Urolinin: The First Linear Peptidic Urotensin-II Receptor Agonist

Article pubs.acs.org/jmc

Urolinin: The First Linear Peptidic Urotensin-II Receptor Agonist Sebastian Bandholtz,†,∥ Sarah Erdmann,†,∥ Jan Lennart von Hacht,†,∥ Samantha Exner,† Gerd Krause,‡ Gunnar Kleinau,§ and Carsten Grötzinger*,† †

Campus Virchow−Klinikum, Department of Hepatology and Gastroenterology and Molecular Cancer Research Center (MKFZ), Charité−Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany ‡ Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany § Institute of Experimental Pediatric Endocrinology, Charité−Universitätsmedizin Berlin, D-13353 Berlin, Germany S Supporting Information *

ABSTRACT: This study investigated the role of individual UII amino acid positions and side chain characteristics important for U-IIR activation. A complete permutation library of 209 UII variants was studied in an activity screen that contained single substitution variants of each position with one of the other 19 proteinogenic amino acids. Receptor activation was measured using a cell-based high-throughput fluorescence calcium mobilization assay. We generated the first complete UII substitution map for U-II receptor activation, resulting in a detailed view into the structural features required for receptor activation, accompanied by complementary information from receptor modeling and ligand docking studies. On the basis of the systematic SAR study of U-II, we created 33 further short and linear U-II variants from eight to three amino acids in length, including D- and other non-natural amino acids. We identified the first high-potency linear U-II analogues. Urolinin, a linear U-II agonist (nWWK-Tyr(3-NO2)-Abu), shows low nanomolar potency as well as improved metabolic stability.

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contribute to VSMC contraction and cell proliferation.8−10 In vivo studies concerning vascular effects mediated by U-II appeared inconclusive because two independent studies using identical procedures showed conflicting results.11,12 Yet, the potency of U-II in vasoconstriction, vasodilation, and inotropism supports a key role of this peptide in the cardiovascular homeostasis. The above-mentioned observations correspond well with reports that U-II might be involved in various diseases, including cardiovascular pathologies such as hypertension and arteriosclerosis. Thus, the U-II system exhibits a remarkable potential for the development of novel therapeutic strategies, especially those related to the treatment of cardiovascular diseases. Such developments require a precise knowledge of the pharmacophore elements within U-II essential for affinity and activity of this peptide. So far, because of its resemblance to somatostatin (SST), it is conceivable that some of the critical structural features already described for SST are shared with UII. Accordingly, both peptides contain the tripeptide Phe-TrpLys followed by one of the hydroxyl group containing residues Thr or Tyr. Moreover, it was demonstrated in SST that an interaction between the aromatic moieties of residues Phe6 and Phe11 stabilizes the orientation of residues Phe7, Trp8, Lys9, and Thr10. Correspondingly, the disulfide bridge in U-II is believed to act in a similar manner. SAR studies of SST showed that this

INTRODUCTION Urotensin II (U-II), a potent mammalian vasoconstrictor peptide, was initially isolated and characterized from the Gillichthys mirabilis (goby) urophysis.1 Later, human U-II cDNA was cloned and U-II appeared as an 11-amino acid peptide with cysteine residues in positions 5 and 10, forming a disulfide bridge through their side chains.2 The most prominent effects of U-II were observed in the mammalian cardiovascular arterial system in which it was frequently identified as a potent vasoconstrictor while the vasoconstricting effect of human U-II with regard to both potency and efficacy varied significantly between species, individuals, and vessels.3 U-II was described as the most potent vasoconstricting agent on the rat thoracic aorta, but in mouse, no matter which vessels were tested, vasoconstriction could not be detected. On the other hand, UII was found to be a potent vasoconstrictor on nonhuman primate arteries.4 The same study demonstrated U-II was a ligand of a GPCR previously known as GPR14.4 In the presence of endothelium, abdominal and small pulmonary arteries showed a strong vasodilation in response to U-II.5 Other investigations in vitro have shown that U-II is a potent inotropic agent on human cardiac muscle6 and that it induces vascular smooth muscle cell (VSMC) proliferation.7 Especially in VSMC, U-II-induced signaling via U-II receptor (U-IIR) is well characterized. After ligand binding, the Gq protein-coupled receptor activates the PLC−PKC cascade, thereby inducing intracellular Ca2+ mobilization and calmodulin/MLC kinase mediated contraction. Furthermore, RhoA and MAPK signaling © 2016 American Chemical Society

Received: February 3, 2016 Published: October 28, 2016 10100

DOI: 10.1021/acs.jmedchem.6b00164 J. Med. Chem. 2016, 59, 10100−10112

Journal of Medicinal Chemistry

Article

activity (EC50 values below 50 nM). However, substitution of Glu1 by Ile, Met, Cys, Ser, and Asn resulted in EC50 values above 50 nM, while substitutions of Thr2 with Lys, His, Leu, Tyr, and Cys similarly increased the EC50 value to about 120− 1800 nM. Exchanges of Pro3 and Asp4 proved to be more restricted: Pro3 could be replaced with His, Ile, Trp, Ala, Gln, Gly, and Asn, whereas Asp4 could be substituted by Ala, Val, Ile, Phe, Leu, Thr, and Gln without prominent reduction of the EC50 value. The exocyclic residue Val11 was found to be even more sensitive to substitution: introduction of most amino acids led to higher EC50 values, while variants containing Cys, Phe, Leu, Pro, Arg, Thr, Trp, or Tyr retained high receptor activity with EC50 values of below 50 nM. Compared to the exocyclic amino acids, most endocyclic substitution variants showed EC50 values higher than 50 nM, many of them above 400 nM. At position Trp7, the two aliphatic hydrophobic amino acids Leu and Met as well as the three aromatic residues His, Phe, and Tyr were tolerated. Lys8 could only be substituted by the two other basic amino acids Arg and His with minimal loss of activity. Correspondingly, Tyr9 could be switched to one of the hydrophobic amino acids Phe, Leu, Trp, and Met as well as Cys. All other substitution variants led to EC50 values above 50 nM in the intracellular Ca2+ mobilization assay. One notable exception was the fourth endocyclic amino acid, Phe at position six, which showed a high flexibility as nearly all substitutions led to EC50 values below 50 nM. Only replacement by the small amino acids Pro and Gly by the two basic amino acids Lys and Arg and by the hydrophilic Thr caused a rise in the EC50 value to between 50 and 400 nM. In native U-II, the two cysteine residues at positions 5 and 10 are known to form a disulfide bond, which induces the cyclic structure of U-II. All exchanges of Cys10 yielded EC50 values above 50 nM, the variants where Phe, Trp, Ala, and Gly were in the range of 50−100 nM. Other changes led to EC50 values up to 5000 nM. Likewise, most substitutions of Cys5 showed EC50 values greater than 50 nM or a complete loss of activity. However, the introduction of a valine residue at this position led to a linear peptide with an EC50 value that was only moderately increased as compared to wild-type U-II (Figure 1). Truncation and Modification Studies. The dispensability of the exocyclic amino acids for receptor activation has been shown before,15 and it corresponds well with the high substitutional flexibility of this structural domain observed in the current study. To further delineate the structure−activity relationship in the peptide, U-II was systematically truncated from either end. Receptor activity was analyzed in an intracellular Ca2+ mobilization assay on HEK293A cells stably expressing the human U-IIR. Wild-type U-II resulted in an EC50 of 1.46 ± 0.6 nM and a ΔF/F0 ratio of 1.82 ± 0.2. Simultaneous removal of the first four amino acids Glu1, Trp2, Pro3, and Asp4 (210, 214) had no influence on either potency or efficacy, while consecutive withdrawal of Val11 (215) resulted in a slight increase of the EC50 to 3.8 ± 1.1 nM, respectively (Table 1). However, the elimination of either Cys5 or Cys10 caused a strong increase of the EC50 of 201 ± 63 nM (216, ΔCys10) and 141 ± 53 nM (217, Cys10). The trimeric peptides Phe-Trp-Lys (219) and Trp-Lys-Tyr (220) were unable to activate the receptor up to a concentration of 10 μM. In contrast, the tetrapeptide Phe-Trp-Lys-Tyr (218) demonstrated the ability to activate the receptor with an EC50 of 179 ± 116 nM yet a reduced intrinsic activity (ΔF/F0 ratio of 1.02 ± 0.2, Table 1).

pharmacophore structure is essential for SST biological activity.13 Earlier SAR studies in fish U-II14 and human UII15,16 had demonstrated that the highly conserved C-terminal portion CFWKYCV represents the minimal sequence necessary for high potency. Furthermore, Lys8 had been described as a critical residue for U-II activity.17 Replacement of the U-II disulfide bridge with lactam bridges of different lengths showed that the biological activity of U-II is dependent upon the size of the cyclic structure.18,19 In addition, a considerable number of peptide or small molecule agonistic analogues and of U-II antagonists has been described.15,18,20−34 However, a full systematic SAR of U-II using the other 19 natural amino acids at each position of the peptide has not been published to date. Therefore, we initiated an SAR study of human U-II using a complete substitution library. Each amino acid position of human U-II was substituted with one of the other natural amino acids, resulting in a library of 209 peptides that were subsequently subjected to activity determination in a Ca2+ mobilization assay. The results of this activity screen were further substantiated by the generation and testing of 33 cyclic and linear U-II variants, ranging from eight to three amino acids in length. Those variants including non-natural amino acids were also analyzed for binding, intracellular signaling, and metabolic stability. Moreover, to enable deeper molecular insights into specific aspects of our studies and main results, we also provide structural information resulting from receptor/ ligand complex models. Thus, we identified the first potent linear U-II analogues. Among them, urolinin is a hexameric UIIR agonist with an EC50 of 4.75 nM and a metabolic stability of 1319 min, 6.3-fold higher than wild-type U-II.

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RESULTS Structure−Activity Relationship in U-II. To characterize the contribution of individual residues in U-II to receptor activation, each of the 11 positions in the human peptide was systematically exchanged for one of the other 19 natural amino acids. The resulting 209-peptide substitution library was analyzed in an intracellular Ca2+ mobilization assay using HEK293A cells overexpressing the human U-II receptor (Figure 1). As expected, the exchange of the exocyclic amino acids Glu1, Thr2, Pro3, Asp4, and Val11 proved to be less critical than a swap of endocyclic residues. Glu1 and Thr2 could be replaced by most amino acids without considerable loss of

Figure 1. Complete substitution analysis of U-II. Heat map diagram of EC50 values from intracellular Ca2+ mobilization assay with wild-type U-II and 209 U-II substitution variants. Values were obtained by stimulation of HEK293A cells transiently transfected with U-IIR. EC50 values from 2−50 nM are colored in dark-blue, 51−400 nM in blue, 401−1000 nM in light-blue, 1001−5000 nM in light-magenta, and those above 5000 nM are colored in magenta. All data represent mean values from at least three independent experiments each done in duplicates. 10101



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Table 1. Identification of FWKY as a Partial U-IIR Agonista

All data are mean values ± SD of duplicate measurements from at least three independent experiments. In cyclic variants, cysteines are shown in bold on gray background. nr = no response.

a

Table 2. Linear U-II Variants Derived from DCFWKYCV and CFWKYCa

All data are mean values ± SD of duplicate measurements from three independent experiments. In cyclic variants, cysteines are shown in bold on gray background.

a

Table 3. Stabilization of NWWKYC with Unnatural Amino Acids and Binding Studiesa

All data are mean values ± standard deviation (SD) or 95% confidence interval (CI) of three independent experiments performed in duplicate. In cyclic variants, cysteines are shown in bold on gray background. D-Amino acids are shown in lowercase characters. The determination of the metabolic stability (t1/2 [min]) was performed in 25% human serum. The data for U-II, 210, 221, 226, 237, 238, and 239 are mean values ± SD from three independent experiments. a

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Figure 2. (A) Ca2+ mobilization assay with U-II and analogues. Representative curves for normalized Ca2+ signaling after stimulation of U-IIR stably transfected HEK293 A cells with U-II, cyclic peptide 210, and the three linear variants (blue) 238, 240, and 241. Data represent mean values from at least three independent experiments each done in duplicates. (B) Binding of [125I-Tyr9]−U-II. Representative saturation binding curve of [125ITyr9]−U-II on U-IIR stably transfected HEK293A cells. Black circles, total binding; black triangles, unspecific binding (in presence of 1 μM U-II); red circles, specific binding (total minus unspecific binding). (C) Representative competition radioligand binding experiments performed on U-IIR stably transfected HEK293A cells. Cells were incubated with a constant concentration of [125I-Tyr9]−U-II and increasing concentrations of U-II, the cyclic peptide 210, and the three linear variants (blue) 238, 240, and 241. Data represent mean values from at least three independent experiments each done in duplicates.

difference. U-II and 210 had a half-life of 209 ± 18 and 372 ± 25 min, respectively, while the half-life of the linear peptides 221 and 226 was 19 min (Table 3). To improve the metabolic stability of the linear U-II variant 226, D-amino acids and non-natural amino acids were incorporated in order to prevent degradation by exo- and endopeptidases. The combined use of D- and structurally related non-natural amino acids resulted in a number of variants with high potency. Peptides 232−234 and 236−242 activated U-IIR with EC50 values below 110 nM, while only the 3-sulfo tyrosine variant 235 provoked a stronger increase of the EC50 to 330 nM. The most stable one was peptide 237 with a half-life of 3120 ± 480 min and an EC50 value of 23.00 ± 7.8 nM. Another peptide (238) also yielded a favorable half-life of 1320 ± 250 min. Compared to the natural cyclic U-II, the metabolic stability of 238 was found to be 6.3-fold higher while it showed a very good potency of 4.75 ± 2.8 nM. This potent and stable linear analogue of U-II is referred to as urolinin. Representative concentration−response curves for the most relevant peptides are shown in Figure 2A. Radioligand Binding. To characterize the binding affinity of urolinin to U-IIR, Tyr9 in U-II was labeled with 125I using a chloramine-T protocol. After HPLC purification, the resulting [125I-Tyr9]−U-II was analyzed in a saturation radioligand binding assay on HEK293A cells overexpressing U-IIR. The Kd value for the radiopeptide [125I-Tyr9]−U-II was found to be 1.1 ± 0.9 nM (Figure 2B). To determine IC50 values for selected peptides, competitive radioligand binding assays were performed. The results of these are summarized in Table 3. UII and the cyclic variant 210 yielded IC50 values of 2.0 ± 1.2 and 0.9 ± 0.2 nM. Surprisingly, IC50 values of all the linear peptides, including those with low nanomolar potencies, were increased by at least a factor of 150. For example, the potent hexameric lead structure 226 showed an IC50 of 1550 nM, with the similarly potent variant 234 having an IC50 of 163 nM. Even the most potent variants 238, 240, and 241 (EC50 between 3.47 and 8.12 nM) showed an IC50 of 600, 429, and 1094 nM, respectively (exemplary curves given in Figure 2C). To investigate whether urolinin was capable of binding to somatostatin receptors (SSTRs), HEK293A cells overexpressing either of the five human SSTR subtypes (1−5) were assayed in a competition radioligand binding experiment with

For further characterization of the two N-terminal amino acids in the truncated U-II variant 210 (first three amino acids deleted), three cyclic variants (211−213, Table 1) and five linear variants (221−225, Table 2) were created. The influence of the two most N-terminal amino acids in the truncated U-II proved to be marginal (exchange of Asp4 against Gln, Thr, or Phe, 211−213, Table 1), and the EC50 was not higher than 4.84 ± 1.5 nM with no significant loss in efficacy. Simultaneous replacement of Asp4 and Cys5 also did not severely impact the potency in most of these variants, with the Gln4/Val4 and Thr4/ Val5 variants (222, 223) resulting in the best EC50 values (9.63 ± 4.8 and 11.2 ± 4.3 nM, respectively). These results confirm the initial finding from the 209 peptide library screen that identified exchanges of Cys5 with Val as highly potent (Figure 1). The positively charged Asp4 as the N-terminal amino acid was less favorable in linear variants: 221, 224, and 225 showed increased EC50 values of up to 155 nM, while intrinsic activity was not altered (Table 2). On the other hand, as an exchange of Phe6 against Trp had preserved high activity in the initial U-II substitution analysis (Figure 1), another six hexameric peptides containing the sequence Trp-Trp-Lys-Tyr were studied (226− 231). Peptide design included substitutions in the N-terminal and the C-terminal part of the peptide while conserving the core sequence WWKY. Most of these variants yielded increased EC50 values of up to 143 nM, yet the most potent peptide proved to be the hexamer NWWKYC, with an EC50 of 10.01 ± 1.9 nM and slightly reduced efficacy (1.56 ± 0.4 nM). Compared to the potency of the corresponding hexameric cyclic peptide 215 (Table 1), this represented a mere 2.6-fold increase, making it a promising short lead structure for further development. Modulation of the Metabolic Stability. For a potential in vivo application, metabolic stability of a peptide is of great concern. As degradation by exopeptidases can be suppressed by cyclization of a peptide, it was intriguing to see whether linearization of a naturally occurring cyclic peptide would diminish its proteolytic stability. To investigate this, peptides were incubated with 25% human serum for different times and were then analyzed for the remaining intact molecule using reversed phase HPLC, allowing calculation of their half-lives (Table 3, Supporting Information, Figure 1). Comparison of metabolic stability of wild-type U-II, cyclic variant 210, and linear variants 221 and 226 revealed an approximately 10-fold 10103



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Figure 3. Competition radioligand binding assay on cells overexpressing somatostatin receptor subtypes 1−5. Radioiodinated somatostatin-14 ([125ITyr11]−SS14) was used in competition binding experiments on cells overexpressing either one of the five human somatostatin receptor subtypes (hSSTR1−5). While somatostatin-14 (SS14, blue curves) is able to compete with radioligand binding in the low nanomolar range (IC50 of 0.76− 4.15 nM), both U-II (in red) and urolinin (238, in black) are 3 orders of magnitude less able to displace [125I-Tyr11]−SS14 (IC50 > 3 μM).

radioiodinated SST ([125I-Tyr11]−SS14). As Figure 3 shows, SST (14mer variant SS14) is able to displace [125I-Tyr11]−SS14 with IC50 values of 0.76−4.15 nM. Urolinin (238) was found to have a similarly low affinity toward SSTRs as wild-type U-II (IC50 values above 3 μM). On SSTR2 and SSTR4, the IC50 of urolinin was more than 10-fold lower than that of U-II (Figure 3, Table 4).

confirmed by radioligand binding assay using the radiopeptide [125I-Tyr9]−U-II and U-II as a competing unlabeled ligand (Figure 4A). Besides intracellular Ca2+ mobilization and cytoskeletal contraction, activation of U-IIR leads to enhanced MAPK signaling within the cell. Thus, MAPK phosphorylation induced by U-II and 237 in VSMC was analyzed by Western blot. As a positive control for peptide-induced MAPK activation via a GPCR, bradykinin was used as an established ligand, causing intracellular signaling by bradykinin B1 receptor activation.35 Primary VSMC were treated with bradykinin, UII, or 237, and lysates were compared by immunoblotting concerning expression and activation of ERK1/2 kinase (Figure 4B). The immunoblots revealed apparent changes of ERK1/2 phosphorylation upon treatment with 500 nM of each ligand, whereas it was barely affected by administration of 5 nM peptide. The amount of total ERK1/2 was not considerably affected by receptor stimulation. The quantification (Figure 4C) confirmed a significant increase of ERK1/2 phosphorylation in bradykinin and U-II treated cells (bradykinin 2.3-fold, U-II 2.4-fold) compared to untreated control. Importantly, a 1.9-fold elevation of ERK1/2 activation stimulated by 237 treatment could also be detected. Hence, an activation of U-IIR by the linear variant 237 comparable to the U-II induced signal could be demonstrated. Insights into Putative Ligand/Receptor Complexes by Homology Modeling and Ligand Docking. We generated structural homology models of U-IIR (Figure 5A) and the ligand U-II. This ligand model was complexed with the receptor (Figure 5B−C) to estimate a putative binding mode and details of receptor/ligand interactions. Binding of U-II shapes a pocket

Table 4. Urolinin Shows No Higher Affinity to Somatostatin Receptors than U-IIa receptor subtype hSSTR1 hSSTR2 hSSTR3 hSSTR4 hSSTR5

SS14 IC50 ± SD (nM) 0.76 0.62 1.15 4.15 3.07

± ± ± ± ±

0.02 0.09 0.71 1.44 3.43

U-II IC50 ± SD (μM)

238 IC50 ± SD (μM)

± ± ± ± ±

6.72 ± 1.69 37.5 ± 11.3 6.07 ± 0.99 >50 28.42 ± 26.80

10.47 2.98 8.64 10.69 7.60

1.46 1.07 2.23 1.14 1.83

a

Results of [125I-Tyr11]-SS14 radioligand competition binding experiments on cells overexpressing human somatostatin receptor subtypes hSSTR1−5. All data represent mean values ± standard deviation (SD) of three independent experiments performed in duplicate.

A Linear U-II Analogue Shows Functionality on Primary Vascular Smooth Muscle Cells. To investigate whether a linear peptide analogue of U-II was functionally active on nontransfected, hence endogenously U-IIR-expressing cells, primary vascular smooth muscle cells from pig aorta (VSMCs) were prepared for further testing. These cells are known to express U-IIR and to show various responses upon U-II stimulation. Presence of U-IIR on these cells was

Figure 4. (A) Binding of [125I-Tyr9]−U-II on pig vascular smooth muscle cells (VSMC). The bound radioligand could be significantly displaced by 1 μM U-II. Data represent mean of six independent experiments, each performed with at least three replicates. (B) Stimulation of VSMC with the peptide ligands bradykinin, U-II, and the linear variant 237 (nWWKY-Abu) induced increased MAPK signaling. After 15 min peptide treatment of VSMC, protein expression levels and ERK1/2 kinase activity were analyzed by immunoblotting and quantified. (C) By treatment with either 5 nM bradykinin (Brady) or urotensin-II (UII), the relative phosphorylation of ERK1/2 was slightly increased, whereas activation was significantly elevated after addition of 500 nM Brady or UII. The linear U-II peptide analogue 237 stimulated the activation of ERK1/2 at a concentration of 500 nM. Relative phosphorylation of ERK1/2 was assessed by immunoblotting, and detected ERK1/2 values were normalized to total ERK1/2 and the untreated control (co). (n = 2−4 independent experiments, each with 3−6 replicates; mean ± SEM; *, p < 0.05). 10104



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Figure 5. Structural homology model of the U-IIR and a complex between U-IIR and U-II. (A) The structural U-IIR homology model (backbone ribbon presentation) in an active conformation was built by using the solved crystal structure of the β2-adrenergic receptor65 as a primary template. Several detailed modifications at the template structure were performed to receive an optimized homology model with respect to individual characteristics of the putative U-IIR structure, like the conformation and spatial location of the ECL2 (see Experimental Section). Docking of the ligand U-II (magenta sticks) was performed by using the already suggested ligand/receptor interaction (U-IIR/U-II) D130/Lys8 as a spatial constraint. (B) Binding of U-II shapes a pocket inside the receptor, visualized as a clipped inner surface. The binding pocket is located between the ECLs and the helices and is covered by particular amino acids (green sticks) (see Table 5). (C) Of note, N-terminal U-II residues outside the cyclic ring are known to have a minor importance for ligand action and are oriented toward the extracellular region. The three functionally important ligand residues Trp7, Lys8, and Tyr9 are located deep inside the binding crevice, close to specific residues in the transmembrane helices like Y305, W275, or D130, which are proposed as direct contact partners. E, extracellular loops; H, transmembrane helix; Ntt, N-terminal tail; Ctt, C-terminal tail.

Of note, U-II residue Val11 that can only be substituted by Phe, Leu, and Arg without strong loss of activity is oriented between the receptor N-terminal tail and extracellular ends of TMH1/TMH7. Our model proposes the residues D47, Y298 (TMH7), and Y111 (TMH2) to be in close spatial vicinity, which may explain the tolerated substitutions at this ligand position. In summary, the proposed model widely matches with important functional−experimental findings based on variations in the amino acid composition of the ligand.

inside the receptor which is located between the ECLs and the extracellular regions of the helices. N-terminal U-II residues outside the ring structure were reported to have minor importance for ligand action (more variations are accepted without changing EC50 values significantly) and they are, according to our designed complex model, oriented toward the extracellular receptor components. The three residues Trp7, Lys8, and Tyr9 are located inside the binding crevice, close to specific residues in the transmembrane helices (Figure 5B,C). In detail, Trp7 is embedded mainly between receptor side chains F127 (TMH3), Y211 (TMH5), L212 (TMH5), and W275 (TMH6). Q278 should participate in this arrangement. In consequence, we postulate a predominantly aromatic and hydrophobic environment for U-II amino acid Trp7, which conforms to the experimentally tolerated side chain variations of, e.g., Leu, Phe, Met, or Tyr. Lys8, which can only be substituted with arginine, interacts directly with D130 (TMH3) and indicates an essential function of the positive/negative charged−charged interaction. Tyrosine at ligand position 9 is located between the aromatic ring side chains of Y298 (TMH7) and Y305 (TMH7), which explains why only aromatic or larger hydrophobic side chain substitutions are able to maintain the full functionality of variants. The side chains of human U-IIR amino acids like Y111, F127, H208, F274, R294, or T301 (Table 5, Figure 5C) are likely additional key players in the justification and interaction with U-II.

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DISCUSSION With this study, we set out to systematically characterize the functional consequences of amino acid exchanges in the natural human U-II peptide. To this end, every amino acid position of human U-II was substituted with one of the other natural amino acids, resulting in a library of 209 peptides that were subsequently subjected to activity screening using a Ca2+ mobilization assay. As expected, we were able to confirm the results of previous analyses regarding the importance of the endocyclic amino acids for receptor activation, while exocyclic amino acids were confirmed to be dispensable.15,36 However, some exchanges, especially of Pro3 and Asp4 in the full-length peptide, led to impaired activity that may be explained with the introduction of an additional negative (Glu) or positive charge (Arg, Lys), compromised disulfide bridge formation (Cys), or altered peptide chain flexibility (Gly, Pro). While previous 10105



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truncation study further confirm their relevance. It had been shown many years ago that removal of the first four and of the last amino acid does not change the activity of U-II activity on cloned U-IIR.15,16 However, further shortening of the molecule was reported to drastically reduce activity (57% of maximum at 500 nM for Ac-FWKY-NH2 and >500 nM for Ac-WKYNH2).16 Although N-terminal acetylation was reported to have no effect on longer U-II forms,38 it may influence activity in truncated variants, as this study identified the uncapped tetrapeptide FWKY-NH2 (218) as a partial agonist with an EC50 of about 180 nM, while shorter peptides did not activate the receptor until concentrations of 10 μM. Many previous studies on structure−activity relationships of U-II14−16,18 have stressed the importance of the endocyclic amino acids and the existence of a ring structure for the activation of the urotensin receptor. Ring removal by substitution of the two cysteine residues with serine was found to lower the affinity to U-IIR to more than 10000 nM.16 Acetamidomethyl modification of the cysteine residues led to a Ki of 864 to 1607 nM on the human receptor and to a Ki of greater than 10000 nM on the rat receptor.36,39 Therefore, the most unexpected result in the substitution analysis was the identification of a noncyclic peptide variant with high receptor activity. While most cysteine substitutions resulted in a dramatic loss of activity, the exchange of Cys5 to Val5 led to a potency similar to wild-type U-II. This was further confirmed by truncated linear variants 221 and 222 with a Cys-Val replacement that showed an EC50 of about 10 nM. Introduction of a tryptophan at position 6 had led to no significant perturbation of the activity, so a number of hexameric variants containing the Trp-Trp-Lys-Tyr core motif were also tested, with peptide 226 being able to activate the receptor at an EC50 value of 10.01 ± 1.9 nM. Early on, the involvement of the U-II/U-IIR system in various disease pathologies had spiked activities to generate compounds for pharmaceutical intervention. In addition to its role in the cardiorenal system, overexpression of U-II and its receptor have recently been demonstrated in tumors and tumor cell lines.40,41 With the derivation of a linear structure from a cyclic wild-type peptide, conditions for metabolic stability drastically change. While cyclic peptides are protected from exopeptidase degradation (in U-II, loss of the exocyclic amino acids has no negative effect on activity15,16,42), linear peptides can be attacked by these proteolytic enzymes present in blood and tissues. Therefore, the initial linear variants showed a 10fold reduced half-life in human serum. To compensate for this disadvantage, we set out to stabilize the peptides using D-amino acids and other non-natural building blocks. As expected, the trimeric motif Trp7-Lys8-Tyr9 had proved rather resistant to substitution by artificial residues, mostly leading to variants with reduced activity. Therefore, the amino acids surrounding this motif were modified, resulting in the identification of an agonistic analogue we called urolinin. This peptide 238 with the sequence nWWK-Tyr(3-NO2)-Abu showed a half-life of 1320 ± 250 min, being 6.3-fold more stable than wild-type urotensinII (209 ± 18 min) while being highly potent (EC50 = 4.75 ± 2.8 nM) in the Ca2+ mobilization assay. In contrast to the high potency and efficacy of many linear UII analogues, their binding affinity (IC50) proved to be at least 2 orders of magnitude lower as for the cyclic variants (e.g., 2 nM for wild-type U-II versus 600 nM for urolinin, 238). A difference in numeric values for affinity and potency is frequently observed with ligands of GPCRs and other receptors. As signaling will depend on the cell’s capacity to

Table 5. Amino Acids Constituting the Potential Ligand Binding Region of the Human U-II Receptora structural localization

U-IIR human

U-IIR rat

U-IIR mouse

TMH2

Ile104 Tyr111

Ile104 Tyr111

Ile103 Tyr110

TMH3

Phe127 Asp130 Phe131



TMH5

His208 Tyr211 Leu212



TMH6

Phe274 Trp275 Gln278



TMH7

Arg294 Asn297 Tyr298 Thr301 Tyr305



a

For comparison, the corresponding positions of mouse and rat orthologues are also shown to highlight high amino acid conservation in this spatial region.

studies employing alanine scans had shown significant loss of activity with the substitution of positions 5, 7, 8, and 9, our results show a more detailed picture with exchanges against amino acids of similar physicochemical properties mostly tolerated (e.g., Ala7 with EC50 > 1000 according to the literature,15,16 yet with an EC50 < 50 nM for substitutions Phe7, His7, Leu7, Met7, and Tyr7 in this study). A number of results from this initial screening was later confirmed with truncated peptides, as in the case of peptides 211−213 that showed an EC50 of no higher than 4.8 ± 1.5 nM, confirming the high potency of the Thr, Phe, and Gln substitutions of Asp4 (Figure 1, Table 2). In addition, Trp7, Lys8, and Tyr9 were confirmed as the key residues for receptor activation.15,36,37 In contrast, Phe6 as another endocyclic amino acid could be substituted by virtually every other amino acid without substantial loss of activity in Ca2+ mobilization, as has been suggested by earlier studies,15,16 while Ala6−U-II had shown impaired potency in the rat aortic assay.36 Modifications of Trp7, Lys8, and Tyr9 demonstrated that substitutions in these positions could only be performed with structurally similar residues to preserve activity. Our homology model and the complex between the human U-IIR and U-II are in agreement with these functional insights. The three residues of significant importance are interacting with essential and conserved amino acids in the transmembrane region inside the binding pocket (for details see also result section). The postulated interaction between U-IIR Asp130 and U-II Arg8 is a functional constraint. Trp7 is involved in directed ligand recognition and justification in a spatial region between TMHs 3−5−6. Tyr9 interacts with aromatic side chains of the receptor in TMH7. Finally, the detailed mode of binding is dependent on very specific interactions in spatially distributed microenvironments inside a binding site located between the helices, close to the ECLs (Figure 5). As experimental data and modeling have stressed the importance of positions 7, 8, and 9, the results of the 10106



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U-II has been shown to possess cardioprotective properties in end-stage renal disease.52 It is intriguing that high serum levels of U-II are able to predict improved patient outcome and survival in chronic kidney disease and acute myocardial infarction.56 Urolinin and the family of related linear agonistic U-II analogues could be a starting point for the development of an orally available agonist. U-II shares a core pharmacophore sequence with another peptide hormone, somatostatin (SST).57 Likewise, U-IIR and SSTRs show high sequence similarity.58 It has been reported previously that U-II and URP at a concentration of 1 μM are able to elicit a calcium signal in CHO cells expressing SSTR subtypes 2 or 5.59 To evaluate the influence of the ring opening in the new U-II analogues on their affinity toward SSTRs, they were tested in competitive binding studies on cells expressing either of the five SSTRs, using [125I-Tyr11]−SS14 as a radioligand. Whereas SST is able to displace [125I-Tyr11]− SS14 with IC50 values of 0.76−4.14 nM, urolinin was found to have a similarly low affinity toward SSTRs as wild-type U-II (IC50 values above 3 μM). On SSTR2 and SSTR4, the IC50 of urolinin was more than 10-fold lower than that of U-II. These results demonstrate that the ring opening did not compromise the discrimination of the linear U-II analogue between U-IIR and SSTRs. As mentioned above, the linear structures presented in this study likely have signaling properties different from the cyclic variants developed so far. While urolinin was able to potently elicit a Ca2+ response in cells expressing the human receptor, it did not induce contraction in the rat aortic ring model (data not shown). In view of the recent appreciation of ligand bias for different downstream signaling pathways in GPCRs,60,61 it is intriguing to speculate about differential activation of G protein-dependent and independent signaling of cyclic versus linear U-II agonists. Different effects of U-II in different tissues may be explained by differential underlying signaling patterns that could potentially be addressed by an agonist that is able to selectively activate only one pathway, preventing unwanted secondary physiological effects (vasoconstriction vs vasodilation). Chatenet et al.24 recently developed urocontrin, a U-IIR ligand that shows differential signaling as it is able to reduce the ex vivo efficacy vasoconstriction in rat aortic rings induced by U-II but not urotensin II-related peptide (URP). Other novel ligands discriminating U-II and URP-mediated biological activities have also been described,25,27 opening the path to a more selective intervention of U-IIR pharmacology and physiology. To obtain a first insight into the downstream signaling pathways activated by a linear U-II analogue, we assessed whether peptide 237 (highest metabolic stability found) would show the same potency as U-II on endogenously U-IIR expressing vascular smooth muscle cells (VSMCs). For this purpose, VSMCs from pig aorta were chosen as a relevant model system. U-IIR is known to activate the PLC−PKC cascade, thus stimulating Ca2+ mobilization and contraction. Moreover, RhoA and MAPK signaling were reported to be involved in proliferation and contraction.9,10 Exposure of VSMC to U-II and analogues was expected to induce activation of extracellular signal-regulated kinase (ERK1/2). We tested whether U-IIR agonists U-II and 237 lead to stimulation of ERK1/2 phosphorylation in primary pig VSMC. Similar to bradykinin-stimulated activation,35 Western blot analysis of VSMC lysates revealed a concentration-dependent ERK1/2 activation upon U-II40,41 and 237 stimulation. More strikingly,

transmit and amplify a stimulus initiated by ligand binding, different cells, receptors, ligands, readouts, and detection systems will result in varying ratios between potency and affinity. Early on, the ability of ligands to induce maximum response with less than full receptor occupancy has been described as receptor reserve and it has been reported in many pharmacologic systems.43,44 Moreover, the two parameters are frequently not detected with the same kinetics; while binding is usually determined under equilibrium conditions, functional assays such as Ca2+ mobilization used in this study record signaling events within a few seconds after agonist addition, hence in a nonequilibrium state of ligand−receptor interaction. From the data set obtained here, a discussion of possible reasons for the unexpectedly reduced binding affinity of linear U-II ligands will have to be speculative. One obvious parameter to consider would be the efficacy of the compounds. However, judged by the ΔF/F0 ratio from Ca2+ mobilization, these linear peptides are neither superagonists exceeding the natural ligand’s maximum response nor partial agonists (80−105% of U-II efficacy, mean efficacy 95%). A concept to explain varying response at similar affinity that has attracted a lot of attention recently is receptor residence time.45,46 Differences in binding kinetics, especially high koff values, were shown to explain alterations in activity with ligands of similar affinity.45,47 However, in the case of linear U-II analogues, neither increased response at maximum concentration nor enhanced potency were observed. Likewise, high efficacy under nonequilibrium conditions (maximum effect measured ∼5 s after compound addition) would suggest higher kon values than for U-II, which would in turn imply drastically reduced koff rates for linear ligands with IC50 values of several hundred nM. On a mechanistic level, a number of concepts may contribute to the phenomenon described above. Linear ligands may differ in their binding mode to the receptor in the way and in the kinetics they induce conformational changes in the receptor, thus leading to similar potency yet reduced affinity. If these analogues, in contrast to their natural cyclic precursor, would stabilize an active state of the receptor with higher signaling capacity, a behavior as observed in this study could result. At higher receptor occupancies, the signaling capacity of the cell may be exhausted, preventing superagonism at saturating concentrations. Clearly, the underlying mechanisms will require a more thorough investigation involving a different set of experimental approaches than used in the current study. Kinetic analysis of ligand−receptor binding using the radioligand method published by Motulsky et al.48,49 or by a recently developed HTRF-based assay50 may be an adequate initial approach to shed light on the mechanics of the pharmacology observed here. Likewise, further analyses using, e.g., NMR spectroscopy will have to determine the consequences of the ring opening and the amino acid substitutions for the secondary structure of urolinin and the other potent linear agonists. The U-II/U-IIR interaction model presented here should facilitate the elucidation of structural rearrangements in the receptor induced by urolinin, as it may serve as a template for an extended modeling approach. However, more functional and structural data will be required for such a study. Until now, most pharmaceutical developments focused on the generation of urotensin receptor antagonists (for a review, see ref 51). This is based on a large body of evidence demonstrating high U-II levels in renal and heart failure in humans.52−55 However, several lines of evidence point to a potential role of U-II agonists for certain therapeutic challenges. 10107



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Two days after transfection, cells were selected in culture medium containing 400 μg/mL G418 over a period of 4 weeks (37 °C, 5% CO2, 95% relative humidity). Functional expression of the selected cell lines was verified in a CellLux assay recording U-IIR-mediated intracellular Ca2+ release upon receptor activation induced by U-IIR, as described below. Calcium Mobilization Assay. HEK293A cells expressing U-IIR were seeded at a density of 50000 cells per well into poly-D-lysinecoated black-wall, clear bottom 96-well well plates (BD Falcon) and cultured overnight. Up to 18−24 h later, cells were loaded with Fluo4AM (Invitrogen). Then the cells were washed two times with C1 buffer (130 mM NaCl, 5 mM KCl, 10 mM HEPES pH 7.4, 2 mM CaCl2, and 10 mM glucose). After the final wash, a 100 μL residual volume remained on the cells. Peptides were dissolved in 10% DMSO to a concentration of 1 mM and were diluted in C1 buffer with 0.1% BSA. They were transferred as 2× solutions in 96-well plates and were dispensed simultaneously from the ligand plate to the cell plate by the robotic system within the imager. Fluorescence was simultaneously recorded by the fluorescence imaging plate reader CellLux (PerkinElmer) in all wells at an excitation wavelength of 488 nm and emission wavelength of 510 nm in 1.5 s intervals over a period of 90 s. Fluorescence data were generated in duplicates, and experiments were repeated for at least three times. All compounds were tested at seven different concentrations over a range of seven orders of magnitude. Duplicate signals were averaged, and signals of C1 buffertreated control wells were subtracted. Signals were normalized to background fluorescence by calculating the ΔF/F0 ratio, where F0 is the basal fluorescence intensity (before compound addition) and ΔF is the difference between peak intensity and F0. The ΔF/F0 ratio at the highest concentration was obtained as a measure of compound efficacy. For calculation of EC50 values, sigmoidal concentration− response curves of ΔF/F0 ratio versus concentration were plotted in GraphPad Prism v5.04. In Vitro Peptide Stability in Serum/Reaction Kinetics. In vitro peptide stability assays were carried out as previously described.63 Briefly, 500 μL of RPMI supplemented with 25% (v/v) of human serum were allocated into a 1.5 mL Eppendorf tube and temperatureequilibrated at 37 ± 1 °C for 15 min before adding peptide stock solution to make a final peptide concentration of 50 μg/mL. The initial time was recorded and at defined time intervals, and 50 μL of the reaction solution was removed and added to 100 μL of 6% aqueous trichloroacetic acid (TCA) for precipitation of serum proteins. The cloudy reaction sample was cooled on ice for 15 min and then spun at 18000g for 2 min to pellet the precipitated serum proteins. The reaction supernatant was then analyzed using RP-HPLC (Agilent 1200 LC System) on a ZORBAX Eclipse XDB-C18, 4.6 mm × 150 mm, 5 μm column (Agilent). A linear gradient from 25% to 80% acetonitrile containing 0.1% TCA was used over 15 min with a flow rate of 1 mL/min at 30 °C. Absorbance was detected at 214 and 280 nm. Fluorescence was detected at an excitation wavelength of 280 nm and emission wavelength of 340 nm. Kinetic analysis was carried out by performing a one-phase exponential decay analysis of the integrated peak area versus time. Iodination of U-II and Tyr11−SS14. U-II and Tyr11−SS14 were iodinated by the chloramine-T method.64 All reagents were freshly prepared. U-II (8 nmol) and Tyr11−SS14, respectively were dissolved in 25 μL of 0.5 M phosphate buffer pH 7.4. To this solution, 1 mCi (0.5 nmol in 3 μL of NaOH, pH 8−11) carrier-free Na125I (PerkinElmer) was added. The radioiodination reaction began with the addition of 4 μL of chloramine-T (14.2 nmol) at a concentration of 1 mg/mL; the reaction was allowed to proceed for 10−15 s at room temperature and was stopped by the addition of 4 μL of 2 mg/mL metabisulfite (Na2S205, 42.1 nmol). Purification of iodinated U-II or Tyr11−SS14 was performed on a RP-HPLC system (Agilent 1200 LC system) with a ZORBAX Eclipse XDB-C18, 4.6 mm × 150 mm, 5 μm column (Agilent). A linear gradient from 30% to 70% acetonitrile containing 0.1% trifluoroacetic acid was used over 20 min with a flow rate of 1 mL/min at 55 °C. Absorbance was detected at 214 and 280 nm. Fluorescence was detected at an excitation wavelength of 280 nm

the endogenous reaction toward 237 was comparable to U-II as well as to published data for VSMC8 and U-IIR-transfected cells.62 Whether the linear U-II agonists presented in this study are able to differentially influence (patho)physiological parameters in vivo needs to be further addressed using suitable animal or organ model systems.

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CONCLUSIONS While characterization of structure−activity relationships in peptides usually can successfully be undertaken using alanine scan and truncation studies, the full high-resolution SAR of U-II interaction with its receptor could be elucidated only with a complete substitution library. As demonstrated with a number of different variants, U-IIR can be activated with high potencies by linear analogues of U-II. Although the ring opening led to decreased metabolic stability, the introduction of D- and other non-natural amino acids resulted in analogues with up to 6.3fold higher serum protease resistance. Out of these structures, urolinin (nWWK-Tyr(3-NO2)-Abu) represents the first linear peptide U-IIR agonist showing nanomolar potency as well as improved metabolic stability.

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EXPERIMENTAL SECTION

Peptides and Synthesis. Peptides were synthesized as carboxyterminal amides at a 2 mmol scale on a LIPS 96 peptide synthesizer (peptides&elephants GmbH, Potsdam, Germany). Synthesis was done in resin-preloaded MultiPep 96 microtiter plates (peptides&elephants GmbH) using Fmoc chemistry on Rink amide AM resin or N-biotinylNFmoc-ethylenediamine-MPB AM resin (Merck Biosciences AG, Darmstadt, Germany). All solvents were of reagent or HPLC grade and were bought from Carl Roth GmbH (Karlsruhe, Germany). Temporary Fmoc protection groups were removed by treatment with 20% piperidine v/v in dimethylformamide. Amino acid coupling was done with 4 equiv activated amino acid solution (0.2 M in N-methyl pyrrolidone). Acid-labile protection groups were removed and peptides were released from the resin by treatment with 90% TFA, 5% triisopropyl silane, 2.5% DTT, and 2.5% HPLC water [v/v]. Peptides were lyophilized, redissolved in TFA, and precipitated by the addition of ice-cold hexane−diethyl ether solution (50/50). For the initial 209 peptide substitution library, peptides were used as raw products from highly parallel synthesis, and identity was confirmed by ESI-MS (Finnigan Surveyor MSQ Plus, Thermo Finnigan, Bremen, Germany). The mean purity of these peptides was 53.9%. All other peptides were HPLC-purified to at least 95% purity and analyzed by HPLC-MS (Dionex HPLC/Finnigan Surveyor MSQ Plus, Thermo Finnigan, Bremen, Germany). Peptide purities, retention factors (k′ values) as well as calculated and determined mass data are summarized in Supporting Information Table 1. Cell Culture and Transient Transfection. HEK293A cells were grown in RPMI 1640 medium buffered with 2.0 g/L NaHCO3 (Biochrom AG, Berlin, Germany) supplemented with 10% FCS, 2 mM L-glutamine, penicillin−streptomycin (10.000−10.000 g/mL) (culture medium) at 37 °C in a humidified 5% CO2 incubator. Cells were split into poly-D-lysine-coated black-wall, clear bottom 96-well well plates (BD Falcon) at a density of 50000 cells per well and cultured for an additional period of 18−24 h. Then cells were transiently transfected with the human U-IIR pcDNA3.1 plasmid (Missouri S&T cDNA Resource Center, Bloomsberg, PA, USA, www. cdna.org) using jetPEI reagent (Polyplus, Illkirch, France) according to the manufacturer’s protocol. Functional fluorescence imaging plate reader (CellLux) assay was conducted another 18−24 h after transfection to measure intracellular Ca2+ release upon receptor activation (described in detail below). Generation of Stable Human U-IIR-Expressing Cell Lines. For the generation of stable cell lines expressing the human U-IIR, HEK293A cells were transfected with human U-IIR pcDNA3.1 using jetPEI transfection reagent according to the manufacturer’s protocol. 10108



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the extracellularly fused T4 lysozyme, (iii) removal of the ADRB2 ligand. The ligand binding region for most of the family A GPCRs is located between the extracellular ends of the transmembrane helices and the extracellular loops.67−70 Moreover, the extracellular loop (ECL) 2 of the ADRB2 template was deleted because of significant differences in length and amino acid composition compared to the sequence of the U-IIR ECL2 (sequence alignment not shown here). The recently published crystal structures of family A GPCRs for peptide ligands, namely the neurotensin (NTSR1, pdbs 4grv, 4xee) and the angiotensin receptors (AT1R, pdb 4zud)71−73 were analyzed concerning the secondary structure and spatial localization of the ECL2. Of note, in rhodopsin and opsin, the ECL2 is located close to the transmembrane helices, nearly parallel to the membrane and closes tightly the binding pocket of the permanently bound ligand retinal, whereby in the aminergic receptors like the ADRB2 and ADRB1 most parts of the ECL2 are different in the fold (opsin β-sheet like versus a helical fold in the aminergic receptors) and localization compared to rhodopsin. In the above-mentioned two solved structures of peptidic ligand GPCRs, the ECL2 shows also a β-sheet like fold, but the spatial localization between the ECL1 and ECL3 is partially different compared to rhodopsin and more oriented toward the extracellular region. This difference to rhodopsin virtually opens the entrance at the extracellular site between the transmembrane helices, which should be of importance to bind larger ligands into a crevice between the helices and loops. Similar properties for the ECL2 can be observed at the nociceptin/orphanin FQ receptor (NOP or ORL-1) in complex with a peptide mimetic (pdb 4ea3)74 or in the δ-opioid receptor (DOR, pdb 4ej4).75 Therefore, we superimposed the neurotensin receptor (NTSR1) crystal structure with the premodified ADRB2 template and merged the ECL2 from the NTSR1 structure into the ADRB2. Additionally, few amino acids of the receptor N-terminus were added manually and oriented as suggested by the neurotensin-1 receptor structure toward the ECL2 and above the ECL1. Our U-IIR homology model based on ADRB2 and NTSR1 (ECL2) structures starts at position W,36 whereby the N-terminus of transmembrane helix 1 was assigned according to available GPCR crystal structures at position 47. Amino acids of this chimeric receptor template were substituted with residues of the human U-IIR, followed by side chain minimization until converging at a termination gradient of 0.1 kcal/mol·Å with constraint backbone atoms of the transmembrane helices, which were finally released in a second minimization step until converging at a termination gradient of 0.05 kcal/mol·Å. This first preliminary model was further refined by molecular dynamic simulations (300 K, 4 ns) and energetic minimizations of the side chain orientations with constrained backbone atoms of helical parts (until converging at a termination gradient of 0.1 kcal/mol·Å). The resulting model was minimized until converging at a termination gradient of 0.05 kcal/mol· Å without any constraint. Ligand Model. The ligand U-II was modeled based on a fragment of the vitronectin crystal structure (pdb 1ssu),76 which included a disulfide bridge between two cysteine residues in a region characterized by sequence similarity in length and amino acid composition of U-II. The U-II model was energetically minimized and used for docking into the U-IIR model. Ligand/Receptor Complex-Assembling. Computational docking of ligands into receptors can be ideally performed under consideration of already known and evidenced receptor/ligand interaction pairs.67 Numerous specific studies on this issue with different methods and also derived receptor/ligand complex models were already published for U-IIR.15,36,77−82 These models reflected available experimental knowledge and suggested putative ligand-binding modes. However, evidence for concrete ligand/receptor pairs or systematic mutagenesis studies in the putative U-IIR ligand binding region are rare. One of the most prominent interaction is assumed between ligand residue Lys8 and receptor residue D130 (TMH3) because both amino acids are experimentally evidenced to be essential for receptor activation and their complementary charges point to a significant role in binding and selectivity. The experimental results in this current study concerning substitutions at ligand position 8 confirmed previous findings. Therefore, we also used this supposed interaction pair as a functional

and emission wavelength of 340 nm and radioactivity was measured with a FlowStar LB 513 radio flow detector (Berthold). Radioligand Binding Assay. HEK293A cells expressing U-IIR or SSTR1−5 were seeded at a density of 60000 cells per well in 96-well flat-bottom cell culture plates and cultured overnight. On the next day, medium was exchanged with 100 μL of binding buffer (50 mM TrisHCl pH 7.4; 5 mM MgCl2, 1 mM CaCl, 0.5% BSA) including a mixture of protease inhibitors (Roche cOmplete). Then 100000 cpm [125I-Tyr9]−U-II or [125I]-Tyr11−SS14, respectively, were used with variable concentrations of competitors. Samples were incubated for 30 min at 37 °C. Cells were washed four times with 4 °C cold washing buffer (50 mM Tris-HCl, pH 7.4, 125 mM NaCl, 0.05% BSA). After buffer removal, 100 μL of 1 N NaOH were added and after 5 min incubation at room temperature transferred to plastic vials for activity measurement in a gamma counter (Wallac 1470 Wizard). Data resulting from competition binding assays were normalized for the nonspecific binding (0%) and specific binding in the absence of competitor (100%). Binding parameters were calculated with GraphPad Prism v5.04 using a nonlinear regression (asymmetric five parameter) model to determine IC50 values. Primary Aortic Smooth Muscle Cells. Pig aortas were isolated from four- to six-month old domestic pigs (Sus scrofa domesticus). Sterile aortic specimens were placed into 50 mL vials containing DMEM basal medium (Merck Millipore) and processed within 4 h. Fat and surrounding tissue were removed by scalpel. For isolation of vascular smooth muscle cells (VSMC), the aortic tissue was minced into small pieces using scalpels and transferred into 6-well plates in 1 mL of complete growth medium (DMEM, 10% FCS, 1% penicillin/ streptomycin all from Merck Millipore). After five to seven days, medium was exchanged and adhered cells were expanded for experiments performed on monolayer cultures within 5−10 passages. Primary Cell Stimulation and Western Blot. Effect of peptide ligands on MAPK signaling was assessed by Western blot analysis of lysates from stimulated pig vascular smooth muscle cells (VSMC). Therefore, VSMC (50000 cells per well) were seeded onto adherent 6well plates and cultured until cells were 80% confluent. After incubation with bradykinin, U-II, or peptide 237 (5 nM and 500 nM in 0.1% FCS/DMEM) for 15 min, cells were lysed using 200 μL/ well cell lysis buffer (100 mM Tris-HCl pH 8.8, 1% SDS) and protease inhibitors (cOmplete, Roche) on ice for 1 h. The protein concentration was determined using the bicinchoninic acid method (Thermo Scientific). Equal amounts of protein (3−5 μg) were treated with Laemmli sample buffer, separated on a 10% SDS polyacrylamide gel, and blotted on a nitrocellulose membrane (GE Healthcare) by applying 3 mA/cm2. Immunolabeling was realized using the specific primary antibody Phospho-ERK1/2 Thr202/Tyr204 (Cell Signaling Technologies, 1:2000) at 4 °C overnight. Specific protein signals were detected by applying horseradish peroxidase-conjugated secondary antibody (Dianova, 1:10000) via a chemiluminescence detection kit (Thermo Scientific) and the ChemiDocTM-MP imaging system (Bio-Rad). Membranes were stripped with 0.5 M glycine pH 2.67 and incubated with antitotal ERK1/2 and anti-GAPDH (both Cell Signaling Technologies, 1:2000), respectively. Blots were analyzed using densitometric quantification using Image Lab Software v4.1 (Bio-Rad). Structural Homology Modeling and Ligand/Receptor Assembling Procedures. Receptor Model. All structural modifications for generating the study-related homology models were performed with the software Sybyl X2.0 (Certara, NJ, USA). The AMBER F99 force field was used for energy minimization and dynamic simulations. Structure images were produced using PyMOL software (DeLano WL, version 1.5, San Carlos, CA, USA). For urotensin-II receptor (U-IIR), no direct structural information is yet available. Our U-IIR homology model was built by using the solved crystal structure of the β2-adrenergic receptor (ADRB2)/Gs complex (pdb 3sn6)65 as a primary template (full-length sequence similarity 30%, Blossum62 matrix). This specific template was chosen because of our purpose to design a receptor-model in an active-state conformation bound with agonistic ligands. Beside usual modifications for template preparations like loop length adjustment,66 additional modifications were: (i) deletion of the G-protein (Gs), (ii) deletion of 10109



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constraint to orient in a first step manually the U-II model superficially into the U-IIR homology model. This complex was modified by molecular dynamic simulations (300 K, 2 ns) with constrained backbone atoms of the transmembrane helices, followed by energetic minimization of the entire complex. The suggested ligand K8/receptor D130 pair was fixed during this procedure by a distance-constraint of 2.5 Å. The resulting complex was minimized without any structural constraint.

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(7) Watanabe, T.; Pakala, R.; Katagiri, T.; Benedict, C. R. Synergistic effect of urotensin II with serotonin on vascular smooth muscle cell proliferation. J. Hypertens. 2001, 19, 2191−2196. (8) Iglewski, M.; Grant, S. R. Urotensin II-induced signaling involved in proliferation of vascular smooth muscle cells. Vasc. Health Risk Manage. 2010, 6, 723−734. (9) Sauzeau, V.; Le Mellionnec, E.; Bertoglio, J.; Scalbert, E.; Pacaud, P.; Loirand, G. Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rhokinase. Circ. Res. 2001, 88, 1102−1104. (10) Tasaki, K.; Hori, M.; Ozaki, H.; Karaki, H.; Wakabayashi, I. Mechanism of human urotensin II-induced contraction in rat aorta. J. Pharmacol. Sci. 2004, 94, 376−383. (11) Bohm, F.; Pernow, J. Urotensin II evokes potent vasoconstriction in humans in vivo. Br. J. Pharmacol. 2002, 135, 25−27. (12) Wilkinson, I. B.; Affolter, J. T.; de Haas, S. L.; Pellegrini, M. P.; Boyd, J.; Winter, M. J.; Balment, R. J.; Webb, D. J. High plasma concentrations of human urotensin II do not alter local or systemic hemodynamics in man. Cardiovasc. Res. 2002, 53, 341−347. (13) Janecka, A.; Zubrzycka, M.; Janecki, T. Somatostatin analogs. J. Pept. Res. 2001, 58, 91−107. (14) Itoh, H.; Itoh, Y.; Rivier, J.; Lederis, K. Contraction of major artery segments of rat by fish neuropeptide urotensin II. Am. J. Physiol. Regul. Integr. Comp. Physiol. 1987, 252, R361−R366. (15) Flohr, S.; Kurz, M.; Kostenis, E.; Brkovich, A.; Fournier, A.; Klabunde, T. Identification of nonpeptidic urotensin II receptor antagonists by virtual screening based on a pharmacophore model derived from structure-activity relationships and nuclear magnetic resonance studies on urotensin II. J. Med. Chem. 2002, 45, 1799−1805. (16) Kinney, W. A.; Almond, H. R., Jr.; Qi, J.; Smith, C. E.; Santulli, R. J.; de Garavilla, L.; Andrade-Gordon, P.; Cho, D. S.; Everson, A. M.; Feinstein, M. A.; Leung, P. A.; Maryanoff, B. E. Structure-function analysis of urotensin II and its use in the construction of a ligandreceptor working model. Angew. Chem., Int. Ed. 2002, 41, 2940−2944. (17) Camarda, V.; Rizzi, A.; Calo, G.; Gendron, G.; Perron, S. I.; Kostenis, E.; Zamboni, P.; Mascoli, F.; Regoli, D. Effects of human urotensin II in isolated vessels of various species; comparison with other vasoactive agents. Naunyn-Schmiedeberg's Arch. Pharmacol. 2002, 365, 141−149. (18) Grieco, P.; Carotenuto, A.; Campiglia, P.; Zampelli, E.; Patacchini, R.; Maggi, C. A.; Novellino, E.; Rovero, P. A new, potent urotensin II receptor peptide agonist containing a Pen residue at the disulfide bridge. J. Med. Chem. 2002, 45, 4391−4394. (19) Grieco, P.; Carotenuto, A.; Patacchini, R.; Maggi, C. A.; Novellino, E.; Rovero, P. Design, synthesis, conformational analysis, and biological studies of urotensin-II lactam analogues. Bioorg. Med. Chem. 2002, 10, 3731−3739. (20) Behm, D. J.; Stankus, G.; Doe, C. P.; Willette, R. N.; Sarau, H. M.; Foley, J. J.; Schmidt, D. B.; Nuthulaganti, P.; Fornwald, J. A.; Ames, R. S.; Lambert, D. G.; Calo’, G.; Camarda, V.; Aiyar, N. V.; Douglas, S. A. The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems. Br. J. Pharmacol. 2006, 148, 173−190. (21) Behm, D. J.; McAtee, J. J.; Dodson, J. W.; Neeb, M. J.; Fries, H. E.; Evans, C. A.; Hernandez, R. R.; Hoffman, K. D.; Harrison, S. M.; Lai, J. M.; Wu, C.; Aiyar, N. V.; Ohlstein, E. H.; Douglas, S. A. Palosuran inhibits binding to primate UT receptors in cell membranes but demonstrates differential activity in intact cells and vascular tissues. Br. J. Pharmacol. 2008, 155, 374−386. (22) Carotenuto, A.; Grieco, P.; Novellino, E.; Rovero, P. UrotensinII receptor peptide agonists. Med. Res. Rev. 2004, 24, 577−588. (23) Carotenuto, A.; Auriemma, L.; Merlino, F.; Yousif, A. M.; Marasco, D.; Limatola, A.; Campiglia, P.; Gomez-Monterrey, I.; Santicioli, P.; Meini, S.; Maggi, C. A.; Novellino, E.; Grieco, P. Lead optimization of P5U and urantide: discovery of novel potent ligands at the urotensin-II receptor. J. Med. Chem. 2014, 57, 5965−5974.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00164. Urotensin U-IIR complex model (PDB) Compound characterization, HPLC assay for peptide stability in serum (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 30 450559488. Fax: +49 30 450559997. E-mail: [email protected]. Author Contributions ∥

S.B., S.E., and J.L.v.H. contributed equally

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grant 03IP614 in the InnoProfile program of the German Federal Ministry of Education and Research (BMBF) to C.G. and by the Else Kröner-FreseniusStiftung (EKFS), project 2014_A114.

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ABBREVIATIONS USED U-II, urotensin-II; U-IIR, urotensin-II receptor REFERENCES

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