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The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)–NS3pro protease. Kinetic parameters (kcat/Km) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4–P3′ residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/Km=11087 M−1·s−1). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4–P4′ positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the Km values, whereas variations at the prime side sequences had higher impact on kcat.

The guanidine compound ME10092 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine), which possesses a strong cardioprotective effect to ischemia-reperfusion, was assessed for different pharmacological actions that may underlie its cardioprotective effect. In the living rat ME10092 decreased the blood pressure and heart rate in a dose-dependent manner. We found ME10092 to bind to 1- and 2-adrenoreceptors with moderate affinity (Kivalues 1–4 μM), and to block adrenaline-elicited contractile responses in isolated guinea pig aortas. Our results indicate that ME10092 possesses a certain anti-oxidant profile. Thus, in a competitive manner and with low affinity it inhibited the bovine milk xanthine oxidase enzyme, as well as NAD(P)H oxidase driven oxyradical formation in membrane fractions isolated from the rat brain. By using electron paramagnetic resonance we here show that, after its systemic administration, ME10092 modulates the nitric oxide (NO) content in several tissues of the rat in a time-dependent manner. However, in vitro ME10092 inhibited the activities of nitric oxide synthases nNOS and eNOS, but not that of iNOS. Our data give evidence that the cardioprotective effect of ME10092 could be mediated through pharmacological mechanisms that include some modulation of NO production, as well as possible inhibition of radical formation during ischemia-reperfusion.

We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors.One of the substances HS028 (cyclic [AcCys11, dichloro‐D‐phenylalanine14, Cys18, Asp‐NH222]‐β‐MSH11–22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014.HS028 antagonised a α‐MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors.Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days.This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.

The potential for the N‐hydroxyguanidine compound PR5 (N‐(3,4‐dimethoxy‐2‐chlorobenzylideneamino)‐N′‐hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models.Administration of 1–10 mg kg−1of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg−1of PR5, respectively).Administration of 3 mg kg−1of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion.Coronary occlusion/reperfusion (10–20 min) increased malondialdehyde (MDA) of rat hearts (0.88±0.13 for sham vs 1.45±0.12 nmol mg−1protein for ischaemic; P<0.05). In rats where 3 mg kg−1PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04±0.12; P<0.05 vs ischaemic).PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326±32 mg for controls vs 137±21 mg for animals treated with 3×3 mg kg−1of PR5 (P<0.01).PR5 reduced the elevation of the ST‐segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion.We conclude that the N‐hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life‐threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N‐hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.

Summary: Four different organic nitroesters, constituting a homologous series based on unbranched polyalcohols, were compared with regard to in vitro relaxation of isolated bovine mesenteric arteries contracted with 2.5 μMphenylephrine. The organic nitroesters included ethylene glycol dinitrate (EGDN), dinitratopropane (DPN), glyceryl trinitrate (GTN), and tetranitratobutane. Glyceryl trinitrate exhibited a biphasic concentration-effect relationship, with pD2values of 11.5 ±; 0.5 and 7.2 ±; 0.2 for the high-pD2and low-pD2component of the relaxation curve, respectively. The high-pD2and low-pD2component contributed 28 and 72 of the maximal response, respectively. EGDN, DPN, and tetranitratobutane induced monophasic concentration-effect curves with pD2values of 7.4 ±;0.1, 7.8 ±;0.2, and 6.9 ±; 0.6, respectively. Stereoisomeric forms of DPN and tetranitratobutane showed no difference with regard to relaxing potency in bovine mesenteric artery. GTN has a partly unique mechanism for vascular smooth muscle relaxation that distinguishes this compound from other related organic nitroesters.

A phage display system for the selection of peptides binding to heterologously expressed human melanocortin receptor 1 on the surface of insect cells has been established. It could be shown that phage particles displaying the natural ligand alpha-melanocyte-stimulating hormone bind selectively to cells expressing this receptor and that these phages exhibit biological activity on mouse B16F1 melanoma cells. Insect cells were superior to other cell lines tested and have been used to select binders from a small library, in which critical determinants (Phe7-Arg8-Trp9) were kept, whereas the flanking regions where allowed to variate freely. One peptide displaying little similarity with native hormone was found that binds to the receptor also in its free form with an affinity of 7 nM. It showed a remarkable selectivity for this receptor, because it binds to the other melanocortin receptor subtypes with a maximum affinity of 21 microM. This is the first time phage display has been used successfully with G-protein-coupled receptors lacking an extracellular binding domain.

We have synthesized and characterized a novel high affinity radioiodinated alpha 1-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido - 3 - [125I]iodophenyl) pentanoyl] - 1 - piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (KD = 130 pM) in a reversible and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an alpha 1-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical alpha 1-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at Mr = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the Mr = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the alpha 1-adrenergic receptor.

We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin1(MC1), MC3, MC4and MC5receptors.We discovered that compounds with 26 membered rings of [Cys4,D‐Nal7,Cys11]α‐MSH(4–11) displayed specific MC4receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys4D‐Nal7Cys11]α‐MSH(4–11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC4receptor with high and the MC1receptor with low relative affinities.HS964 and HS014 have 12 and 17 fold MC4/MC3receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys4,Cys10]α‐MSH analogues SHU9119 and HS9510.HS964 is the first substance showing higher affinity for the MC5receptor than the MC1receptor.HS014, which was the most potent and selective MC4receptor ligand (Ki3.2 nM, which is ∼300 fold higher affinity than for α‐MSH), was also demonstrated to antagonize α‐MSH stimulation of cyclic AMP in MC4receptor transfected cells.We found that a compound with a 29 membered ring of [Cys3,Nle10,D‐Nal7,Cys11]α‐MSH(3–11) (HS010) had the highest affinity for the MC3receptor.This is the first study to describe ligands that are truly MC4selective and a ligand having a high affinity for the MC3receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4and MC5receptors.

THE actions of sympathomimetic amines have attracted considerable attention for some time. Ahlquist1–3introduced the concept of adrenergic α and β receptors to explain the differences in the effects of some of these amines on various organ systems and then Lands et al.4,5suggested the existence of two types of β receptors, β1and β2. It has been claimed that there is a pharmacological difference between α receptors at adrenergic neurones and those in smooth muscle6–9. I have previously shown that the inhibitory α receptor of the cholinergic neurone in guinea pig ileum is pharmacologically different from the excitatory α receptor located in the smooth muscle of the guinea pig ileocaecal sphincter and rabbit aorta10–13. Langer14suggested that there are two kinds of α receptors, α1and α2(see also ref. 15). Adopting this nomenclature, it was proposed that α1receptors are present when the relative affinity of phenylephrine for the receptors is stronger than that of clonidine, and α2receptors when the relative affinities are reversed13, α1Receptors have thus been shown to be present in smooth muscle cells of rabbit aorta and pulmonary artery, and in guinea pig ileocaecal sphincter; and inhibitory α2receptors in cholinergic neurones of guinea pig ileum and in adrenergic neurones of rabbit pulmonary artery and rat vas deferens. I report here the results of further studies supporting the theory of two types of α receptors.

Motivation: Proteochemometrics is a novel technology for the analysis of interactions of series of proteins with series of ligands. We have here customized it for analysis of large datasets and evaluated it for the modeling of the interaction of psychoactive organic amines with all the five known families of amine G protein-coupled receptors (GPCRs). Results: The model exploited data for the binding of 22 compounds to 31 amine GPCRs, correlating chemical descriptions and cross-descriptions of compounds and receptors to binding affinity using a novel strategy. A highly valid model (q2 = 0.76) was obtained which was further validated by external predictions using data for 10 other entirely independent compounds, yielding the high q2ext = 0.67. Interpretation of the model reveals molecular interactions that govern psychoactive organic amines overall affinity for amine GPCRs, as well as their selectivity for particular amine GPCRs. The new modeling procedure allows us to obtain fully interpretable proteochemometrics models using essentially unlimited number of ligand and protein descriptors. Contact: jarl.wikberg@farmbio.uu.se Supplementary information: Supplementary data are available at Bioinformatics online.