Screening Platform toward New Anti-HIV Aptamers Set on Molecular Docking and Fluorescence Quenching Techniques.

By using a new rapid screening platform set on molecular docking simulations and fluorescence quenching techniques, three new anti-HIV aptamers targeting the viral surface glycoprotein 120 (gp120) were selected, synthesized, and assayed. The use of the short synthetic fluorescent peptide V35-Fluo mimicking the V3 loop of gp120, as the molecular target for fluorescence-quenching binding affinity studies, allowed one to measure the binding affinities of the new aptamers for the HIV-1 gp120 without the need to obtain and purify the full recombinant gp120 protein. The almost perfect correspondence between the calculated Kd and the experimental EC50 on HIV-infected cells confirmed the reliability of the platform as an alternative to the existing methods for aptamer selection and measuring of aptamer-protein equilibria.

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.

Urotensin-II-Targeted Liposomes as a New Drug Delivery System towards Prostate and Colon Cancer Cells.

Urotensin-II (UT-II) and its receptor (UTR) are involved in the occurrence of different epithelial cancers. In particular, UTR was found overexpressed on colon, bladder, and prostate cancer cells. The conjugation of ligands, able to specifically bind receptors that are overexpressed on cancer cells, to liposome surface represents an efficient active targeting strategy to enhance selectivity and efficiency of drug delivery systems. The aim of this study was to develop liposomes conjugated with UT-II (LipoUT) for efficient targeting of cancer cells that overexpress UTR. The liposomes had a mean diameter between 150 nm and 160 nm with a narrow size distribution (PI ≤ 0.1) and a doxo encapsulation efficiency of 96%. Moreover, the conjugation of UT-II to liposomes weakly reduced the zeta potential. We evaluated UTR expression on prostate (DU145, PC3, and LNCaP) and colon (WIDR and LoVo) cancer cells by FACS and western blotting analysis. UTR protein was expressed in all the tested cell lines; the level of expression was higher in WIDR, PC3, and LNCaP cells compared with LoVo and DU145. MTT cell viability assay showed that LipoUT-doxo was more active than Lipo-doxo on the growth inhibition of cells that overexpressed UTR (PC3, LNCaP, and WIDR) while in LoVo and DU145 cell lines, the activity was similar to or lower than that one of Lipo-doxo, respectively. Moreover, we found that cell uptake of Bodipy-labeled liposomes in PC3 and DU145 was higher for LipoUT than the not-armed counterparts but at higher extent in UTR overexpressing PC3 cells (about 2-fold higher), as evaluated by both confocal and FACS. In conclusion, the encapsulation of doxo in UT-II-targeted liposomes potentiated its delivery in UTR-overexpressing cells and could represent a new tool for the targeting of prostate and colon cancer.

Urokinase receptor derived peptides as potent inhibitors of the formyl peptide receptor type 1-triggered cell migration.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration. We and others have previously documented that the uPAR(84-95) sequence, interacts with the formyl peptide receptors (FPR)s, henceforth inducing cell migration of several cell lines, including leukocytes, and the synthetic shorter peptide (Ser88-Arg-Ser-Arg-Tyr92, SRSRY) retains chemotactic activity in vitro and in vivo. Recently, we have developed the head-to-tail cyclic analog [SRSRY], a new potent and stable inhibitor of monocyte trafficking. This prompted us to develop novel cyclic and linear analogs of [SRSRY] with the aim to broaden the knowledge about structure-activity relationships of peptide [SRSRY]. Herein we report their synthesis, effects on cell migration, conformational and docking analyses which served to envisage a new pharmacophore model for inhibitors of FPR1-triggered cell migration.

The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.

Discovery of Novel Potent and Selective Agonists at the Melanocortin-3 Receptor.

The melanocortin receptors 3 and 4 control energy homeostasis, food-intake behavior, and correlated pathophysiological conditions. The melanocortin-4 receptor (MC4R) has been broadly investigated. In contrast, the knowledge related to physiological roles of the melanocortin-3 receptor (MC3R) is lacking because of the limited number of known MC3R selective ligands. Here, we report the design, synthesis, biological activity, conformational analysis, and docking with receptors of two potent and selective agonists at the human MC3 receptor.

Cyclization of the urokinase receptor-derived ser-arg-ser-arg-tyr Peptide generates a potent inhibitor of trans-endothelial migration of monocytes.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.

Lead optimization of P5U and urantide: discovery of novel potent ligands at the urotensin-II receptor.

We have optimized 1 (P5U) and urantide, two important ligands at the h-UT receptor, designing several analogues by the exchange of the Tyr9 residue with different unnatural aromatic amino acids. This study allowed us to discover novel ligands with improved activity. In particular, the replacement of the Tyr9 residue by (pCN)Phe or (pNO2)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 Tyr9 in 1 sequence with the Btz or the (3,4-Cl)Phe residues led to superagonists 6 (UPG-100) and 10 (UPG-92) with pEC50 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.

Urotensin II receptor determines prognosis of bladder cancer regulating cell motility/invasion.

BACKGROUND: Non Muscle Invasive Bladder Transitional Cancer (NMIBC) and Muscle Invasive Bladder Transitional Cancer (MIBC)/invasive have different gene profile and clinical course. NMIBC prognosis is not completely predictable, since the relapse rate is higher than 20%, even in the form of MIBC. The aim of this study is to evaluate if UTR expression can discriminate between NMIBC and MIBC and predict the risk of relapses in NMIBCs. METHODS: We have investigated upon urotensin-II (UII) receptor (UTR) expression in vivo in 159 patients affected by NMIBC. The biological role of UTR was also investigated in vitro. UTR expression was evaluated in a tissue-micro-array, consisting of normal, NMIBC and invasive bTCC samples. RESULTS: UTR discriminated between NMIBC and MIBC and showed a significant correlation between low UTR expression and shorter disease free survival in NMIBC. The superagonist UPG84 induced growth suppression at nM concentrations on 3/4 cell lines. Bladder cancer cell treatment with the antagonist urantide or the knock-down of UTR with a specific shRNA significantly blocked both the motility and invasion of bladder cancer cells. CONCLUSIONS: The evaluation of UTR expression can discriminate between NMIBC at high and low risk of relapse. Moreover, our data suggest that UTR is involved in the regulation of motility, invasion and proliferation of bladder cancer cells. High UTR expression is an independent prognostic factor of good prognosis for NMIBC regulating motility and invasion of bladder cancer cells.