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. 2020 Aug 20;11(9):1717–1722. doi: 10.1021/acsmedchemlett.0c00223

Insights into the Molecular Determinants Involved in Urocontrin and Urocontrin A Action

Etienne Billard 1, David Chatenet 1,*
PMCID: PMC7488282  PMID: 32944139

Abstract

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In the past few years, we have identified two allosteric modulators of the urotensinergic system with probe-dependent action, termed Urocontrin (UC) and Urocontrin A (UCA). Such action is atypical and important since it will allow us to understand the specific function of the functionally selective cognate ligands of this system, namely urotensin II and urotensin II-related peptide. Delineating the molecular determinants involved in this particular behavior would represent an important step toward designing small molecules suitable for pharmacologic and/or therapeutic intervention. Hence, we undertook an exploratory research by replacing the Trp4 residue of URP with several para-substituted phenylalanine amino acids in order to get a grasp on the required nature, distance, and orientation of the side chain of this residue for allosteric modulatory action. We found that the position of the second aromatic group is crucial, and we identified two new allosteric modulators: [Trip4]URP and [Phe(pPy-4)4]URP with probe-dependent action.

Keywords: Urotensin II-related peptide, human UT, allosteric modulator, aortic ring bioassay, binding, urocontrin

Over the last two decades, the urotensinergic system, composed of the G protein-coupled receptor, UT, and two endogenous peptide ligands, urotensin II (UII, hUII = H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and urotensin II-related peptide (URP, H-Ala-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH), has garnered significant attention as a promising novel target for treatment of atherosclerosis, metabolic syndrome, pulmonary arterial hypertension, and congestive heart failure.1−4 As a matter of fact, several UT antagonists such as Urantide,5 SB611812,6,7 SB657510,2,3 KR-36676,8 and KR-369969 attenuated cardiac hypertrophy and fibrosis10 and improved myocardial contractility in various preclinical studies. However, despite these demonstrations of an active role of this system in several cardiovascular animal models, UT antagonist candidates fail to meet expectations in humans. One hypothesis explaining this lack of success might rely on an insufficient understanding of the specific roles of UII and URP which appear to be functionally selective.11,12 Over the years, a growing body of evidence suggests that UII and URP, despite bearing a common cyclic core sequence (c[Cys-Phe-Trp-Lys-Tyr-Cys]-), are in fact able to elicit common but also distinct biological actions, therefore dismissing their possible redundancy. While both peptides promote aortic contraction in isolated rat aortic rings13,14 as well as activate similar G-proteins on UT transfected human embryonary kidney cell line (HEK-293),15 they modulate in a distinct way transcription initiation,16 astrocyte proliferation,17 polyphosphoinositide turnover,17 ANP release,18 and myocardial contraction.18 Ligand-specific modulation of UT seems therefore essential for dissecting their biological functions and consequently their respective roles in the etiology of specific cardiovascular diseases.13−17,19 Thus, the ability to better understand UT functional selectivity could give a clear advantage in the design of properly targeted drugs and would certainly be of great therapeutic value.

UII and URP are closely related in terms of sequence: they share a common hexapeptide cyclic core but a distinct N-terminal domain. Structure–activity relationship (SAR) studies have demonstrated that equivalent substitutions on the common intracyclic amino-acids in hUII and URP can lead to crucial differences in biological activity.12,20,21 With both peptides adopting a similar ring conformation, it is suggested that their different biological activities result from different binding modalities within the orthosteric site of UT rather than alternate tridimensional structures. Accordingly, two ionic bridges between Glu1 and Lys196 and between Asp4 and Arg193 were noticed in the hUII/hUT complex only.23 It is in fact the variable N-terminal domain of the two ligands that triggers specific conformational changes within the UT binding pocket, promoting two different modes of binding between UII and URP.23,24 Prior to this work, our team underwent a SAR study on the intracyclic Trp4 of URP and discovered two compounds: [Bip4]URP25 and [Pep4]URP,22 respectively named Urocontrin (UC) and Urocontrin A (UCA), that are able to antagonize the ex-vivo rat and monkey aortic contractions caused by hUII without affecting URP-mediated ones. Also, in HEK 293-hUT, both derivatives can increase the dissociation rate of 125I-hUII without affecting 125I-URP.22,25 Because of this probe dependence effect, they have been described as allosteric modulators (the firsts of this system) and therefore represent an innovative scaffold to explore UII/URP specific actions.

Thus, it appeared actually crucial to deepen our comprehension of the interactions that such compounds have with UT, by exploring the molecular determinants needed to fit in this special “urocontrin-allosteric cavity” with a well-addressed SAR study. From the above-mentioned observations on UII and URP, we stated that a library of URP analogs bearing a para-substituted phenylalanine in position 4 (Figure 1) would be a good starting point to create UT peptidic allosteric modulators. With this scaffold, substitutions were chosen to examine three major parameters: (1) the impact of spatial orientation (D-amino acids), (2) the spacing between aromatic moieties, and (3) the nature of the aromatic substitution. The generated library (Table 1) produced several new allosteric modulators that allowed us to determine crucial fitting parameters for this deep and linear pocket. Taken together, these results represent an interesting opening to the design of probe-dependent hUT smart-ligands.

Figure 1.

Figure 1

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Chemical structures of the different nonproteinogenic amino acids introduced at the fourth position in URP.

Table 1. Sequences and Molecular Weights of URP and Related Analogs.

Peptide Sequence Calculated Observeda
URP H-Ala-[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH 1017.43 1017.56
[D-Phe(pI)4]URP H-Ala-[Cys-Phe-D-Phe(pI)-Lys-Tyr-Cys]-Val-OH 1105.32 1105.12
[D-Bip4]URP H-Ala-[Cys-Phe-D-Bip-Lys-Tyr-Cys]-Val-OH 1055.45 1055.24
[D-Pep4]URP H-Ala-[Cys-Phe-D-Pep-Lys-Tyr-Cys]-Val-OH 1079.45 1079.62
[Trip4]URP H-Ala-[Cys-Phe-Trip-Lys-Tyr-Cys]-Val-OH 1131.48 1131.50
[Tyr(OBn)4]URP H-Ala-[Cys-Phe-Tyr(OBn)-Lys-Tyr-Cys]-Val-OH 1085.46 1085.49
[Phe(pThio-3)4]URP H-Ala-[Cys-Phe-Phe(pThio-3)-Lys-Tyr-Cys]-Val-OH 1061.41 1061.62
[Phe(pPy-3)4]URP H-Ala-[Cys-Phe-Phe(pPy-3)-Lys-Tyr-Cys]-Val-OH 1056.45 1056.77
[Phe(pPy-4)4]URP H-Ala-[Cys-Phe-Phe(pPy-4)-Lys-Tyr-Cys]-Val-OH 1056.45 1056.33
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a

MALDI mass spectral analysis [M + H]+. The observed m/z of the monoisotope compared with the calculated monoisotopic mass. D-Amino acids are indicated in bold italic letters.

Results and Discussion

In the present study, we first addressed how inversion of configuration of the nonproteinogenic amino acids in the two UT allosteric modulators ([D-Bip4]URP and [D-Pep4]URP) as well as their synthetic precursor ([D-Phe(pI)4]URP) (Figure 1) affects their respective pharmacological profiles. Competition experiments (Table 2; Figure 2) reveal that [D-Phe(pI)4]URP (pIC50 = 8.50 ± 0.11), [D-Bip4]URP (pIC50 = 8.61 ± 0.11), and [D-Pep4]URP (pIC50 = 7.92 ± 0.12) retain a binding affinity similar to that of URP (pIC50 = 8.29 ± 0.08), as no statistical differences were found. Indeed, supporting our results, previous structure–activity relationship studies have described that inversion of configuration of the Trp residue in URP26 or hUII27 is relatively well tolerated, with both analogs, i.e. [D-Trp4]URP and [D-Trp7]hUII, retaining significant activity and affinity for UT while inversion of configuration of the three other intracyclic residues (Phe, Lys, and Tyr) led to a complete loss of binding. However, [Phe(pI)4]URP, [Bip4]URP, and [Pep4]URP exhibit respectively a 5 times,24 31 times,25 and 19 times22 decrease in affinity compared with URP. The D-orientation at this position in a para-substituted phenylalanine scaffold is therefore beneficial for the affinity.

Table 2. Binding Affinity and Contractile Activity of URP and Related Analogs.

  Binding Affinity Aortic ring contraction
Peptide pIC50 pEC50 Emaxa
URP 8.29 ± 0.08 8.16 ± 0.08 107 ± 3
[D-Phe(pI)4]URP 8.61 ± 0.11 7.54 ± 0.10* 114 ± 5
[D-Bip4]URP 8.50 ± 0.11 7.32 ± 0.13** 97 ± 7
[D-Pep4]URP 7.92 ± 0.12 7.55 ± 0.15* 99 ± 8**
[Trip4]URP 6.87 ± 0.10*** 8.11 ± 0.38 9 ± 1***
[Tyr(OBn)4]URP 8.10 ± 0.12 <5.5 80%b
[Phe(pThio-3)4]URP 7.44 ± 0.11*** <5.5 20%b
[Phe(pPy-3)4]URP 6.99 ± 0.11*** <5.5 24%b
[Phe(pPy-4)4]URP 6.81 ± 0.15*** <5.5 5%b
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a

The maximum efficacy is defined as a percentage of the KCl-induced contraction (40 mM) divided by the hUII-induced tissue-response (10–6 M).

b

Maximum efficacy at 3 × 10–6 M. All values are expressed as mean ± SEM. Statistical analysis, using URP values as controls, were performed using unpaired Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Experiments were conducted at least 3 times and on tissues from different animals or on different cellular passages.

Figure 2.

Figure 2

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Representative displacement curves of URP and related analogs using HEK-293-hUT cells and 125I-URP as a radioligand. Data represent the mean ± SEM of at least four independent experiments each performed in duplicate.

Next, we evaluated the propensity of these three derivatives to trigger rat aortic ring contraction. [D-Bip4]URP, [D-Pep4]URP, and [D-Phe(pI)4]URP behave as full and potent agonists with a similar potency (Table 2; Figure 3) but approximately half a log less powerful than URP (pEC50 = 8.16 ± 0.08). Interestingly, [D-Phe(pI)4]URP (pEC50 = 7.54 ± 0.10; *P < 0.05) only exhibited a 4-fold decrease in potency compared to URP, while its L-counterpart, i.e. [Phe(I)4]URP, is 72 times less potent than URP.24 This is especially striking considering that [Bip4]URP is a very weak agonist and [Pep4]URP is devoid of agonistic activity in this assay.22,25 Thus, inversion of configuration at position 4 of UC and UCA is therefore detrimental for their allosteric modulatory action since it allows the restoration of a contractile activity. It is worth mentioning that [D-Trp4]URP is able to fully antagonize UII-induced contraction,26 whereas the present ligands behave as full agonists, implying that the L/D-tryptophan and L/D-para-substituted phenylalanine allow the exploration of very distinct bioactive conformations.

Figure 3.

Figure 3

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Vasoconstrictor effects of URP and related analogs on rat thoracic aorta rings.

Because [Phe(pI)4]URP is an agonist,24 and considering that both [Bip4]URP and [Pep4]URP act as allosteric modulators, we postulated that the minimal requirement in order to obtain ligands with such properties is an aromatic ring in the para position of phenylalanine. To address this question, two analogs, i.e. [Trip4]URP and [Tyr(OBn)4]URP (Figure 1) were generated. The Tyr(OBn) moiety is composed by two phenyl rings separated by a linker formed by two atoms (−CH2–O−), like Pep, but unlike it, there is no hyperconjugation between the two aromatics, and TyrOBn is neither linear nor constrained. Conversely, in the 4-(4-phenyl)-phenyl–phenylalanine (Trip) amino acid, the three aromatics are linear and totally conjugated, and the second ring could be seen as an isostere of the ethynyl function of Pep. The major difference could therefore consist in a greater length of the Trip side chain.

While [Tyr(OBn)4]URP (pIC50= 8.10 ± 0.12) exhibits a binding affinity similar to that of URP (pIC50 = 8.29 ± 0.08), [Trip4]URP affinity is significantly lower (pIC50= 6.87 ± 0.10; ***P < 0.001; Table 2 and Figure 2). Comparison between the binding affinity of [Trip4]URP and [Tyr(OBn)4]URP with the one previously reported for [Pep4]URP (pIC50= 6.60 ± 0.18)22 suggests that the rigidity of the para-substitution in position 4 but not the length of it alters UT binding.

Surprisingly, [Tyr(OBn)4]URP acted as a very weak (pEC50 < 5.5) but seemingly full agonist (80% of contraction recorded at 3 μM) (Table 2; Figure 3. By linking this activity with the affinity observed in the competition binding assay, it seems that the freedom of rotation of the second cycle offers ease to fit into the binding pocket but the lack of rigidity might prevent an adequate receptor activation. Since this derivative, which could have represented an interesting UCA alternative, exerts a very high contractile action at 1 μM (53%), no further investigation of its potential antagonistic activity was performed. [Trip4]URP retains a potency (pEC50 = 8.11 ± 0.38) similar to URP (pEC50 = 8.16 ± 0.08) but is a partial agonist with a very low efficacy (Emax = 9 ± 1%; Table 2; Figure 3).

When tested as an antagonist, [Trip4]URP (10–6 M), like UC and UCA, reduced hUII- but not URP-induced contraction (Emax= 79 ± 7% (*P < 0.05) and 99 ± 7%, respectively), and this effect was concentration-dependent (Table 3; Figure 4). However, when compared with the reduction of hUII-induced contraction observed with UC (Emax= 61 ± 7%)25 or UCA (Emax= 28 ± 3%)22 at the same concentration, it appears that [Trip4]URP has a weaker antagonist ability but still conserved a probe-dependent action. Since the antagonistic activity and probe-dependent action is retained but its potency appears to be lower than UCA, the Pep residue might be of optimal length for the development of UT allosteric modulators with probe-dependent action. Moreover, while the side chains of Bip, Pep, and Trip are linear, only the phenyl ring in UCA possesses freedom of rotation, since ortho-hydrogens create steric hindrance that limits the freedom of rotation in biphenyl scaffolds, imposing an angle of 44.4°2829 (Figure 5). Considering that UCA is devoid of agonistic property, to the opposite of UC and [Trip4]URP, it seems reasonable to conclude that a correct positioning of the second aromatic ring is mandatory for an absence of agonistic activity.

Table 3. Antagonist Contractile Activitiesa.

    Aortic ring contraction hUII
 Aortic ring contraction URP
Peptide M Emaxb pEC50 Emaxb pEC50
No treatment   101 ± 4 8.59 ± 0.12 107 ± 3 8.16 ± 0.08
[Trip4]URP 10–6 79 ± 7* 8.47 ± 0.27 99 ± 7 7.92 ± 0.23
  3 × 10–6 65 ± 3*** 8.68 ± 0.17 95 ± 4 8.41 ± 0.10
  10–5 55 ± 2** 8.65 ± 0.14 92 ± 7 8.18 ± 0.22
[Phe(pThio-3)4]URP 10–6 105 ± 7 8.12 ± 0.26 115 ± 4 7.89 ± 0.12
[Phe(pPy-3)4]URP 10–6 94 ± 4 8.68 ± 0.18 102 ± 6 8.07 ± 0.23
[Phe(pPy-4]4]URP 10–6 70 ± 6*** 8.85 ± 0.27 96 ± 7 8.53 ± 0.22
  3 × 10–6 25 ± 4*** 8.35 ± 0.64 94 ± 10 8.16 ± 0.37
  10–5 9 ± 1c d 92 ± 13 8.19 ± 0.41
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a

Effect of aortic ring pretreatment with various analogs (30 min) on hUII- or URP-induced contraction.

b

Maximum efficacy (Emax) is expressed as a percentage of the KCl-induced contraction (40 mM) divided by the tissue-response induced by hUII (10–6 M).

c

Basal contractile activity of [Phe(p4-Pyr)4]URP at 10–5 M.

d

No subsequent contraction by hUII was observed. All values are expressed as mean ± SEM. Statistical analyses were performed using unpaired Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, versus values obtained for UII or URP. Each replicate (n) was conducted on tissue from different animals.

Figure 4.

Figure 4

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Effects of [Trip4]URP (A) or [Phe(pPy-4)4]URP (B) on hUII- (top) or URP-induced contraction (bottom).

Figure 5.

Figure 5

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Schematic representation of Trip (left) and Pep (right) side-chains. In Trip, ortho-hydrogenes create a sterical barrier that limits the freedom of rotation of the phenyls rings.

Finally, we investigated whether the substitution of the phenyl ring in para by a heteroaromatic would impact the binding, contractile activity, or antagonist propensity of UC. Three derivatives, [Phe(pThio-3)4]URP, [Phe(pPy-3)4]URP, and [Phe(pPy-4)4]URP (Figure 1), have been prepared, and all of them retained a lower affinity for UT than URP (Table 1, Figure 2). While the introduction of the pyridine rings in [Phe(pPy-3)4]URP (pIC50 = 6.99 ± 0.11; ***P < 0.01) and [Phe(pPy-4)4]URP (pIC50 = 6.81 ± 0.15; ***P < 0.01) is more detrimental for their binding affinity compared to URP, it appears slightly better than the one reported for UC (pIC50 = 6.47 ± 0.12). Interestingly, [Phe(pThio-3)4]URP (pIC50 = 7.44 ± 0.11; ***P < 0.001) binds UT with an affinity lower than URP but significantly higher than UC, [Phe(pPy-3)4]URP, and [Phe(pPy-4)4]URP (*P < 0.05). Hence since thiophene is a π-electron rich heteroaromatic while pyridine is a π-electron deficient, the electronic density and/or repartition in the bicycle can thus play a role in their binding. We believe that H-bonding is not a pertinent descriptor for the binding since [Phe(pThio-3)4]URP exhibits a behavior different than [Phe(pPy-3)4]URP despite similar H-bond potential (Figure 6).

Figure 6.

Figure 6

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Schematic representation of the substituted residue and their neighboring Lys5. In [Phe(pThio-3)4]URP (left) and [Phe(pPy-3)4]URP (center) a H-bond might be expected according to the position of the heteroatom. Such interaction is not permitted in [Phe(pPy-4)4]URP (right).

Despite all of them retaining significant binding affinities, these compounds were very weak contractile agonists (Table 2, Figure 3, [Phe(pThio-3)4]URP, and [Phe(pPy-3)4]URP exhibiting less than 25% of contraction at the maximal tested dose while [Phe(pPy-4)4]URP was almost devoid of any contractile activity (5% contraction at 3 μM). Hence the substitution of the phenyl cycle of UC has little to no effect on UT agonism. We then evaluated the ability of these 3 compounds to antagonize hUII- and URP-induced contraction. Tested at 1 μM, [Phe(pThio-3)4]URP and [Phe(pPy-3)4]URP were unable to modulate hUII and URP vasoconstriction. Interestingly, [Phe(pPy-4)4]URP reduced in a concentration-dependent manner hUII- but not URP-mediated contraction (Table 3; Figure 4). At 10–5 M, this compound almost totally suppresses hUII-evoked contraction (9%) without modulating URP vasocontractile function (92 ± 13% versus 107 ± 3% for URP alone). When compared to the antagonistic activity of UC25 at similar concentration (for instance 3 μM), this compound is not better than UC. Hence, the substitution of the phenyl in UC by a pyridine-4-yl does not impact its pharmacological profile. Considering that the introduction of an electron-rich thiophene moiety leads to an inactive compound and that the activity of the electron-deficient ring pyridine-substituted analogues is only a function of the position of the heteroatom, the electronic density is not a key-factor in their allosteric modulation or probe-dependent effect. Since the nitrogen and sulfur heteroatoms can play a H-bond acceptor role, a plausible reason could be that the heteroatom in [Phe(pThio-3)4]URP and [Phe(pPy-3)4]URP establish a H-bond with the tertiary ammonium of the neighboring Lys5 (Figure 6), as the structure obtained from nuclear magnetic resonance of URP reveals numerous nuclear Overhauser effect contacts between the side chains of Trp4 and Lys5.23 In [Phe(pPy-4)4]URP, the orientation of the heteroatom prevents such interaction, and the two residues Phe(pPy-4)4 and Lys5 would behave as in UC, explaining the similar properties of these two compounds.

Overall, we have characterized two new allosteric modulators, [Trip4]URP and [Phe(pPy-4]4]URP, that retain similar affinity and capacity to reduce UII-evoked contraction albeit to different extents. Throughout this study, we have identified several molecular determinants that influence the modulatory action of UC and UCA including the L-orientation and rigidity of the para substituted Phe residue. We explored several substitutions in the UC scaffold and found that the second aromatic is not tolerant to 3-heteroaromatics but tolerant to the 4-aromatic Py-4, as [Phe(pPy-4)4]URP acts as an allosteric modulator. In the case of [Pep4]URP, the freedom of rotation of the second phenyl ring seems to confer ideal properties, as [Trip4] behaves similarly as UC but not UCA. We have also observed that affinity as measured with a competitive binding assay is not a relevant parameter to preclude the allosteric modulatory effect of a ligand. Indeed, because these compounds are able to establish a probe-dependence effect, they behave as noncompetitive antagonists. As demonstrated through dissociation experiments, UC25 and UCA22 are able to accelerate the dissociation rate of membrane-bound radiolabeled UII but not URP mimicking what is observed in the aortic ring contraction assay. We propose therefore that establishing a concentration–response regimen of an allosteric modulator in order to establish an “effective dissociation 50: ED50” would offer a crucial descriptive parameter in order to compare and understand the activity of these allosteric modulators. Accordingly, as we have now identified numerous compounds that act as allosteric modulators through the substitution of the residues Phe330 or Tyr631 or modification of the peptide backbone of URP/UII4–11,32−34 efforts will have to be put toward understanding the precise mechanisms subtending their probe-dependent actions.

Materials and Methods

Solid-phase peptide synthesis, palladium catalyzed cross-coupling reaction, aortic ring experiments, radioidination, and competition binding experiments as well as statistical analysis were performed as described in our previous work.30

Acknowledgments

We thank the Canadian Institutes of Health Research (CIHR, MOP-142184) and the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2015-04848) for funding.

Glossary

Abbreviations

Bip

biphenylalanine

HEK-293

human embryonary kidney-293

MALDI

matrix-assisted laser desorption/ionization

OBn

O-benzyl

Pep

(phenylethynyl)-phenylalanine

Phe(pI)

para-iodo-phenylalanine

Py

pyridine

SAR

structure–activity relationship

Thio

thiophene

Trip

triphenylalanine

UC

urocontrin

UCA

urocontrin A

UII

urotensin II

URP

urotensin II-related peptide

UT

urotensin II receptor.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

References

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