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• W3191569443 abstract "Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially. Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially. In humans, the urotensinergic system, composed of a class 1A G protein-coupled receptor (GPCR, hUT), 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), continues to represent a promising target for the treatment of several pathologies (1Vaudry H. Leprince J. Chatenet D. Fournier A. Lambert D.G. Le Mevel J.C. Ohlstein E.H. Schwertani A. Tostivint H. Vaudry D. International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, urotensin II-related peptide, and their receptor: From structure to function.Pharmacol. Rev. 2015; 67: 214-258Crossref PubMed Scopus (69) Google Scholar, 2Nassour H. Iddir M. Chatenet D. Towards targeting the urotensinergic system: Overview and challenges.Trends Pharmacol. Sci. 2019; 40: 725-734Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Notably, multiple studies in animal models have suggested that UT antagonists may represent potential therapeutic agents for treating atherosclerosis (3Watson A.M. Olukman M. Koulis C. Tu Y. Samijono D. Yuen D. Lee C. Behm D.J. Cooper M.E. Jandeleit-Dahm K.A. Calkin A.C. Allen T.J. Urotensin II receptor antagonism confers vasoprotective effects in diabetes associated atherosclerosis: Studies in humans and in a mouse model of diabetes.Diabetologia. 2013; 56: 1155-1165Crossref PubMed Scopus (34) Google Scholar, 4You Z. Genest Jr., J. Barrette P.O. Hafiane A. Behm D.J. D'Orleans-Juste P. Schwertani A.G. Genetic and pharmacological manipulation of urotensin II ameliorate the metabolic and atherosclerosis sequalae in mice.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1809-1816Crossref PubMed Scopus (42) Google Scholar, 5Zhao J. Xie L.D. Song C.J. Mao X.X. Yu H.R. Yu Q.X. Ren L.Q. Shi Y. Xie Y.Q. Li Y. Liu S.S. Yang X.H. Urantide improves atherosclerosis by controlling C-reactive protein, monocyte chemotactic protein-1 and transforming growth factor-beta expression in rats.Exp. Ther. Med. 2014; 7: 1647-1652Crossref PubMed Scopus (15) Google Scholar, 6Zhao J. Yu Q.X. Kong W. Gao H.C. Sun B. Xie Y.Q. Ren L.Q. The urotensin II receptor antagonist, urantide, protects against atherosclerosis in rats.Exp. Ther. Med. 2013; 5: 1765-1769Crossref PubMed Scopus (27) Google Scholar), pulmonary arterial hypertension (7Lee J.H. Park B.K. Oh K.S. Yi K.Y. Lim C.J. Seo H.W. Lee B.H. A urotensin II receptor antagonist, KR36676, decreases vascular remodeling and inflammation in experimental pulmonary hypertension.Int. Immunopharmacol. 2016; 40: 196-202Crossref PubMed Scopus (21) Google Scholar, 8Pehlivan Y. Dokuyucu R. Demir T. Kaplan D.S. Koc I. Orkmez M. Turkbeyler I.H. Ceribasi A.O. Tutar E. Taysi S. Kisacik B. Onat A.M. Palosuran treatment effective as bosentan in the treatment model of pulmonary arterial hypertension.Inflammation. 2014; 37: 1280-1288Crossref PubMed Scopus (10) Google Scholar, 9Wang Y. Tian W. Xiu C. Yan M. Wang S. Mei Y. Urantide improves the structure and function of right ventricle as determined by echocardiography in monocrotaline-induced pulmonary hypertension rat model.Clin. Rheumatol. 2019; 38: 29-35Crossref PubMed Scopus (6) Google Scholar), metabolic syndrome (4You Z. Genest Jr., J. Barrette P.O. Hafiane A. Behm D.J. D'Orleans-Juste P. Schwertani A.G. Genetic and pharmacological manipulation of urotensin II ameliorate the metabolic and atherosclerosis sequalae in mice.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1809-1816Crossref PubMed Scopus (42) Google Scholar), and heart failure (10Bousette N. Hu F. Ohlstein E.H. Dhanak D. Douglas S.A. Giaid A. Urotensin-II blockade with SB-611812 attenuates cardiac dysfunction in a rat model of coronary artery ligation.J. Mol. Cell Cardiol. 2006; 41: 285-295Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Bousette N. Pottinger J. Ramli W. Ohlstein E.H. Dhanak D. Douglas S.A. Giaid A. Urotensin-II receptor blockade with SB-611812 attenuates cardiac remodeling in experimental ischemic heart disease.Peptides. 2006; 27: 2919-2926Crossref PubMed Scopus (49) Google Scholar, 12Oh K.S. Lee J.H. Yi K.Y. Lim C.J. Park B.K. Seo H.W. Lee B.H. A novel urotensin II receptor antagonist, KR-36996, improved cardiac function and attenuated cardiac hypertrophy in experimental heart failure.Eur. J. Pharmacol. 2017; 799: 94-102Crossref PubMed Scopus (17) Google Scholar). However, in spite of such promise, clinical studies of UT candidate antagonists have had limited success due to a lack of efficacy in humans (1Vaudry H. Leprince J. Chatenet D. Fournier A. Lambert D.G. Le Mevel J.C. Ohlstein E.H. Schwertani A. Tostivint H. Vaudry D. International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, urotensin II-related peptide, and their receptor: From structure to function.Pharmacol. Rev. 2015; 67: 214-258Crossref PubMed Scopus (69) Google Scholar, 2Nassour H. Iddir M. Chatenet D. Towards targeting the urotensinergic system: Overview and challenges.Trends Pharmacol. Sci. 2019; 40: 725-734Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 13Billard E. Iddir M. Nassour H. Lee-Gosselin L. Poujol de Molliens M. Chatenet D. New directions for urotensin II receptor ligands.Pept. Sci. 2018; e24056Google Scholar, 14Kim S.K. Li Y. Park C. Abrol R. Goddard 3rd., W.A. Prediction of the three-dimensional structure for the rat urotensin II receptor, and comparison of the antagonist binding sites and binding selectivity between human and rat receptors from atomistic simulations.ChemMedChem. 2010; 5: 1594-1608Crossref PubMed Scopus (22) Google Scholar). Our current knowledge remains insufficient to clearly assess its therapeutic potential, and accordingly, a deeper understanding of UT pharmacology is critically needed to accelerate development of UT ligands exhibiting efficacy in humans. While the two endogenous ligands share a common bioactive core, the distinct N-terminal domain of UII isoforms appears to be involved in specific topological changes associated with UT activation (2Nassour H. Iddir M. Chatenet D. Towards targeting the urotensinergic system: Overview and challenges.Trends Pharmacol. Sci. 2019; 40: 725-734Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 15Brancaccio D. Merlino F. Limatola A. Yousif A.M. Gomez-Monterrey I. Campiglia P. Novellino E. Grieco P. Carotenuto A. An investigation into the origin of the biased agonism associated with the urotensin II receptor activation.J. Pept. Sci. 2015; 21: 392-399Crossref PubMed Scopus (15) Google Scholar, 16Merlino F. Billard E. Yousif A.M. Di Maro S. Brancaccio D. Abate L. Carotenuto A. Bellavita R. d'Emmanuele di Villa Bianca R. Santicioli P. Marinelli L. Novellino E. Hebert T.E. Lubell W.D. Chatenet D. et al.Functional selectivity revealed by N-methylation scanning of human urotensin II and related peptides.J. Med. Chem. 2019; 62: 1455-1467Crossref PubMed Scopus (12) Google Scholar, 17Chatenet D. Letourneau M. Nguyen Q.T. Doan N.D. Dupuis J. Fournier A. Discovery of new antagonists aimed at discriminating UII and URP-mediated biological activities: Insight into UII and URP receptor activation.Br. J. Pharmacol. 2013; 168: 807-821Crossref PubMed Scopus (28) Google Scholar). Contingent on their interactions with UT, UII and URP probably induce distinct UT conformational changes that lead to divergent signaling profiles with both common and distinct biological activities (2Nassour H. Iddir M. Chatenet D. Towards targeting the urotensinergic system: Overview and challenges.Trends Pharmacol. Sci. 2019; 40: 725-734Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 17Chatenet D. Letourneau M. Nguyen Q.T. Doan N.D. Dupuis J. Fournier A. Discovery of new antagonists aimed at discriminating UII and URP-mediated biological activities: Insight into UII and URP receptor activation.Br. J. Pharmacol. 2013; 168: 807-821Crossref PubMed Scopus (28) Google Scholar, 18Chatenet D. Nguyen Q.T. Letourneau M. Dupuis J. Fournier A. Urocontrin, a novel UT receptor ligand with a unique pharmacological profile.Biochem. Pharmacol. 2012; 83: 608-615Crossref PubMed Scopus (20) Google Scholar, 19Doan N.D. Nguyen T.T. Letourneau M. Turcotte K. Fournier A. Chatenet D. Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart.Br. J. Pharmacol. 2012; 166: 243-257Crossref PubMed Scopus (29) Google Scholar, 20Jarry M. Diallo M. Lecointre C. Desrues L. Tokay T. Chatenet D. Leprince J. Rossi O. Vaudry H. Tonon M.C. Prezeau L. Castel H. Gandolfo P. The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte activity through common and distinct mechanisms: Involvement in cell proliferation.Biochem. J. 2010; 428: 113-124Crossref PubMed Scopus (47) Google Scholar, 21Prosser H.C. Forster M.E. Richards A.M. Pemberton C.J. Urotensin II and urotensin II-related peptide (URP) in cardiac ischemia-reperfusion injury.Peptides. 2008; 29: 770-777Crossref PubMed Scopus (38) Google Scholar). Recent years have witnessed the emergence of useful molecules, with probe-dependent actions, that could shed light on the respective roles and importance of UII and URP under normal and pathological conditions (13Billard E. Iddir M. Nassour H. Lee-Gosselin L. Poujol de Molliens M. Chatenet D. New directions for urotensin II receptor ligands.Pept. Sci. 2018; e24056Google Scholar, 17Chatenet D. Letourneau M. Nguyen Q.T. Doan N.D. Dupuis J. Fournier A. Discovery of new antagonists aimed at discriminating UII and URP-mediated biological activities: Insight into UII and URP receptor activation.Br. J. Pharmacol. 2013; 168: 807-821Crossref PubMed Scopus (28) Google Scholar, 18Chatenet D. Nguyen Q.T. Letourneau M. Dupuis J. Fournier A. Urocontrin, a novel UT receptor ligand with a unique pharmacological profile.Biochem. Pharmacol. 2012; 83: 608-615Crossref PubMed Scopus (20) Google Scholar, 22Billard E. Hebert T.E. Chatenet D. Discovery of new allosteric modulators of the urotensinergic system through substitution of the urotensin II-related peptide (URP) phenylalanine residue.J. Med. Chem. 2018; 61: 8707-8716Crossref PubMed Scopus (8) Google Scholar, 23Billard E. Letourneau M. Hebert T.E. Chatenet D. Insight into the role of urotensin II-related peptide tyrosine residue in UT activation.Biochem. Pharmacol. 2017; : 100-107Crossref PubMed Scopus (7) Google Scholar, 24Douchez A. Billard E. Hebert T.E. Chatenet D. Lubell W.D. Design, synthesis, and biological assessment of biased allosteric modulation of the urotensin II receptor using achiral 1,3,4-Benzotriazepin-2-one turn mimics.J. Med. Chem. 2017; 60: 9838-9859Crossref PubMed Scopus (13) Google Scholar, 25Dufour-Gallant J. Chatenet D. Lubell W.D. De novo conception of small molecule modulators based on endogenous peptide ligands: Pyrrolodiazepin-2-one gamma-turn mimics that differentially modulate urotensin II receptor-mediated vasoconstriction ex vivo.J. Med. Chem. 2015; 58: 4624-4637Crossref PubMed Scopus (17) Google Scholar, 26Merlino F. Yousif A.M. Billard E. Dufour-Gallant J. Turcotte S. Grieco P. Chatenet D. Lubell W.D. Urotensin II((4-11)) azasulfuryl peptides: Synthesis and biological activity.J. Med. Chem. 2016; 59: 4740-4752Crossref PubMed Scopus (21) Google Scholar, 27Strack M. Billard E. Chatenet D. Lubell W.D. Urotensin core mimics that modulate the biological activity of urotensin-II related peptide but not urotensin-II.Bioorg. Med. Chem. Lett. 2017; 27: 3412-3416Crossref PubMed Scopus (7) Google Scholar). Promoting specific GPCR signaling events with biased agonists or allosteric modulators is a potentially innovative way to treat numerous conditions including cardiovascular disease, diabetes as well as neuropsychiatric/neurodegenerative disorders (28Kenakin T. The potential for selective pharmacological therapies through biased receptor signaling.BMC Pharmacol. Toxicol. 2012; 13: 3Crossref PubMed Scopus (39) Google Scholar). Hence, this notion has been introduced into various drug discovery programs through theoretical predictions based on known signaling components of cells and from studies in knockout animals (reviewed in (29Kenakin T. Functional selectivity and biased receptor signaling.J. Pharmacol. Exp. Ther. 2011; 336: 296-302Crossref PubMed Scopus (389) Google Scholar)). However, there are numerous instances where it is still not yet possible to predict what type of signaling bias represents a superior therapeutic approach. In these cases, empirical testing of exemplar molecules in animal models is a way forward. Such tools are currently unavailable in the context of the urotensinergic system. Pepducins are lipidated cell-penetrating peptides composed of a lipid moiety attached to a peptide corresponding to an amino acid segment from one of the cytoplasmic loops of a GPCR of interest (reviewed in (30Chaturvedi M. Schilling J. Beautrait A. Bouvier M. Benovic J.L. Shukla A.K. Emerging paradigm of intracellular targeting of G protein-coupled receptors.Trends Biochem. Sci. 2018; 43: 533-546Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 31Dimond P. Carlson K. Bouvier M. Gerard C. Xu L. Covic L. Agarwal A. Ernst O.P. Janz J.M. Schwartz T.W. Gardella T.J. Milligan G. Kuliopulos A. Sakmar T.P. Hunt 3rd., S.W. G protein-coupled receptor modulation with pepducins: Moving closer to the clinic.Ann. N. Y. Acad. Sci. 2011; 1226: 34-49Crossref PubMed Scopus (39) Google Scholar)). Following establishment of an equilibrium between the inner and outer leaflets of the lipid bilayer, pepducins interact with their cognate GPCR leading to stabilization of a restricted subset of its inactive and active conformational states (32Carlson K. McMurry T. Hunt 3rd., S.W. Pepducins: Lipopeptide allosteric modulators of GPCR signaling.Drug Discovery Today. Tech. 2012; 9: e1-e70Crossref Scopus (18) Google Scholar, 33Covic L. Gresser A.L. Talavera J. Swift S. Kuliopulos A. Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 643-648Crossref PubMed Scopus (267) Google Scholar, 34Covic L. Misra M. Badar J. Singh C. Kuliopulos A. Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation.Nat. Med. 2002; 8: 1161-1165Crossref PubMed Scopus (239) Google Scholar). Hence, such compounds can function as allosteric agonists or positive/negative allosteric modulators, making them useful for the study of GPCR signaling, as reported for protease-activated receptors (33Covic L. Gresser A.L. Talavera J. Swift S. Kuliopulos A. Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 643-648Crossref PubMed Scopus (267) Google Scholar, 34Covic L. Misra M. Badar J. Singh C. Kuliopulos A. Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation.Nat. Med. 2002; 8: 1161-1165Crossref PubMed Scopus (239) Google Scholar), chemokine receptors (35Kaneider N.C. Agarwal A. Leger A.J. Kuliopulos A. Reversing systemic inflammatory response syndrome with chemokine receptor pepducins.Nat. Med. 2005; 11: 661-665Crossref PubMed Scopus (114) Google Scholar, 36Quoyer J. Janz J.M. Luo J. Ren Y. Armando S. Lukashova V. Benovic J.L. Carlson K.E. Hunt 3rd, S.W. Bouvier M. Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E5088-5097Crossref PubMed Scopus (108) Google Scholar), and β-adrenergic receptors (37Carr 3rd, R. Du Y. Quoyer J. Panettieri Jr., R.A. Janz J.M. Bouvier M. Kobilka B.K. Benovic J.L. Development and characterization of pepducins as Gs-biased allosteric agonists.J. Biol. Chem. 2014; 289: 35668-35684Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), as well as for the potential treatment of various diseases including inflammatory diseases, cardiovascular pathologies, and cancer (38Tressel S.L. Koukos G. Tchernychev B. Jacques S.L. Covic L. Kuliopulos A. Pharmacology, biodistribution, and efficacy of GPCR-based pepducins in disease models.Methods Mol. Biol. 2011; 683: 259-275Crossref PubMed Scopus (71) Google Scholar). Here, we describe the design and pharmacological characterization of two pepducins derived from the second (hUT-Pep2) and third ([Trp1, Leu2]hUT-Pep3) intracellular loops of hUT. Our results demonstrate that both hUT-derived pepducins, while noncytotoxic, mediate ERK1/2 phosphorylation and IP1 accumulation. Using BRET-based biosensors, we observed that [Trp1, Leu2]hUT-Pep3 induced Gq, Gi, G13, β-arrestin 1, β-arrestin 2 activation, and epidermal growth factor receptor (EGFR) transactivation while UT-Pep2 only activated Gi, G13, β-arrestin-2 and EGFR transactivation. Interestingly, while only [Trp1, Leu2]hUT-Pep3 was able to induce proliferation in HEK 293-hUT cells, both pepducins were able to cause proliferation of neonatal rat cardiac fibroblasts. Further, while both hUT-derived pepducins were unable to induce rat aortic ring contraction on their own, they could modulate hUII- and URP-mediated contraction to different extents. These new molecular tools represent unique UT-targeted ligands that can be used to interrogate the involvement of specific pathways in hUT-associated diseases and to develop pharmacological agents with fewer side effects and a unique and more precise action for the treatment of various pathologies. Pepducins are composed of a synthetic peptide, mimicking an intracellular GPCR loop, to which a hydrophobic moiety, most commonly the fully saturated C16 fatty acid palmitate, is conjugated at their N-termini (39O'Callaghan K. Kuliopulos A. Covic L. Turning receptors on and off with intracellular pepducins: New insights into G-protein-coupled receptor drug development.J. Biol. Chem. 2012; 287: 12787-12796Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Based on a predicted structure for hUT (14Kim S.K. Li Y. Park C. Abrol R. Goddard 3rd., W.A. Prediction of the three-dimensional structure for the rat urotensin II receptor, and comparison of the antagonist binding sites and binding selectivity between human and rat receptors from atomistic simulations.ChemMedChem. 2010; 5: 1594-1608Crossref PubMed Scopus (22) Google Scholar, 15Brancaccio D. Merlino F. Limatola A. Yousif A.M. Gomez-Monterrey I. Campiglia P. Novellino E. Grieco P. Carotenuto A. An investigation into the origin of the biased agonism associated with the urotensin II receptor activation.J. Pept. Sci. 2015; 21: 392-399Crossref PubMed Scopus (15) Google Scholar), pepducins derived from the sequence of hUT intracellular loops 2 and 3 (Table 1) were synthesized using solid-phase peptide synthesis. hUT-Pep2 is identical to the ICL2 sequence found in hUT. The second pepducin ([Trp1, Leu2]hUT-Pep3) comprises the hUT ICL3 sequence; however, the first two N-terminal amino acids were replaced by the corresponding residues from rat UT ICL3 (-Arg-Arg-in hUT and -Trp-Leu- in rUT). This modification was necessary since the pepducin derived from hUT ICL3, comprising seven positively charged residues, was highly cytotoxic from 10−7 M, which prevented further use (Fig. 1A). Nonetheless, hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, up to 10−5 M, did not significantly affect HEK 293 cell viability (Fig. 1A). However, at 10−4 M, both derivatives were as cytotoxic as unmodified hUT-Pep3 at 10−7 M. Since these derivatives translocate to the inner leaflet of the plasma membrane, we tested the possibility that they promoted LDH release into the culture media. As shown in Figure 1B, none of these compounds, tested at the highest concentration (10−5 M) that did not lead to cell death, triggered LDH release. Altogether, our results demonstrated that both pepducins did not reduce cell viability at the concentrations used. Similar results were obtained in a second stably transfected CHO-hUT cell line (data not shown).Table 1Amino acid sequences and analytical data of compounds hUII, URP, and hUT-derived pepducinsCpd nameSequenceMWaMolecular weight calculated on a CEM Liberty Blue peptide synthesizer software. calcMSbMALDI-TOF mass spectral analysis (m/z). foundhUIIH-Glu-Thr-Pro-Asp-[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH1387.61388.1URPH-Ala-[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH1016.41016.4hUT-Pep2Palm-Arg-Pro-Leu-Asp-Thr-Val-Gln-Arg-Pro-Lys-Gly-Tyr-NH21666.01667.1[Trp1, Leu2]hUT-Pep3Palm-Trp-Leu-Ser-Gln-Arg-Ala-Ser-Phe-Lys-Arg-Ala-Arg-Arg-NH21899.31899.9Abbreviation: Palm, palmitoyl.Values represent the observed m/z of the monoisotope [M + H]+ ions.a Molecular weight calculated on a CEM Liberty Blue peptide synthesizer software.b MALDI-TOF mass spectral analysis (m/z). Open table in a new tab Abbreviation: Palm, palmitoyl. Values represent the observed m/z of the monoisotope [M + H]+ ions. Conformational analysis of hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 was performed using solution NMR in DPC micelle suspensions, a solvent system commonly used in NMR and CD studies to mimic the zwitterionic membrane environment (40Saviello M.R. Malfi S. Campiglia P. Cavalli A. Grieco P. Novellino E. Carotenuto A. New insight into the mechanism of action of the temporin antimicrobial peptides.Biochemistry. 2010; 49: 1477-1485Crossref PubMed Scopus (38) Google Scholar). Figures of the NOESY spectra of hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 are shown in the supporting information (Figs. S1 and S2). hUT-Pep2 yielded well-resolved NMR spectra and measured NMR parameters indicated a folded structure (Tables S1 and S2). In particular, medium-range Nuclear Overhauser effects (NOEs, Table S2) between Hα(i) and HN(i+2) pointed to β-turn structures confirmed by other diagnostic parameters, such as amide temperature coefficients and 3JHN-HA coupling constants. The palmitate moiety turned out to be mostly unstructured, as could be inferred by the degeneracy of the proton chemical shifts of almost all the methylene groups. However, the amide bond connecting palmitate and peptide is in trans configuration as suggested by the intense NOE contact between the Cα protons of the palmitate and NH of Arg1. Actually, 135 NOEs (Table S2) were used in the structure calculation of hUT-Pep2, which corresponds to 11.2 NOEs per residue, a ratio similar to what is found in structured protein (41Doreleijers J.F. Rullmann J.A. Kaptein R. Quality assessment of NMR structures: A statistical survey.J. Mol. Biol. 1998; 281: 149-164Crossref PubMed Scopus (93) Google Scholar). Among those, 22 medium range NOEs in line with folded (β-turn) peptides were observed. Restrained MD calculations provided the well-defined structures shown in Figure 2A. Starting from the N-terminus, two type I β-turns along residues 1–4 and 3–6 and three distorted (type IV) β-turns along residues 4–7, 5–8, and 6–9 can be observed in the simulated structures of hUT-Pep2. Interestingly, the obtained structure can be easily overlapped with the corresponding ICL2 segment of a hUT model recently generated (Fig. 2B) (15Brancaccio D. Merlino F. Limatola A. Yousif A.M. Gomez-Monterrey I. Campiglia P. Novellino E. Grieco P. Carotenuto A. An investigation into the origin of the biased agonism associated with the urotensin II receptor activation.J. Pept. Sci. 2015; 21: 392-399Crossref PubMed Scopus (15) Google Scholar). hUT-Pep2 therefore preserves the conformational propensities of the corresponding ICL2 segment when embedded in the wild-type receptor. Unfortunately, NMR spectra of [Trp1, Leu2]hUT-Pep3, despite complete assignment of all the proton resonances (Table S3), showed many broad and overlapping signals particularly in the amide proton region that prevented measure of many NMR parameters (3JHN-HA coupling constants and most of the NOEs), and therefore NMR-based calculation of its 3D structure. However, retrievable NMR parameters from the spectra (a few NOEs, up-field shifts of the Hα resonances, and amide temperature coefficients, Tables S3 and S4) suggest a high tendency of [Trp1, Leu2]hUT-Pep3 to fold into a helix. Using circular dichroism (CD) analyses, we were able to observe that [Trp1, Leu2]hUT-Pep3, under any condition tested, was able to assume folded structural motifs (Fig. 2C). In 0.1 mg mL−1 SDS solution (red), it appeared to assume a β-sheet conformation (lower peak at 216 nm), while in TFE solutions it appeared to fold as an α-helix (dark green, TFE 80%, lower peaks at 205 and 216 nm). In 0.5 mg mL−1 DPC solution, it appeared to assume a less structured α-helix conformation, which correlates with our NMR data, while in water a nonstructured shape (random coil) was adopted. Worth noting, a pepducin derived from the ICL3 of PAR1 studied in DPC solutions also showed a stable helical conformation (42Zhang P. Leger A.J. Baleja J.D. Rana R. Corlin T. Nguyen N. Koukos G. Bohm A. Covic L. Kuliopulos A. Allosteric activation of a G protein-coupled receptor with cell-penetrating receptor mimetics.J. Biol. Chem. 2015; 290: 15785-15798Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Together, these results suggest that both pepducins likely adopt conformations closely related to those found within the wild-type receptor. hUT-mediated production of the inositol 1,4,5-trisphosphate metabolite inositol monophosphate (IP1) was quantified in CHO-hUT cells using the IP-One terbium immunoassay. In agreement with previously reported data (43Brule C. Perzo N. Joubert J.E. Sainsily X. Leduc R. Castel H. Prezeau L. Biased signaling regulates the pleiotropic effects of the urotensin II receptor to modulate its cellular behaviors.FASEB J. 2014; 28: 5148-5162Crossref PubMed Scopus (35) Google Scholar), we observed that hUII induced a time-dependent increase in IP1 that reached a plateau after 40 min (Fig. 3A). Concentration–response curves, constructed after 40 min incubation, revealed that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 induced IP1 production in CHO cells stably expressing hUT (Fig. 3B) but not untransfected CHO-K1 cells after a similar period of incubation (Fig. S3). Compared with hUII (pEC50 = 12.54 ± 0.06; Emax = 97 ± 1), hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 appeared to be less potent (pEC50 < 6)" @default.
• W3191569443 created "2021-08-16" @default.
• W3191569443 creator A5006175603 @default.
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• W3191569443 creator A5072339386 @default.
• W3191569443 date "2021-09-01" @default.
• W3191569443 modified "2023-10-16" @default.
• W3191569443 title "Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands" @default.
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• W3191569443 doi "https://doi.org/10.1016/j.jbc.2021.101057" @default.

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