Corrigendum to "Activation of Melanocortin Receptors MC1 and MC5 Attenuates Retinal Damage in Experimental Diabetic Retinopathy".

[This corrects the article DOI: 10.1155/2016/7368389.].

The Amphibian Antimicrobial Peptide Temporin B Inhibits In Vitro Herpes Simplex Virus 1 Infection.

The herpes simplex virus 1 (HSV-1) is widespread in the population, and in most cases its infection is asymptomatic. The currently available anti-HSV-1 drugs are acyclovir and its derivatives, although long-term therapy with these agents can lead to drug resistance. Thus, the discovery of novel antiherpetic compounds deserves additional effort. Naturally occurring antimicrobial peptides (AMPs) represent an interesting class of molecules with potential antiviral properties. To the best of our knowledge, this study is the first demonstration of the in vitro anti-HSV-1 activity of temporin B (TB), a short membrane-active amphibian AMP. In particular, when HSV-1 was preincubated with 20 µg/ml TB, significant antiviral activity was observed (a 5-log reduction of the virus titer). Such an effect was due to the disruption of the viral envelope, as demonstrated by transmission electron microscopy. Moreover, TB partially affected different stages of the HSV-1 life cycle, including the attachment and the entry of the virus into the host cell, as well as the subsequent postinfection phase. Furthermore, its efficacy was confirmed on human epithelial cells, suggesting TB as a novel approach for the prevention and/or treatment of HSV-1 infections.

Activation of Melanocortin Receptors MC 1 and MC 5 Attenuates Retinal Damage in Experimental Diabetic Retinopathy.

We hypothesize that melanocortin receptors (MC) could activate tissue protective circuit in a model of streptozotocin- (STZ-) induced diabetic retinopathy (DR) in mice. At 12-16 weeks after diabetes induction, fluorescein angiography (FAG) revealed an approximate incidence of 80% microvascular changes, typical of DR, in the animals, without signs of vascular leakage. Occludin progressively decreased in the retina of mice developing retinopathy. qPCR of murine retina revealed expression of two MC receptors, Mc1r and Mc5r. The intravitreal injection (5 µL) of the selective MC1 small molecule agonist BMS-470539 (33 µmol) and the MC5 peptidomimetic agonist PG-901 (7.32 nM) elicited significant protection with regular course and caliber of retinal vessels, as quantified at weeks 12 and 16 after diabetes induction. Mouse retina homogenate settings indicated an augmented release of IL-1α, IL-1ß, IL-6, MIP-1α, MIP-2α, MIP-3α, and VEGF from diabetic compared to nondiabetic mice. Application of PG20N or AGRP and MC5 and MC1 antagonist, respectively, augmented the release of cytokines, while the agonists BMS-470539 and PG-901 almost restored normal pattern of these mediators back to nondiabetic values. Similar changes were quantified with respect to Ki-67 staining. Finally, application of MC3-MC4 agonist/antagonists resulted to be inactive with respect to all parameters under assessment.

Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage.

BACKGROUND AND PURPOSE: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. EXPERIMENTAL APPROACH: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP--MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. KEY RESULTS: All shocked rats treated with saline died within 30-35 min. Treatment with NDP--MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. CONCLUSIONS AND IMPLICATIONS: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

Antimicrobial properties of alpha-MSH and related synthetic melanocortins.

The natural antimicrobial peptides are ancient host defense effector molecules, present in organisms across the evolutionary spectrum. Several properties of alpha-melanocyte stimulating hormone (alpha-MSH) suggested that it could be a natural antimicrobial peptide. Alpha-MSH is a primordial peptide that appeared during the Paleozoic era, long before adaptive immunity developed and, like natural antimicrobial molecules, is produced by barrier epithelia, immunocytes, and within the central nervous system. alpha-MSH was discovered to have antimicrobial activity against two representative pathogens, Staphylococcus aureus and Candida albicans. The candidacidal influences of alpha-MSH appeared to be mediated by increases in cell cyclic adenosine monophosphate (cAMP). The cAMP-inducing capacity of alpha-MSH likely interferes with the yeast's own regulatory mechanisms of this essential signaling pathway. It is remarkable that this mechanism of action in yeast mimics the influences of alpha-MSH in mammalian cells in which the peptide binds to G-protein-linked melanocortin receptors, activates adenylyl cyclase, and increases cAMP. When considering that most of the natural antimicrobial peptides enhance the local inflammatory reaction, the anti-inflammatory and antipyretic effects of alpha-MSH confer unique properties to this molecule relative to other natural antimicrobial molecules. Synthetic derivatives, chemically stable and resistant to enzymatic degradation, could form the basis for novel therapies that combine anti-inflammatory and antimicrobial properties.

Urotensin-II receptor antagonists.

Urotensin-II (U-II) is a "somatostatin-like" cyclic neuropeptide which was originally isolated from goby fish urophysis, and subsequently identified in other species, including man. The interest in human U-II (hU-II) has grown enormously in the last few years, following the identification of a specific human receptor (formerly identified as the GPR14/SENR orphan receptor), now referred to as UT receptor. The U-II/UT system seems to play an important role in cardiovascular functions. hU-II vasoconstrictive potency is reported to be an order of magnitude greater than that of endothelin-1 (ET-1), which would make it the most potent mammalian vasoconstrictor identified to date. hU-II also exerts potent inotropic effects in the human heart in vitro. On the basis of its spectrum of activities, hU-II has been suggested to modulate cardiovascular homeostasis and possibly to be involved in certain cardiovascular pathologies. Central nervous effects of U-II have also been described, in particular, intracerebroventricular administration promotes anxiogenic-like behaviors in rodents. Furthermore, UT receptor overexpression has been observed in some tumor cell lines. Therefore, specific and selective UT receptor antagonists provide useful tools for investigating the (patho)physiological role(s) of the U-II/UT receptor system. In this review we aim to provide an overview of the research in the area of UT receptor antagonists as well as the progress in understanding the role of the U-II/UT system in human (patho)physiology.

A novel route to synthesize Freidinger lactams by micowave irradiation.

The incorporation of a Freidinger-like lactam structure into the backbone of peptides has been proven to be an useful strategy in the design of a variety of conformationally restricted targets. Several different strategies have been developed toward Freidinger lactams but no one resulted to be completely facile. Here, we report an efficient strategy that involves the iodo-derivatives in side chain of an appropriate amino acid used as electrophilic agent, and the standard solid phase peptide synthesis assisted by microwave irradiation. The methodology developed could be useful to perform Freidinger-like lactams with defined stereochemistry for routine use in solid phase peptide chemistry.

Three-dimensional structure of the alpha-MSH-derived candidacidal peptide [Ac-CKPV]2.

Previous research has shown that the immunomodulatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH) and its carboxy-terminal tripeptide KPV (Lys-Pro-Val alpha-MSH11-13) have antimicrobial influences. By inserting a Cys-Cys linker between two units of KPV, we designed the dimer [Ac-CKPV]2 that showed excellent candidacidal effects in pilot tests and was the subject of further investigations. [Ac-CKPV]2 was active against azole-resistant Candida spp. Therefore, the molecule appeared a promising candidate for therapy of fungal infections and was the subject of a structural study. 1H-NMR and restrained mechanic and dynamic calculations suggest that the peptide adopts an extended backbone structure with a beta-turn-like structure. These results open a pathway to development of additional novel compounds that have candidacidal effects potentially useful against clinical infections.

Recent structure-activity studies of the peptide hormone urotensin-II, a potent vasoconstrictor.

Human Urotensin-II is a potent vasoconstrictor and binds with high affinity to GPR14 receptor, recently cloned and renamed UT receptor. U-II vasoconstrictive potency is reported to be an order of magnitude greater than that of endothelin-1 (ET-1), which would make it the most potent mammalian vasoconstrictor identified to date. Urotensin-II is a neuropeptide "somatostatin-like" cyclic peptide, which was originally isolated from fish spinal cords, and which has recently been cloned from human. Human U-II is composed of only 11 amino acids residues, while fish and frog U-II possess 12 and 13 amino acids residues, respectively. The cyclic region of U-II, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. This review focuses on recent structure-activity relationships studies performed on Urotensin-II with the aim to provide the required structural elements to design new ligands as agonists and antagonists for UT receptor.

Extensive structure-activity studies of lactam derivatives of MT-II and SHU-9119: their activity and selectivity at human melanocortin receptors 3, 4, and 5.

The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nm) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-II and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nm, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.

Synthesis and biological evaluation on hMC3, hMC4 and hMC5 receptors of gamma-MSH analogs substituted with L-alanine.

To elucidate the molecular basis of the interaction of the native dodecapeptide gamma-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced l-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of gamma-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l-Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in gamma-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with l-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of gamma-MSH.

Oxabicyclo[3.2.1]octenes in organic synthesis--direct ring opening of oxabicyclo[3.2.1] systems employing silyl ketene acetals in concentrated solutions of lithium perchlorate-diethyl ether: application to the synthesis of the C(19)-C(27) fragment of rifamycin S.

[figure: see text] The direct opening at the bridgehead of oxabicyclo[3.2.1]octenes employing silyl ketene acetals in 4.0-5.0 M lithium perchlorate in diethyl ether has been realized, which gives rise to highly functionalized cycloheptadienes that can be further manipulated for use in natural product synthesis. The bridgehead opening reaction has been employed in the construction of the C(19)-C(27) fragment of Rifamycin S.

Preparation of 'side-chain-to-side-chain' cyclic peptides by Allyl and Alloc strategy: potential for library synthesis.

Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side-chain-to-side-chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibe method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh3)4/PhSiH3)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium-mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.

Novel esters of glaucarubolone as inducers of terminal differentiation of promyelocytic HL-60 cells and inhibitors of 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesion formation in mouse mammary organ culture.

In an effort to discover new chemotherapeutic/chemopreventive agents from natural sources, brusatol (1) was found to induce HL-60 cellular differentiation, accompanied by strong antiproliferative and cytotoxic effects. A series of natural and semisynthetic quassinoids (1-48) was designed to effect both antiproliferative and differentiation-inducing properties. Compounds were assessed in vitro using the HL-60 promyelocytic cell model. Changes in activity due to structural modification of the core structure glaucarubolone (24) were consistent with activities reported in other cell systems. However, the following were novel SAR findings: (1) semisynthetic analogues with a hydroxylated ring at the beta-position of the ester side chain at C-15 were able to induce cellular differentiation at concentrations lower than those inducing cell growth arrest, and (2) quassinoids inhibiting DNA synthesis with greater efficacy than reducing cellular viability possessed alkyl substitutions at the alpha-position of the C-15 ester side chain. Analogues from this latter group and brusatol (1) and bruceantin (2) inhibited dimethylbenz(a)anthracene-induced preneoplastic lesion formation in a mouse mammary organ culture. The novel finding of 1 and glaucarubolone analogues as potent inducers of differentiation leads to potential novel applications in the field of cancer.

Synthesis and structure-activity of antisense peptides corresponding to the region for CaM-binding domain of the inducible nitric oxide synthase.

Nitric oxide synthase (NOS) catalyses the conversion of L-arginine to nitric oxide (NO) which plays an important role in the regulation of cellular functions and intracellular communications. Three distinct isoforms of NOS have so far been identified, two constitutive and one inducible. All three mammalian isoforms of NOS contain putative CaM-binding domains with the canonical composition. In this paper we report the synthesis and the inhibitory activity on rat neuronal and lung inducible NOS of antisense peptides corresponding to the antisense strand read in 3' to 5' (CALM 1) or 5' to 3' (CALM 2) direction of the region encoding for the CaM-binding domain of the inducible NOS isoform (residues 503-522). CALM 1 inhibited, at all the concentrations tested (0.01-1 mM), both the inducible and constitutive NOS (IC(50) 98 microM and 56 microM, respectively), while CALM 2 (0.01-1 mM) was ineffective on both isoforms. The acetylation of CALM 1 at its amino terminal (CALM 8) completely abolished its inhibitory activity. We also synthesized and analysed the activity of amino terminal truncated analogues (CALM 3-7) of CALM 1, which selectively inhibited the inducible isoform, although less potently than the parent compound. The pentapeptides (CALM A-D) deriving from the cleavage of CALM 1 were ineffective, except the pentapeptide CALM C corresponding to the residues 513-517, which was as potent as the parent compound (IC(50) 65 microM).

Baeyer-Villiger oxidation promoted by reaction of peracids with cyclic oxocarbenium ions generated in situ from internal hemiketals.

[reaction: see text] Reactive cyclic oxocarbenium ions, generated in situ from internal hemiketals, undergo Baeyer-Villiger oxidation upon exposure to m-chloroperbenzoic acid leading, after hydrolysis of the resultant lactones, to acyclic fragments for use in natural product synthesis.

Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands.

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.

D-Amino acid scan of gamma-melanocyte-stimulating hormone: importance of Trp(8) on human MC3 receptor selectivity.

In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide gamma-melanocyte-stimulating hormone (gamma-MSH). The native gamma-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the gamma-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp(8) analogue, there is an increase in binding affinity by about 1 order of magnitude (IC(50) = 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe(6) analogue is about 10-fold more potent (IC(50) = 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, and MC5R revealed that the native peptide shows potent activity at the MC3R (EC(50) = 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg(10) (EC(50) = 35 nM) and DPhe(11) (EC(50) = 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp(8) compound (EC(50) = 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr(1) peptide is selective for the MC3R (EC(50) = 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10), but not Arg(7), result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of gamma-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10) (but not Arg(7)), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far.