Lead Optimization of P5U and Urantide: Discovery of Novel Potent Ligands at the Urotensin-II Receptor Alfonso Carotenuto, † Luigia Auriemma, † Francesco Merlino, † Ali Munaim Yousif, † Daniela Marasco, †,‡ Antonio Limatola, † Pietro Campiglia, ∥ Isabel Gomez-Monterrey, † Paolo Santicioli, § Stefania Meini, § Carlo A. Maggi, § Ettore Novellino, † and Paolo Grieco* ,†,‡ † Department of Pharmacy, University of Naples “Federico II”, I-80131 Naples, Italy ‡ CIRPEB: Centro Interuniversitario di Ricerca sui Peptidi Bioattivi , University of Naples “Federico II”, DFM-Scarl, Institute of Biostructures and Bioimaging-CNR, 80134, Naples, Italy § Department of Pharmacology, Menarini Ricerche, Via Rismondo 12/A, I-50131, Florence, Italy ∥ Department of Pharmacy, University of Salerno, I-84084 Fisciano, Salerno Italy * S Supporting Information ABSTRACT: We have optimized 1 (P5U) and urantide, two important ligands at the h-UT receptor, designing several analogues by the exchange of the Tyr 9 residue with different unnatural aromatic amino acids. This study allowed us to discover novel ligands with improved activity. In particular, the replacement of the Tyr 9 residue by (pCN)Phe or (pNO 2 )Phe within the urantide sequence led to compounds 13 (UPG-83) and 15 (UPG-95), respectively, which showed pure antagonist activity toward UT receptor in a rat aorta bioassay. More interestingly, the replacement of the Tyr 9 in 1 sequence with the Btz or the (3,4-Cl)Phe residues led to superagonists 6 (UPG-100) and 10 (UPG-92) with pEC 50 values at least 1.4 log higher than that of 1, being the most potent UT agonists discovered to date. Compounds 10 and 13 showed also a good stability in a serum proteolytic assay. These ligands represent new useful tools to further characterize the urotensinergic system in human physiopathology. ■ INTRODUCTION Urotensin-II (U-II), a somatostatin-like neuropeptide, is a cyclic peptide originally isolated in the teleost fish Gillichthys mirabilis in the 1960s. 1 Subsequently, it has been demonstrated that U-II is also expressed in tetrapods and that its gene is located in the central nervous system (CNS). 2 The human U-II (hU-II) consists of 11 amino acids, H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp- Lys-Tyr-Cys]-Val-OH, and the whole sequence is recognized as the natural ligand of an orphan rat G-protein coupled receptor, first named GPR14. 3,4 Subsequently, a human G-protein coupled receptor with 75% similarity to the orphan rat receptor was replicated and finally renamed the UT receptor by IUPHAR. 5 The U-II precursor has proved to be widely expressed in various vertebrate species, including frogs, rats, mice, pigs, monkeys, and humans. 6 In 2003, a paralogue of U-II, known as urotensin-related peptide (URP), was isolated in mammals. 7 The U-II and URP genes are mostly expressed in motoneurons located in discrete brainstem nuclei and in the ventral horn of the spinal cord. 6 U-II and URP mRNAs have also been detected, albeit in lower concentrations, in different peripheral tissues, including the pituitary, heart, spleen, thymus, pancreas, kidney, small intestine, adrenal gland, and prostate. 6 Also, UT receptor is widely distributed in the CNS and in different organs and peripheral tissues, including cardiovascular system, kidney, bladder, prostate, and adrenal gland. 3,8−10 This extensive expression turned out to be very important in understanding the multiple pathophysiological effects in which the hU-II/UT receptor interaction is involved, such as cardiovascular disorders (heart failure, cardiac remodelling, hypertension), smooth muscle cell proliferation, renal disease, diabetes, and tumor growth. 11 It has recently been reported that U-II plays an important role in pulmonary hypertension, 12 modulates erectile function through eNOS, 13 and regulates cell proliferation in prostate cancer. 14 Furthermore, it has been demonstrated that U-II is implicated in immune inflammatory diseases 15 and in many effects on the CNS. 16 Hence, hU-II analogues could be therapeutically appealing in diverse pathological disorders. 17 The N-terminus portion of urotensin isopeptides is highly variable across animal species, 18 whereas the C-terminal region, structurally organized in a cyclic sequence by a disulfide bridge, c[Cys-Phe-Trp-Lys-Tyr-Cys], is well-conserved from species to species, outlining its primary role in the biological activity. 19 In fact, the conserved C-terminal octapeptide cyclic portion of U-II [hU-II(4−11)] retains both biological and binding properties. Received: February 10, 2014 Article pubs.acs.org/jmc © XXXX American Chemical Society A dx.doi.org/10.1021/jm500218x | J. Med. Chem. XXXX, XXX, XXX−XXX