2-{N-[(2,4,5-trichlorophenoxy) acetyl]-N-methylamino}-3-pyrrolidinepropanamide analogs as potential antagonists of Urotensin II receptor.

THE PURPOSE OF THE ARTICLE: To identify novel small molecule antagonists of Urotensin II receptor with acceptable pharmacological profile. MATERIALS AND METHODS: Structure-activity-relationship (SAR) studies on 2-{N-[(2,4,5-trichlorophenoxy) acetyl]-N-methylamino}-3-pyrrolidinepropanamide series were conducted and shortlisted compounds were synthesized and evaluated in in vitro cell-based assays. Human and mouse Urotensin II receptor overexpressing CHO cells were used for calcium release and radioligand binding assays. Initial molecules in this series had solubility and inter-species variability issue in the calcium release assay. We, therefore, conducted SAR to overcome these 2 issues and molecules with accepted in vitro profile were evaluated further in mouse pressor response model to generate the in vivo proof of concept for UII receptor antagonization. RESULTS AND CONCLUSIONS: We report herewith identification of 2-{N-[(2,4,5-trichlorophenoxy)acetyl]-N-methylamino}-3-pyrrolidinepropanamides series to obtain novel small molecule antagonists of Urotensin II receptor with acceptable pharmacological profile.

International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, urotensin II-related peptide, and their receptor: from structure to function.

Urotensin II (UII) is a cyclic neuropeptide that was first isolated from the urophysis of teleost fish on the basis of its ability to contract the hindgut. Subsequently, UII was characterized in tetrapods including humans. Phylogenetic studies and synteny analysis indicate that UII and its paralogous peptide urotensin II-related peptide (URP) belong to the somatostatin/cortistatin superfamily. In mammals, the UII and URP genes are primarily expressed in cholinergic neurons of the brainstem and spinal cord. UII and URP mRNAs are also present in various organs notably in the cardiovascular, renal, and endocrine systems. UII and URP activate a common G protein-coupled receptor, called UT, that exhibits relatively high sequence identity with somatostatin, opioid, and galanin receptors. The UT gene is widely expressed in the central nervous system (CNS) and in peripheral tissues including the retina, heart, vascular bed, lung, kidney, adrenal medulla, and skeletal muscle. Structure-activity relationship studies and NMR conformational analysis have led to the rational design of a number of peptidic and nonpeptidic UT agonists and antagonists. Consistent with the wide distribution of UT, UII has now been shown to exert a large array of biologic activities, in particular in the CNS, the cardiovascular system, and the kidney. Here, we review the current knowledge concerning the pleiotropic actions of UII and discusses the possible use of antagonists for future therapeutic applications.

Urotensin core mimics that modulate the biological activity of urotensin-II related peptide but not urotensin-II.

A novel approach for the synthesis of head-to-tail cyclic peptides has been developed and used to prepare two mimics of the urotensin II-related peptide (URP) cyclic core. Mimics 1 and 2 (c[Trp-Lys-Tyr-Gly-ψ(triazole)-Gly] and c[Phe-Trp-Lys-Tyr-Gly-ψ(triazole)-Gly]) were respectively prepared using a combination of solid- and solution-phase synthesis. The silyl-based alkyne-modifying (SAM) linker enabled installation of C-terminal alkyne and N-terminal azide moieties onto linear peptide precursors, which underwent head-to-tail copper-catalyzed azide-alkyne cycloaddition (CuAAC) in solution. In an aortic ring contraction assay, neither 1 nor 2 exhibited agonist activity; however, both inhibited selectively URP- but not UII-mediated vasoconstriction. The core phenylalanine residue was shown to be important for enhancing modulatory activity of the urotensinergic system.

Urotensin-II peptidomimetic incorporating a non-reducible 1,5-triazole disulfide bond reveals a pseudo-irreversible covalent binding mechanism to the urotensin G-protein coupled receptor.

The urotensin-II receptor (UTR) is a class A GPCR that predominantly binds to the pleiotropic cyclic peptide urotensin-II (U-II). U-II is constrained by a disulfide bridge that induces a ß-turn structure and binds pseudo-irreversibly to UTR and is believed to result in a structural rearrangement of the receptor. However, it is not well understood how U-II binds pseudo-irreversibly and the nature of the reorganization of the receptor that results in G-protein activation. Here we describe a series of U-II peptidomimetics incorporating a non-reducible disulfide bond structural surrogate to investigate the feasibility that native U-II binds to the G protein-coupled receptor through disulfide bond shuffling as a mechanism of covalent interaction. Disubstituted 1,2,3-triazoles were designed with the aid of computational modeling as a non-reducible mimic of the disulfide bridge (Cys5-Cys10) in U-II. Solid phase synthesis using CuAAC or RuAAC as the key macrocyclisation step provided four analogues of U-II(4-11) incorporating either a 1,5-triazole bridge (5, 6) or 1,4-triazole bridge (9, 10). Biological evaluation of compounds 5, 6, 9 and 10 was achieved using in vitro [125I]UII binding and [Ca2+]i assays at recombinant human UTR. Compounds 5 and 6 demonstrated high affinity (KD ∼ 10 nM) for the UTR and were also shown to bind reversibly as predicted and activate the UTR to increase [Ca2+]i. Importantly, our results provide new insight into the mechanism of covalent binding of U-II with the UTR.

Conformation and Dynamics of Human Urotensin II and Urotensin Related Peptide in Aqueous Solution.

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K8Y9 or F6W7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F6W7K8 or W7K8Y9. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

An investigation into the origin of the biased agonism associated with the urotensin II receptor activation.

The urotensin II receptor (UTR) has long been studied mainly for its involvement in the cardiovascular homeostasis both in health and disease state. Two endogenous ligands activate UTR, i.e. urotensin II (U-II) and urotensin II-related peptide (URP). Extensive expression of the two ligands uncovers the diversified pathophysiological effects mediated by the urotensinergic system such as cardiovascular disorders, smooth muscle cell proliferation, renal disease, diabetes, and tumour growth. As newly reported, U-II and URP have distinct effects on transcriptional activity, cell proliferation, and myocardial contractile activities supporting the idea that U-II and URP interact with UTR in a distinct manner (biased agonism). To shed light on the origin of the divergent activities of the two endogenous ligands, we performed a conformational study on URP by solution NMR in sodium dodecyl sulfate micelle solution and compared the obtained NMR structure of URP with that of hU-II previously determined. Finally, we undertook docking studies between URP, hU-II, and an UT receptor model.

Urantide conformation and interaction with the urotensin-II receptor.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have previously identified the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor (UTR) antagonist described to date. Urantide may have potential clinical value in the treatment of atherosclerosis. In the present study, we studied the conformational preferences of urantide in DPC micelles and developed a urantide/UTR interaction model. This model can help the design of novel peptides and small molecules as UTR antagonists.

Expanding Structure-Activity Relationships of Human Urotensin II Peptide Analogues: A Proposed Key Role of the N-Terminal Region for Novel Urotensin II Receptor Modulators.

While the urotensinergic system plays a role in influencing various pathologies, its potential remains untapped because of the absence of therapeutically effective urotensin II receptor (UTR) modulators. Herein, we developed analogues of human urotensin II (hU-II) peptide in which, along with well-known antagonist-oriented modifications, the Glu1 residue was subjected to single-point mutations. The generated library was tested by a calcium mobilization assay and ex vivo experiments, also in competition with selected ligands. Interestingly, many derivatives showed noncompetitive modulation that was rationalized by the lateral allostery concept applied to a G protein-coupled receptor (GPCR) multimeric model. UPG-108 showed an unprecedented ability to double the efficacy of hU-II, while UPG-109 and UPG-111 turned out to be negative allosteric modulators of UTR. Overall, our investigation will serve to explore and highlight the expanding possibilities of modulating the UTR system through N-terminally modified hU-II analogues and, furthermore, will aim to elucidate the intricate nature of such a GPCR system.

New insight into the binding mode of peptides at urotensin-II receptor by Trp-constrained analogues of P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-D-Trp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp(7) residue was replaced by the highly constrained L-Tpi and D-Tpi residues. The replacement of the Trp(7) by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation-activity relationships previously reported on UT receptor ligands.

Urotensin II and its receptor in the killifish gill: regulators of NaCl extrusion.

The peptide urotensin II (UII) and its receptor (UT) mediate cardiovascular and renal effects in both mammals and fishes. In both groups, vasopressor and diuretic responses predominate, although, in mammals, some secondary vasodilatation is found, mediated by secondary release of nitric oxide or prostacyclin. In fishes, gill extrusion of NaCl is inhibited by UII, but a single study has determined that UT is expressed in gill vasculature, not on the epithelium that mediates the transport. To begin to clarify the pathways involved in UII inhibition of gill transport, we have cloned the cDNA encoding UII and UT from the euryhaline killifish (Fundulus heteroclitus L.) gill and spinal cord, quantified UT mRNA expression in various tissues and measured relative expression in gill tissue from fish acclimated to seawater (SW) vs fresh water (FW). We have also localized UT in the gill epithelium, and measured the effect of UII on ion transport across the opercular epithelium. We found that both UII and UT are synthesized in the gill of F. heteroclitus and that gill UT mRNA levels are ~80% higher in SW- vs FW-acclimated individuals. In addition, UII inhibits NaCl transport across the opercular epithelium in a concentration-dependent manner, and this inhibition is at least partially mediated by both nitric oxide and a prostanoid.

Characterization of a corticotropin-releasing factor (CRF)/diuretic hormone-like peptide from tunicates: insight into the origins of the vertebrate CRF family.

The corticotropin-releasing factor (CRF) superfamily of peptides includes the four paralogous vertebrate peptide lineages of CRF, urotensin-1/urocortin/sauvagine, urocortin 2 and urocortin 3, as well as the diuretic hormones (DH) and peptides of the arthropods. However, there are considerable sequence differences between the group of vertebrate peptides and those of the arthropods, notably insects. Because of the likely incidence of the formation of paralogous forms within the insects and the great variation in primary structures among these peptides, establishing the structure of the ancestral vertebrate version has not been possible. We screened the genomes of the tunicates, Ciona intestinalis and Ciona savignii, in silico, using the various conserved motifs of both the vertebrate CRF paralogues and the insect diuretic hormone sequences to identify the structure of the Ciona CRF/DH-like peptide genes. A single peptide gene was found in both genomes that possessed motifs reflective of both groups of peptides. These structures suggest a single CRF-like peptide was inherited by vertebrates and possibly chordates. Moreover, the conserved structure of the CRF peptide may have become constrained once it became associated with the regulation of the hypothalamus-pituitary-adrenal/interrenal axis.

Development of Novel 111-In-Labelled DOTA Urotensin II Analogues for Targeting the UT Receptor Overexpressed in Solid Tumours.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), 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 tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111In-DOTA-hUII or 111In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools.

Human urotensin II promotes hypertension and atherosclerotic cardiovascular diseases.

Human urotensin II (U-II), the most potent vasoconstrictor undecapeptide identified to date, and its receptor (UT) are involved in the pathogenesis of systemic and pulmonary hypertension. Here, we review recent advances in our understanding of the pathophysiology of U-II with particular reference to its role in atherosclerotic cardiovascular diseases. Single-nucleotide polymorphisms of U-II gene (S89N) are associated with onset of essential hypertension, type II diabetes mellitus, and insulin resistance in the Asian population. Plasma U-II levels are elevated in patients with vascular endothelial dysfunction-related diseases such as essential hypertension, diabetes mellitus, atherosclerosis, ischemic heart disease, and heart failure. Chronic infusion of U-II enhances atherosclerotic lesions in the aorta in apolipoprotein E-knockout mice. In human atherosclerotic plaques from the aorta and coronary and carotid arteries, U-II is expressed at high levels in endothelial cells (ECs) and lymphocytes, whereas UT is expressed at high levels in vascular smooth muscle cells (VSMCs), ECs, monocytes, and macrophages. U-II stimulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in human ECs as chemoattractant for monocytes, and accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages. U-II produces reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate oxidase activation in human VSMCs, and stimulates VSMC proliferation with synergistic effects when combined with ROS, oxidized LDL, and serotonin. Clinical studies demonstrated increased plasma U-II levels in accordance with the severity of carotid atherosclerosis in patients with essential hypertension and that of coronary artery lesions in patients with ischemic heart disease. Here, we summarize the key roles of U-II in progression of hypertension and atherosclerotic cardiovascular diseases.

Identification of new agonists of urotensin-II from a cyclic peptide library.

Urotensin-II (UT-II) is thought to be involved in the regulation of cardiovascular homeostasis and pathology. A head-to-tail cyclic hexapeptide library based on UT-II sequence was designed, synthesized, and evaluated by the activity on the UT-II receptor (GPR-14). A new synthetic sequence, WK[Xaa] (Xaa: amino acid with aromatic side chain), was identified as a characteristic minimum fragment activating hUT-II receptor instead of the WK[Y] sequence. Compound 1 showed an agonistic activity with an EC(50) value of 6.94 nM. The conformational investigation suggested that 1 did not have typical secondary structure in the message sequence. Structural analyses may enable us to investigate the active conformation of UT-II and lead to the identification of new ligands for GPR-14.

Three-dimensional model of the human urotensin-II receptor: docking of human urotensin-II and nonpeptide antagonists in the binding site and comparison with an antagonist pharmacophore model.

Human urotensin-II (hU-II) is a cyclic peptide that plays a central role in cardiovascular homeostasis and is considered to be the most potent mammalian vasoconstrictor identified to date. It is a natural ligand of the human urotensin-II (hUT-II) receptor, a member of the family of rhodopsin-like G-protein-coupled receptors. To understand the molecular interactions of hU-II and certain antagonists with the hUT-II receptor, a model of the hUT-II receptor in an active conformation with all its connecting loops was constructed by homology modeling. The initial model was placed in a pre-equilibrated lipid bilayer and re-equilibrated by several procedures of energy minimization and molecular dynamics simulations. Docking studies were performed for hU-II and for a series of nonpeptide hUT-II receptor antagonists in the active site of the modeled receptor structure. Results of the hU-II docking study are in agreement with our previous work and with experimental data showing the contribution of the extracellular loops II and III to ligand recognition. The docking of hU-II nonpeptide antagonists allows identification of key molecular interactions and confirms a previously reported hU-II antagonist pharmacophore model. The results of the present studies will be used in structure-based drug design for developing novel antagonists for the hUT-II receptor.

Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction.

Urotensin II (UII) and UII-related peptide (URP) are paralog neuropeptides whose existence and distribution in mouse have not yet been investigated. In this study, we showed by HPLC/RIA analysis that the UII-immunoreactive molecule in the mouse brain corresponds to a new UII(17) isoform. Moreover, calcium mobilization assays indicated that UII(17) and URP were equally potent in stimulating UII receptor (UT receptor). Quantitative RT-PCR and in situ hybridization analysis revealed that in the CNS UII and URP mRNAs were predominantly expressed in brainstem and spinal motoneurons. Besides, they were differentially expressed in the medial vestibular nucleus, locus coeruleus and the ventral medulla. In periphery, both mRNAs were expressed in skeletal muscle, testis, vagina, stomach, and gall bladder, whereas only URP mRNA could be detected in the seminal vesicle, heart, colon, and thymus. By contrast, the UT receptor mRNA was widely expressed, and notably, very high amounts of transcript occurred in skeletal muscle and prostate. In the biceps femoris muscle, UII-like immunoreactivity was shown to coexist with synaptophysin in muscle motor end plate regions. Altogether these results suggest that (i) UII and URP may have many redundant biological effects, especially at the neuromuscular junction; (ii) URP may more specifically participate to autonomic, cardiovascular and reproductive functions.

Structure-activity relationship study of position 4 in the urotensin-II receptor ligand U-II(4-11).

In the present study we describe the synthesis and biological evaluation of 24 analogues of the urotensin II (U-II) fragment U-II(4-11) substituted in position 4 with coded and non-coded aromatic amino acids. All of the new analogues behaved as full U-II receptor (UT) agonists. Our results indicated that aromaticity is well tolerated, size, length and chirality of the side chain are not important, while substituents with a nitrogen atom are preferred. Thus acylation of U-II(5-11) with small groups bearing nitrogen atoms could be instrumental in future studies for the identification of novel potent UT receptor ligands.

Nonpeptide Urotensin-II receptor agonists and antagonists: review and structure-activity relationships.

Human Urotensin-II (hU-II) is a cyclic 11-amino acid peptide that plays a role in cardiovascular homeostasis. Its receptor is a member of the class A of G-protein-coupled receptors, called GPR14. In recent years, several nonpeptide ligands have been reported in the literature. Most were identified by high-throughput screening and optimized by medicinal chemistry methods. Other nonpeptide ligands were discovered starting from the 3D structure of hU-II or other ligands. They were identified by a virtual screening approach based on a 3D pharmacophore or by structural similarity with others cyclic peptides. In this review, nonpeptide agonists and antagonists are presented in relation to structure-activity relationships.

Structure-activity relationships of urotensin II and URP.

Urotensin II (U-II) and urotensin II-related peptide (URP) are the endogenous ligands for the orphan G-protein-coupled receptor GPR14 now renamed UT. At the periphery, U-II and/or URP exert a wide range of biological effects on cardiovascular tissues, airway smooth muscles, kidney and endocrine glands, while central administration of U-II elicits various behavioral and cardiovascular responses. There is also evidence that U-II and/or URP may be involved in a number of pathological conditions including heart failure, atherosclerosis, renal dysfunction and diabetes. Because of the potential involvement of the urotensinergic system in various physiopathological processes, there is need for the rational design of potent and selective ligands for the UT receptor. Structure-activity relationship studies have shown that the minimal sequence required to retain full biological activity is the conserved U-II(4-11) domain, in particular the Cys5 and Cys10 residues involved in the disulfide bridge, and the Phe6, Lys8 and Tyr9 residues. Free alpha-amino group and C-terminal COOH group are not necessary for the biological activity, and modifications of these radicals may even increase the stability of the analogs. Punctual substitution of native amino acids, notably Phe6 and Trp7, by particular residues generates analogs with antagonistic properties. These studies, which provide crucial information regarding the structural and conformational requirements for ligand-receptor interactions, will be of considerable importance for the design of novel UT ligands with increased selectivity, potency and stability, that may eventually lead to the development of innovative drugs.

Automated synthesis of the generic peptide labelling agent N-succinimidyl 4-[(18)F]fluorobenzoate and application to (18)F-label the vasoactive transmitter urotensin-II as a ligand for positron emission tomography.

INTRODUCTION: The objectives of this work were to develop an automated production of N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]-SFB) and to test whether the vasoactive peptide urotensin-II (U-II) could be labelled by conjugation with [(18)F]-SFB. METHODS: A TRACERlab MX(FDG) synthesizer including an HPLC unit was used. The MS Excel synthesis sequence and the standard disposable FDG cassette were modified to allow the synthesis of [(18)F]-SFB. U-II was subsequently conjugated with [(18)F]-SFB, and the resulting (18)F-labelled peptides were characterised using in vitro ligand binding assays. RESULTS: [(18)F]-SFB was successfully synthesised in the TRACERlab MX(FDG) in 44.3+/-2.5% (n=25) radiochemical yield in 98 min. [(18)F]-SFB (8-12 GBq) has been produced with specific activities in the range of 250-350 GBq/mumol and a radiochemical purity >95%. [(18)F]-SFB was subsequently used to label U-II. Two radiolabelled products, [(18)F]-(Glu(1))-U-II and [(18)F]-(Lys(8))-U-II, were formed in an isolated radiochemical yield from [(18)F]-SFB of 5.2+/-0.3% and 29.0+/-3.7%, respectively (n=7). Radioligand binding assays revealed that [(18)F]-(Glu(1))-U-II had retained subnanomolar affinity. Binding to human skeletal muscle (n=3) was concentration dependent and saturable with K(d)=0.84+/-0.51 nM, B(max)=0.69+/-0.14 fmol/mg protein and Hill slope (nH)=1.03+/-0.12. CONCLUSIONS: [(18)F]-SFB has been synthesised using the TRACERlab MX(FDG) module, allowing production of up to 8-12 GBq of [(18)F]-SFB with specific activities of 250-350 GBq/mumol. [(18)F]-SFB was used for the labelling of U-II. In vitro characterisation demonstrated that [(18)F]-(Glu(1))-U-II had retained desirable binding properties and may be suitable as a positron emission tomography radioligand for the imaging of the U-II receptor.