Francesco Merlino

Novel α-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity

Previous investigations indicate that α-melanocyte-stimulating hormone (α-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broad-spectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(2′)-7, Phe-12]-MSH(6–13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8–13 is a key factor in antimicrobial activity. Synthetic analogues of α-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity.

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.

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.

Urotensin II(4–11) Azasulfuryl Peptides: Synthesis and Biological Activity

Cyclic azasulfuryl (As) peptide analogs of the urotensin II (UII, 1, H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) fragment 4-11 were synthesized to explore the influences of backbone structure on biological activity. N-Aminosulfamides were inserted as surrogates of the Trp(7) and Lys(8) residues in the biologically relevant Trp-Lys-Tyr triad. A combination of solution- and solid-phase methods were used to prepare novel UII((4-11)) analogs 6-11 by routes featuring alkylation of azasulfuryl-glycine tripeptide precursors to install various side chains. The pharmacological profiles of derivatives 6-11 were tested in vitro using a competitive binding assay and ex vivo using a rat aortic ring bioassay. Although the analogs exhibited weak affinity for the urotensin II receptor (UT) without agonistic activity, azasulfuryl-UII((4-11)) derivatives 7-9 reduced up to 50% of the effects of UII and urotensin II-related peptide (URP) without affecting their potency.

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. Copyright

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.

Urotensin-II Ligands: An Overview from Peptide to Nonpeptide Structures

Urotensin-II was originally isolated from the goby urophysis in the 1960s as a vasoactive peptide with a prominent role in cardiovascular homeostasis. The identification of human isoform of urotensin-II and its specific UT receptor by Ames et al. in 1999 led to investigating the putative role of the interaction U-II/UT receptor in multiple pathophysiological effects in humans. Since urotensin-II is widely expressed in several peripheral tissues including cardiovascular system, the design and development of novel urotensin-II analogues can improve knowledge about structure-activity relationships (SAR). In particular, since the modulation of the U-II system offers a great potential for therapeutic strategies related to the treatment of several diseases, like cardiovascular diseases, the research of selective and potent ligands at UT receptor is more fascinating. In this paper, we review the developments of peptide and nonpeptide U-II structures so far developed in order to contribute also to a more rational and detectable design and synthesis of new molecules with high affinity at the UT receptor.

Treatment with a Urokinase Receptor-derived Cyclized Peptide Improves Experimental Colitis by Preventing Monocyte Recruitment and Macrophage Polarization

Background:Leukocyte migration across the blood barrier and into tissues represents a key process in the pathogenesis of inflammatory bowel diseases. The urokinase receptor (urokinase-type plasminogen activator receptor) is a master regulator of leukocyte recruitment. We recently found that cyclization of the urokinase-type plasminogen activator receptor–derived peptide Ser-Arg-Ser-Arg-Tyr [SRSRY] inhibits transendothelial migration of monocytes. Now, we have explored the effects of [SRSRY] administration during experimental colitis. Methods:The effects of [SRSRY] on cytokine profile, cytoskeletal organization, and cell migration were investigated using phorbol-12-myristate acetate–differentiated THP-1 cells exposed to polarizing stimuli. In vivo, [SRSRY] was intraperitoneally administered during dextran sodium sulfate– or 2,4,6-trinitrobenzene sulfonic acid–induced colitis in wild-type or urokinase-type plasminogen activator receptor knockout mice. Levels of pro-inflammatory cytokines and inflammatory monocytes in mucosal infiltrates were assessed by enzyme-linked immunosorbent assay and flow cytometry, respectively. Results:[SRSRY] prevents M0 to M1 transition and migration of M1 polarized macrophages. In vivo, [SRSRY] reduces intestinal inflammation diminishing body weight loss and disease activity index. These beneficial effects are accompanied by a reduction of interleukin 1&bgr;, interleukin 6, and tumor necrosis factor &agr;, an increase of interleukin 10, and an abridged recruitment of inflammatory monocytes to the inflamed tissue. Conclusions:Altogether, these findings indicate that [SRSRY] may be considered as a new drug useful for the pharmacological treatment of chronic inflammatory diseases, such as inflammatory bowel diseases.

SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage

Abstract Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.