binding

Aortic ring contraction

n◦

Peptide

n

pIC50

n

pEC50

Emaxa

2

URP

5

7.71 ± 0.09

22

8.16 ± 0.08

107 ± 3

14

[Phe(pI)3]URP

4

8.25 ± 0.12**

3

7.50 ± 0.23**

53 ± 5***

15

[Bip3]URP

4

8.27 ± 0.18*

3

7.54 ± 0.44*

25 ± 3***

16

[Pep3]URP

4

7.57 ± 0.19

4

7.49 ± 0.32**

28 ± 3***

17

[D-Phe(pI)3]URP

4

Discovery of New Allosteric Modulators of the Urotensinergic System

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