Urotensin II (UII) is a potent peptide hormone with a complex species and vessel-dependent vascular profile. UII and the homologous UII-related peptide (URP) bind to the g-protein coupled urotensin II receptor (UT) with high affinity. The peptide ligands and receptor have been detected in numerous human and rat tissues including heart, brain and kidney. The kidney is a major source of UII, which appears to act as both an endocrine and paracrine mediator of renal function. UII has been shown to influence renal blood flow, glomerular filtration rate and sodium handling in the renal tubules. More speculative actions of UII as a pro-fibrotic mediator include the activation of fibroblasts and promotion of collagen synthesis. Abnormally elevated UII, URP and UT expression has been highlighted in a number of cardio-renal disease states; particularly end stage renal disease, diabetes and diabetic nephropathy (DN). This work aims to investigate the role of the UII system in the development and progression of CKD using an experimental model of CKD in rodents. The first aim of the current work involved establishing the surgical 5/6th subtotal nephrectomy (SNx) model of chronic kidney disease (CKD) in the laboratory and forming a profile of UII expression in late stage experimental CKD to complement UII clinical data which are exclusively from patients in the later stages of disease. UII/URP and UT were substantially over-expressed in the kidneys of SNx rats in late stage CKD. This novel insight complements the clinical profile of CKD/DN where over expression of the UII system is routinely reported. In a second study the 5/6th SNx rat model was used to explore the effects of chronic UT receptor antagonism on the progression of CKD. Although there were no discernible differences in kidney mass or histological profile between the treatment groups at the end of the study, there was a small delay in the development of albuminuria and in the onset of systolic blood pressure elevation in the UT antagonist treated cohort. The study did not produce clear-cut evidence defining the potential therapeutic value of UT-antagonism in the treatment of CKD. Despite this the results are encouraging and suggest that the role of UT-inhibition in CKD is worth considering further.

The role and functions of Urotensin II (UII), Urotensin II-related peptide (URP) and proangiotensin-12 (PA12) are currently ambiguous, either due their relatively new identification and isolation from their host species, or due to contrasting and conflicting reports observing the physiological and pathophysiological role of these spasmogens within the mammalian cardiovascular system. Accordingly, we sought to determine the true physiological functions of these peptides in both healthy and diseased states. The initial task was to reveal potential reasons for the contrasting responses to UII, and to define the role of UII within the isolated rat heart. UII and URP retain a highly conserved cyclic region, shown to be necessary in receptor binding and activation, with the high inter-species variance within the N-terminus reported to be of little importance. Our research revealed UII to be highly species-specific, stimulating potent, sustained vasodilation of the coronary arteries in response to the native form infused, while non-native UII peptides had either no effect, or caused significant vasoconstriction. UII-induced vasodilative effects were found to be mediated by nitric oxide and prostaglandin activity combined. Reviewing publications to date it was evident that many studies employed UII foreign to the host species, reporting potentially untrue effects, based on our findings. Recent studies have identified UII as a potent agent in developing and promoting atherosclerosis and coronary artery disease through UII-induced mitogenic activity and promoting foam cell formation. Hence, we observed the effect of infusing the native species of UII and URP into a model of cardiac ischemia-reperfusion. Both preconditioning the heart with UII or URP, or infusing UII or URP upon reperfusion caused significant coronary vasodilation following ischemia, and significantly attenuated ischemic-induced myocardial injury. These studies indicated elevating UII and URP provided a level of cardioprotection, not only when administered into healthy hearts prior to an ischemic event, but also in hearts having already undergone ischemia and the resultant endothelial damage. PA12 was the third peptide tested in the current thesis. Being newly identified and suggested to be a new component of the renin-angiotensin system (RAS) it was important to define the physiological role of PA12 upon the cardiovasculature, as the RAS is heavily associated with the development and progression of cardiovascular disease. Utilising the Langendorff isolated rat heart technique, PA12 was found to cause potent vasoconstriction of the coronary arteries, mediated by the angiotensin II type 1 receptor (AT₁R). Furthermore, using subjecting the perfusate samples to radioimmunoassay and RP-HPLC revealed PA12 was converted to AngII. Both PA12-induced vasoconstriction and generation of AngII were found to be dependent upon chymase activity, with inhibition of ACE1 having little effect. Myography was employed to further study the vascular response to PA12 throughout the rat arterial system from the common carotid to the femoral arteries. PA12-induced vasoconstriction displayed a potency gradient, with greatest constriction observed in vessels closest to the heart, with potency reduced and eventually lost further from the heart. PA12-induced vasoactivity was shown to be dependent upon both chymase and ACE1 activity, with ACE1 regulating PA12 activity with greater potency. The intracellular pathways stimulated in response to PA12 were defined using western blotting, with PA12 stimulating phosphorylation of ERK1/2, JNK, p38 and PKCα/β₁₁, but having no influence on PKCδ/θ. Stimulation of these pathways is consistent with the observed PA12-induced vasoconstriction, and also indicates that PA12 activation of AT₁R and the subsequent cytokines, could potentially stimulate hypertrophy, apoptosis, cell growth and differentiation, and inflammation, promoting cadiovascular remodelling and progressing atherosclerosis, hypertension and other vascular diseases if not sufficiently regulated. Taken together, these studies indicate PA12 may have a primary role within the local, tissue-based RAS, providing an alternate substrate to angiotensin I, while ACE1 is the primary regulatory enzyme within the circulation. Our findings also display the chymase-dependent PA12/AT₁R pathway as potential novel targets for pharmacological inhibition of RAS activity to ameliorate hypertension and maladaptive vascular remodelling.

The role and functions of Urotensin II (UII), Urotensin II-related peptide (URP) and proangiotensin-12 (PA12) are currently ambiguous, either due their relatively new identification and isolation from their host species, or due to contrasting and conflicting reports observing the physiological and pathophysiological role of these spasmogens within the mammalian cardiovascular system. Accordingly, we sought to determine the true physiological functions of these peptides in both healthy and diseased states. The initial task was to reveal potential reasons for the contrasting responses to UII, and to define the role of UII within the isolated rat heart. UII and URP retain a highly conserved cyclic region, shown to be necessary in receptor binding and activation, with the high inter-species variance within the N-terminus reported to be of little importance. Our research revealed UII to be highly species-specific, stimulating potent, sustained vasodilation of the coronary arteries in response to the native form infused, while non-native UII peptides had either no effect, or caused significant vasoconstriction. UII-induced vasodilative effects were found to be mediated by nitric oxide and prostaglandin activity combined. Reviewing publications to date it was evident that many studies employed UII foreign to the host species, reporting potentially untrue effects, based on our findings. Recent studies have identified UII as a potent agent in developing and promoting atherosclerosis and coronary artery disease through UII-induced mitogenic activity and promoting foam cell formation. Hence, we observed the effect of infusing the native species of UII and URP into a model of cardiac ischemia-reperfusion. Both preconditioning the heart with UII or URP, or infusing UII or URP upon reperfusion caused significant coronary vasodilation following ischemia, and significantly attenuated ischemic-induced myocardial injury. These studies indicated elevating UII and URP provided a level of cardioprotection, not only when administered into healthy hearts prior to an ischemic event, but also in hearts having already undergone ischemia and the resultant endothelial damage. PA12 was the third peptide tested in the current thesis. Being newly identified and suggested to be a new component of the renin-angiotensin system (RAS) it was important to define the physiological role of PA12 upon the cardiovasculature, as the RAS is heavily associated with the development and progression of cardiovascular disease. Utilising the Langendorff isolated rat heart technique, PA12 was found to cause potent vasoconstriction of the coronary arteries, mediated by the angiotensin II type 1 receptor (AT₁R). Furthermore, using subjecting the perfusate samples to radioimmunoassay and RP-HPLC revealed PA12 was converted to AngII. Both PA12-induced vasoconstriction and generation of AngII were found to be dependent upon chymase activity, with inhibition of ACE1 having little effect. Myography was employed to further study the vascular response to PA12 throughout the rat arterial system from the common carotid to the femoral arteries. PA12-induced vasoconstriction displayed a potency gradient, with greatest constriction observed in vessels closest to the heart, with potency reduced and eventually lost further from the heart. PA12-induced vasoactivity was shown to be dependent upon both chymase and ACE1 activity, with ACE1 regulating PA12 activity with greater potency. The intracellular pathways stimulated in response to PA12 were defined using western blotting, with PA12 stimulating phosphorylation of ERK1/2, JNK, p38 and PKCα/β₁₁, but having no influence on PKCδ/θ. Stimulation of these pathways is consistent with the observed PA12-induced vasoconstriction, and also indicates that PA12 activation of AT₁R and the subsequent cytokines, could potentially stimulate hypertrophy, apoptosis, cell growth and differentiation, and inflammation, promoting cadiovascular remodelling and progressing atherosclerosis, hypertension and other vascular diseases if not sufficiently regulated. Taken together, these studies indicate PA12 may have a primary role within the local, tissue-based RAS, providing an alternate substrate to angiotensin I, while ACE1 is the primary regulatory enzyme within the circulation. Our findings also display the chymase-dependent PA12/AT₁R pathway as potential novel targets for pharmacological inhibition of RAS activity to ameliorate hypertension and maladaptive vascular remodelling.

Urotensin-II (U-II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U-II on [Ca2+]i, was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase-positive and had morphological features of motoneurons. U-II induced [Ca2+]i, increases in these neurons with a threshold of 10-9 M, and a maximal effect at 10-6 M with an estimated EC50 of 6.2 × 10-9 M. The [Ca2+]i increase induced by U-II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+-free medium. Omega-conotoxin GVIA (10-7 M), a N-type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega-conotoxin GVIIC (10-7 M) and the L-type Ca2+ channel blocker, verapamil (10-5 M) had minimal effects. Down-regulation of protein kinase C by 4-α-phorbol 12-myristate 13-acetate (10-6 M) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10-6 M) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10-6 M) or Rp-8-pCPT-cGMPS (3 × 10-5 M) did not modify U-II-induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10-6 M) and Rp-cAMPS (3 × 10-5 M) reduced the response to 25 ± 3% and 42 ± 8%, respectively. Present results demonstrate that U-II modulates [Ca2+]i, in rat spinal cord neurons via protein kinase A cascade.

La surexpression de récepteurs couplés aux protéines G (RCPG) dans certains cancers est mise à profit en médecine nucléaire pour développer des radioligands capables de diagnostiquer la présence de tumeurs. A titre d’exemple, des analogues de la somatostatine marqués à l’indium-111 (111In-OctreoScan) sont utilisés pour le diagnostic de tumeurs neuroendocrines. L’urotensine II (UII), qui présente des homologies structurales avec la somatostatine, est considérée comme le neuropeptide vasoactif le plus puissant découvert à ce jour. L’UII interagit avec un unique RCPG de très haute affinité appelé UT, classiquement couplé à la voie Gαq/PLC/IP3/Ca2+. L’UII exerce notamment des activités pro-mitotiques et chémoattractantes et une expression élevée de l'UT a été rapportée dans plusieurs types de tumeurs solides humaines provenant des poumons, de l'intestin, de la prostate ou du sein. Ces données suggèrent que l'UT pourrait être une cible prometteuse pour concevoir des analogues urotensinergiques radiomarqués à finalités diagnostiques voire thérapeutiques. Deux analogues urotensinergiques capables de lier des isotopes radioactifs (le DOTA-UII et le DOTA-urantide) ont été synthétisés et radiomarqués avec succès avec l’111In. L'incubation de l’111In-DOTA-UII dans du plasma humain a révélé que seulement 30% du radioligand étaient dégradés après 3 heures d’incubation. L'administration de concentrations croissantes de DOTA-UII et de DOTA-urantide sur des cellules HEK-293 exprimant l'UT induit une augmentation dose-dépendante de la concentration cytosolique de calcium, avec une puissance et une efficacité similaires à celles obtenues avec l'UII (EC50: 1,26 10-8 M et 2,09 10-8 M, UII et DOTA-UII, respectivement) et urantide (EC50: 1,82 10-8 M et 1,52 10-8 M, urantide et DOTA-urantide, respectivement). Alors que la fixation sur l’UT du DOTA-UII ou l’UII entraîne l'internalisation du complexe ligand-récepteur (ELISA et immunocytochimie) dans les cellules HEK-293 exprimant l'UT, l’urantide et le DOTA-urantide restent inactifs. L'injection intraveineuse de l’111In-DOTA-UII chez des souris C57BL/6 a révélé un léger signal principalement restreint dans les reins, indiquant une clairance rapide du peptide. Des résultats similaires ont été obtenus avec des souris dont le gène codant l’UT a été invalidé (mUTS2R-/-) ou des souris exprimant constitutivement la forme humaine de l’UT (mUTS2R-/- hUTS2R+/+). Enfin, l’111In-DOTA-UII a été injecté chez des souris Nudes porteuses de xénogreffes hétérotopiques de cellules humaines A549 (adénocarcinome pulmonaire) ou DLD-1 (adénocarcinome colorectal), exprimant fonctionnellement l’UT, comme nous l’avons préalablement vérifié par analyses western blot, par immunohistochimie et par des tests de migration/prolifération cellulaire. Dans les deux cas, l'imagerie TEMP/TDM n'a toutefois pas révélé de signal exploitable dans les tumeurs, suggérant que la clairance du radioligand est trop importante pour permettre l'accumulation du radiotraceur et la détection des tumeurs. L’ensemble de nos résultats démontre que la conjugaison de DOTA dans les analogues urotensinergiques n'altère pas l'activation de l'UT. Cependant, d'autres investigations sont nécessaires pour diminuer la clairance rénale et améliorer l'imagerie tumorale et ainsi permettre, à terme, de concevoir des radioligands urotensinergiques à finalités diagnostiques voire théranostiques. / Overexpression of G protein-coupled receptors (GPCRs) in tumor is widely used to develop GPCR-targeting radioligands for solid tumor imaging. For example, somatostatin analogue labeled with 111Indium (111In-OctreoScan) is used for the diagnosis of neuroendocrine tumors. The vasoactive neuropeptide urotensin II (UII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumors from lung, gut, prostate or breast, suggesting that UT is a valuable target to design radiolabeled UII analogues for cancer diagnosis. Two urotensinergic analogues (DOTA-UII and DOTA-urantide) both containing the DOTA chelating group capable of complexing radioactive metal isotopes have been synthetized and radiolabeled with 111Indium. Incubation of 111In-DOTA-UII in human plasma revealed that only 30% of the radioligand was degraded after a 3h incubation period. Administration of graded concentrations of both DOTA-UII and DOTA-urantide in the vicinity of HEK293 cells expressing UT induced a dose-dependent increase in cytosolic calcium concentration, with similar potency and efficacy to that obtained with UII and urantide. These results demonstrated that conjugation of DOTA in urotensinergic analogues did not affect UT activation. DOTA-UII was also able to promote UT internalization in HEK293 cells expressing UT, while DOTA-urantide was ineffective. Intravenous injection of 111In-DOTA-UII in C57BL/6 mice revealed a slight signal mostly restricted in kidney, and similar results were obtained with knock-out mice or constitutively expressing human UT mice. Finally, 111In-DOTA-UII was injected into nude mice bearing heterotopic xenografts of human A549 cells (lung adenocarcinoma) or DLD-1 cells (colorectal adenocarcinoma) both expressing functional UT. In both cases, SPECT-CT imaging showed the absence of tumor uptake and significant renal and bladder uptakes, suggesting fast tracer clearance from the organism. However, further investigations will be necessary to decrease renal clearance and to improve tumor imaging.

Ce travail de thèse a porté sur l’étude structurale de peptides urotensinergiques humains par DC, RMN etmodélisation moléculaire. L’hUII (11 aa) et son analogue l’URP (8 aa) sont considérés comme les peptides vasoactifs les plus puissants connus à ce jour et sont impliqués dans divers systèmes biologiques, notamment le système cardiovasculaire et la prolifération des cellules tumorales. Ces deux peptides sont des ligands endogènes d'un RCPG, l’UT. Ils peuvent exercer des actions physiologiques communes mais aussi divergentes. Afin d’apporter des éléments permettant une meilleure compréhension de leurs activités biologiques, nous avons, dans un premier temps, déterminé la structure 3D de trois agonistes (hUII4-11, URP,P5U) et d’un antagoniste (urantide) dans un milieu micellaire mimant les membranes des cellules eucaryotes, le DPC. Dans les quatre peptides, nous avons observé la présence de deux conformations majoritaires du pont disulfure, RHStaple et LHHook, qui sont connues pour être essentielles à l’activité biologique. Nous avons mis en évidence une différence de nature de coude entre les agonistes (coude β de type I) et l’antagoniste (coude β de type II’). Nos analyses ont également permis de montrer l’existence de variations d’orientation des chaînes latérales des résidus F6, Y9 et plus spécialement celle de W7 entre les agonistes etl’antagoniste. Le groupe indole du D-W7 présente ainsi une rotation de 180°. Dans un deuxième temps, nous avons mis en évidence un impact de la concentration sur la conformation de l’hUII qui n’est pas observé pour l’URP. Ce phénomène d’auto-association pourrait avoir une influence sur l’interaction avec le récepteur et être à l’origine des divergences d’activités biologiques entre l’hUII et l’URP. / This work aims to characterize the structure of human urotensinergic peptides by CD, NMR and molecular modelling. hUII (11 aa) and its analogue URP (8 aa) are considered as the most potent vasoactive peptides known so far and are involved in various biological systems, including the cardiovascular system and tumor cell proliferation. These two peptides are endogenous ligands of a GPCR, UT, and exert common but also divergent physiological actions. In order to gain a better understanding of their biological activities, we determined the structures of three agonists (hUII4-11, URP, P5U) and one antagonist (urantide), in DPC micelles, a cellular eukaryotic mimetic membrane. For all peptides, we observed the presence of two major forms of the disulfide bridge, RHStaple and LHHook, which are known to be essential for biological activity. We showed a difference in the turn nature between agonists (type I β turn) and the antagonist (type II’ β turn). Our analyses also revealed that, in agonists and antagonist, the side chain orientations of residues F6, Y9 and more specifically W7 were different. Indeed, the indole group of D-W7 exhibited a 180° rotation. Secondly, we showed that, contrary to URP, the conformation of hUII was dependent on concentration. This selfassembly phenomenon may impact the interaction with the receptor and be responsible for the differential biological activities of hUII and URP.

Les pyrrolodiazépinones ont des activités biologiques intéressantes sur différents récepteurs biologiques, ce qui en font une cible de choix pour développer de nouvelles petites molécules biologiquement actives. Une méthodologie en solution a été développée pour synthétiser des pyrrolo[3,2-e][1,4]diazépin-2-ones, qui utilise la réaction de Pictet-Spengler pour former le cycle diazépinone, comme réaction clé. Il a été démontré que le pyrrolo[3,2-e][1,4]diazépin-2-one mime un tour-γ inverse par l’analyse de cristaux par rayon X. Cette méthodologie a été transposée sur trois types de support, soit la résine de Merrifield, de Wang et un support soluble (TAP). Le système urotensinergétique joue un rôle dans certaines pathologies du système cardiovasculaire, comme l’hypertension artérielle, l’insuffisance cardiaque et l’athérosclérose. Le système urotensinergétique est exprimé dans le système circulatoire, extractoire et le système nerveux central et comprend l’UII, l’URP et le récepteur UT. L’UII et l’URP humains sont composés respectivement des séquences d’acides aminés : H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH et H-Ala-c[Cys-Phe-Trp-LysTyr-Cys]-Val-OH. L’UII est le peptide vasoconstricteur le plus puissant connu à ce jour, dont l’URP est son isoforme. Les deux peptides ont des effets biologiques différents et on peut supposer qu’ils jouent un rôle distinct dans certaines pathologies. Il a été démontré que la partie active de l’UII est composée du tripeptide : Trp-Lys-Tyr. Dans l’URP, il a été démontré que ce tripeptide forme un tour-γ inverse, ce qui fait du récepteur UT une bonne cible biologique pour tester une librairie de pyrrolo[3,2-e][1,4]diazépin-2-ones, reprenant le tripeptide Trp-Lys-Tyr. Dernièrement, l’équipe du professeur David Chatenet a mis au point un peptide, l’urocontrin en remplaçant le segment Trp par un groupement biphénylalanine, qui a démontré un comportement spécifique comme antagoniste du récepteur UT. La Librairie de pyrrolo[3,2-e][1,4]diazépin-2-ones est basée sur la séquence TrpLys-Tyr de l’UII et de l’URP et de la séquence Trp-Lys-Bip de l’urocontrin. La synthèse de la librairie est faite sur la résine de Wang. La chaîne latérale de Tyr est mimée en utilisant la tyramine, Lys et Orn sont utilisés et la chaîne latérale de Trp a été reproduite II en utilisant le biphényle (comme dans l’urocontrin), le 1-naphthyle et le 2-naphthyle, sont introduits en employant les aldéhydes respectifs dans la réaction de Pictet-Spengler, ce qui donne les pyrrolo[3,2-e][1,4]diazépin-2-ones insaturés et les saturés S- et R-. L’évaluation de l’activité biologique des pyrrolo[3,2-e][1,4]diazépin-2-ones obtenues sur le récepteur UT se fait par des tests in vitro et ex vivo. Les tests in vitro consistent en un essai de liaisons sur des cellules CHO exprimant le récepteur UT en employant hUII-125I, comme contrôle radiomarqé. Les tests ex vivo sont effectués sur des aortes de rats pour mesurer la capacité à induire des contractions ou de moduler les contractions induites par hUII et URP. Certains R-pyrrolo[3,2-e][1,4]diazépin-2-ones causent une réduction de 50% du signal radioactivité du hUII-125I. Les pyrrolo[3,2-e][1,4]diazépin-2-ones ne montrent guère d’activité ex vivo, mais ils ont la capacité de moduler les contractions induites par l’hUII et l’URP. Par exemple, l’analogue Lys R-saturé avec le biphényle inhibe toutes les contractions de l’aorte à 14 µM avec un pKb de 5,54 à 4 µM, sans influencer les contractions de l’aorte induites par l’URP. Les pyrrolo[3,2-e][1,4]diazépin-2-ones ont une sélectivité pour le système urotensinergétique et sont inactifs sur le récepteur de l’endotheline-1. Les pyrrolo[3,2-e][1,4]diazépin-2-ones sont les premières petites molécules qui peuvent moduler l’activité biologique de l’UII et URP et offrir un potentiel intéressant comme outil pour étudier le système urotensinergétique. / The pyrrolodiazepinones have interesting biological activities on various biological receptors, which makes them a prime target for developing new biologically active small molecules. A methodology in solution had been developed for synthesizing pyrrolo[3,2-e][1,4]diazepin-2-ones, which utilized the Pictet-Spengler condensation as the key reaction to form the diazepinone ring. Pyrrolo[3,2-e][1,4]diazepin-2-ones were found to mimic an inverse γ-turn conformation by X-ray crystallographic analysis. The methodology was subsequently implemented on three types of support: Merrifield resin, Wang resin and the soluble TAP support. The urotensinergic system plays a role in certain diseases of the cardiovascular system, such as hypertension, heart failure and atherosclerosis. The urotensinergic system is expressed in the circulatory system, excretory and central nervous systems and includes the endogenous ligands urotensin II (UII) and urotensin II-related peptide (URP), and the urotensin receptor UT. The ligands UII and human URP are composed of the respective amino acid sequences: H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH and H-Ala-c[Cys-Phe-Lys-Tyr-Trp-Cys]-Val-OH. The peptide UII is the most potent vasoconstrictor known to date. The two peptides have different biological effects and may exhibit distinct roles in certain diseases. Their common Trp-Lys-Tyr sequence is believed to play an important role in the activity of UII and URP, and has been suggested to adopt an inverse γ-turn conformation. Notably, the laboratory of Professor David Chatenet developed the UT receptor antagonist peptide urocontrin by replacing the Trp residue by biphenylalanine (Bip) in URP. A library of pyrrolo[3,2-e][1,4]diazepin-2-one analogs was thus designed to mimic the inverse γ-turn sequence and targeted against UT. The pyrrolo[3,2-e][1,4]diazepin-2-one library was designed based on the Trp-Lys-Tyr sequence of UII and URP, and Trp-Lys-Bip sequence of urocontrin. The synthesis of the pyrrolo[3,2-e][1,4]diazepin-2-one library was achieved on Wang resin. The side chain of Tyr was mimicked using tyramine, Lys and Orn were used as the basic amino acid component, and the side chain of Trp was replicated using biphenyl (as in urocontrin) 1-naphthyl and 2-naphthyl groups that were introduced by employing their respective aldehydes in a Pictet-Spengler reaction, which furnished unsaturated and saturated S- and R-pyrrolo[3,2-e][1,4]diazepin-2-ones. Evaluation of the biological activity of the pyrrolo[3,2-e][1,4]diazepin-2-ones on the UT receptor was performed in vitro and ex vivo. Tests in vitro measured binding in CHO-cells which expressed UT by employing hUII-125I as radiolabeled control. In rat aorta, ex vivo tests measured capacity to induce contraction, or modulate the contractions induced by hUII and URP. Certain R-pyrrolo[3,2-e][1,4]diazepin-2-ones caused an up to 50% reduction of the radioactive signal of hUII-125I. Pyrrolo[3,2-e][1,4]diazepin-2-ones exhibited little activity ex vivo; however, they modulated contractions induced by hUII and URP. For example, the saturated R-analog possessing lysine and a biphenyl side chain inhibited completely hUII-induced contractions of the aorta at 14 µM with a pKb of 5.54 at 4 µM, without influencing URP-induced contractions. Pyrrolo[3,2-e][1,4]diazepin-2-ones were selective for the urotensinergic system and inactive on the related receptor endothelin-1. Pyrrolo[3,2-e][1,4]diazepin-2-ones represent the first small molecules that can differently modulate the biological activities of UII and URP, and offer interesting potential as tools for studying the urotensinergic system.