A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease.

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, Km, kcat, and kcat/Km for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters Km, kcat, and kcat/Km for Ac-nKRR-amc substrate were 100 µM, 0.112 s(-1), and 1120 M(-1)·s(-1), respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.

Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U).

The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (kcat) and a lower Km value than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities.

Dermal melanocortin receptor rebound in diffuse systemic sclerosis after anti-TGFß1 antibody therapy.

Disturbed transforming growth factor beta (TGFß) signalling leads to enhanced synthesis of extracellular matrix (ECM), which is manifested as systemic sclerosis (SSc), but this may be attenuated by the melanocortin system. Here, we report of rebound reaction in the gene expression of melanocortin receptor (MCR) subtypes and of the precursor of these receptors' ligands, the pro-opio-melanocortin protein (POMC), in the acute skin lesion of diffuse systemic sclerosis (dSSc) after treatment with a recombinant human anti-TGFß1 antibody. Biopsies, taken from the leading edge of the skin lesion, before and after treatment of a patient with recent onset dSSc, were examined. Before treatment, increased levels of TGFß mRNA and suppressed levels of POMC mRNA and MCR subtypes MC(1-3, 5) R mRNAs were seen in the lesion, compared with healthy controls. After treatment, there was a rebound expression of POMC, MC(2, 3, 5) R mRNAs. As the melanocortin system regulates collagen and melanin production, our findings add a new understanding to the pathogenetic mechanisms involved in the acute skin lesion of dSSc, which is characterized by enhanced ECM formation and changes in skin pigmentation.

Quantitative measurement of the levels of melanocortin receptor subtype 1, 2, 3 and 5 and pro-opio-melanocortin peptide gene expression in subsets of human peripheral blood leucocytes.

Levels of the melanocortin receptor (MCR) 1, 2, 3 and 5 subtypes and pro-opio-melanocortin (POMC) protein mRNA were measured by the real-time quantitative reverse transcriptase polymerase chain reaction method in CD4+ T helper (Th) cells, CD8+ T cytotoxic cells, CD19+ B cells, CD56+ natural killer (NK) cells, CD14+ monocytes and CD15+ granulocytes from healthy donors. We found high levels of all of the MC1, 2, 3 and 5R subtype mRNA in Th cells and moderate levels in NK cells, monocytes and granulocytes. POMC peptide mRNA was found in all examined leucocyte subsets, but only low levels were present in granulocytes. Our findings suggest a co-ordinating role for MCR subtypes and their naturally occurring ligands in the co-operation between innate and adaptive immunity. Moreover, our findings are compatible with earlier finding of MCR-mediated tolerance induction in Th cells.

In vivo measurements of the cerebral perfusion and cardiovascular effects of the novel guanidine ME10092 in the non-human primate, Papio ursinus.

The novel guanidines N-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine (ME 10092) and N-(3,4-dimethoxy-2-chlorobenzylideneamino)-N1-hydroxyguanidine (PR5) were recently reported to exhibit promising cardioprotective activities in myocardial ischaemia and reperfusion in rats. The current study investigated for the first time pharmacological effects of ME10092 in the primate, viz. the Cape baboon Papio ursinus. The effects of ME10092 (1 and 2 mg/kg doses) on the cerebral blood flow, heart rates and the systolic and diastolic blood pressure were investigated after intravenous injection to the baboon under anaesthesia. The cerebral perfusion effects of ME10092 were assessed using Single Photon Emission Computed Tomography according to the split-dose approach and 99mTc-hexamethyl-propylene amine oxime as brain perfusion tracer. The observation that the recovery times from the anaesthesia were unacceptably prolonged excluded doses beyond 2 mg/kg. The data indicate that no cerebral perfusion changes were induced at both the 1 and 2 mg/kg doses of ME10092. Both these doses of ME10092 showed blood pressure and heart rate effects, with the latter being more significant. Decreases in heart rate were seen directly after ME10092 administration reaching levels of about 20% for the 2 mg/kg dose and about 15% for the 1 mg/kg dose at around 6 min post drug administration. A transient decrease in both systolic and diastolic blood pressure was observed for the higher dose. The blood pressure data further suggest an attenuation of the anaesthesia induced increase in pressure usually present in non-intervention studies. ME10092 clearly exhibits mycocardial effects in the non-human primate, similar to the effects previously observed in the ischaemia-reperfusion rat model, where ME10092 showed strong protection.

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.

Melanocortin receptors: ligands and proteochemometrics modeling.

The melanocortin receptors exist in five subtypes, MC(1-5)R. These receptors participate in important regulations of the immune system, central behavior, and endocrine and exocrine glands. Here we provide a short review on MCR subtype selective peptides and organic compounds with activity on the MCRs, developed in our laboratory. Also provided is an overview of our new proteochemometric modeling technology, which has been applied to model the interaction of MSH peptides with the MCRs.

Alcohol-preferring AA rats show a derangement in their central melanocortin signalling system.

The AA (Alko, Alcohol) rats are selectively bred for their preference of alcohol to water, contrasting to ANA rats that avoid alcohol. They also exhibit a lower growth rate compared to ANA rats, as well as differences in their response to substances affecting food intake. The melanocortin (MC) system is involved in the regulation of feeding behaviour and in mechanisms underlying drug addiction and tolerance. Recently, administration of an MC receptor agonist proved to reduce alcohol intake in AA rats. We predicted that the ratio of endogenous MC receptor agonists (proopiomelanocortin, POMC) and antagonists (agouti-related protein, AgRP) would differ from ANA rats, and that subsequent differences in MC receptor levels would be detectable. We used in situ hybridization to detect an increased ratio of POMC/AgRP mRNA in the arcuate nucleus (Arc) of AA rats. Receptor autoradiography indicated that MC3 receptor binding differed in the nucleus accumbens and several hypothalamic nuclei, possibly reflecting differences in MC peptide transmission in the AA rats. Our results support the claim that AA rats have a high ratio of POMC/AgRP expression, and that this observation is accompanied by differences in MC3 receptor levels.

MC(1) receptors are constitutively expressed on leucocyte subpopulations with antigen presenting and cytotoxic functions.

The expression of melanocortin MC(1) receptors on human peripheral lymphocyte subsets was analysed by flow cytometry using rabbit antibodies selective for the human MC(1) receptor and a panel of monoclonal antibodies against lymphocyte differentiation markers. The MC(1) receptor was found to be constitutively expressed on monocytes/macrophages, B-lymphocytes, natural killer (NK) cells and a subset of cytotoxic T-cells. Interestingly T-helper cells appeared to be essentially devoid of MC(1) receptors. The results were confirmed by RT-PCR which indicated strong expression of MC(1) receptor mRNA in CD14(+), CD19(+) and CD56(+) cells. However, only a faint RT-PCR signal was seen in CD3(+) cells, in line with the immuno-staining results that indicated that only part of the CD3(+) cells (i.e. some of the CD8(+) cells) expressed the MC(1) receptor. The MC(1) receptors' constitutive expression on immune cells with antigen-presenting and cytotoxic functions implies important roles for the melanocortic system in the modulation of immune responses.

Gamma(2)-melanocyte-stimulating hormone suppression of systemic inflammatory responses to endotoxin is associated with modulation of central autonomic and neuroendocrine activities.

Central autonomic and neuroendocrine activities are important components of the host response to bacterial inflammation. We demonstrate that intravenous infusion of gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH), a potent autonomic regulating peptide, prevents lipopolysaccharide (LPS)-induced hypotension and tachycardia, and modulates the ACTH response to endotoxin. In the hypothalamic paraventricular nucleus, a major neuroendocrine and autonomic center, gamma(2)-MSH inhibits LPS-induced increases in CRF mRNA levels, but does not suppress LPS-augmented arginine vasopressin heteronuclear RNA expression. In the locus coeruleus, a brainstem noradrenergic center, gamma(2)-MSH inhibits LPS-induced increases in tyrosine hydroxylase mRNA levels. Gamma(2)-MSH inhibits LPS-induced IL-1beta gene expression in the brain, pituitary and thymus, and prevents increases in plasma NO levels. These findings reveal that gamma(2)-MSH attenuates systemic inflammatory responses to endotoxin and suggest that modulation of central autonomic and neuroendocrine activities by gamma(2)-MSH contributes to its anti-inflammatory effects.

The MC4 receptor mediates alpha-MSH induced release of nucleus accumbens dopamine.

Strong evidence suggests a functional link between the melanocortin and dopamine systems. alpha-Melanocyte stimulating hormone (alpha-MSH) induced grooming behaviour, which can be blocked by dopamine receptor antagonists, is associated with increased dopaminergic transmission in the striatal regions. Whether this effect is mediated specifically by melanocortin (MC) receptors has not previously been established. Using in vivo microdialysis on anesthesized rats we have shown that alpha- MSH administered into the ventral tegmental area induced a significant increase in dopamine and DOPAC levels in the nucleus accumbens. This increase was completely blocked by pre-treatment with the MC4 receptor selective antagonist HS131, indicating that the effects of alpha-MSH on dopamine transmission may be mediated by the MC4 receptor.

The gamma(2)-MSH peptide mediates a central analgesic effect via a GABA-ergic mechanism that is independent from activation of melanocortin receptors.

Using the latency for tail-flick after thermal stimulation we have assessed the effects of alpha-, gamma(1)- and gamma(2)-MSH on nociceptive threshold in the mice. Intracisternal injections of gamma(2)-MSH induced a distinct analgesia, while gamma(1)-MSH in the same doses gave only a minor analgesia. Intracisternal alpha-MSH instead gave a short-term hyperalgesia. The effect of gamma(2)-MSH was not blocked by any of the MC(4)/MC(3)receptor antagonist HS014, naloxone or by the prior intracisternal administrations of gamma(1)-MSH. However, the gamma(2)-MSH analgesic response was completely attenuated by treating animals with the GABA(A)antagonist bicuculline. The gamma(2)-MSH analgesic effect was moreover additive to the analgesia afforded by muscimol and ethanol, but not to that afforded by diazepam. In addition both gamma(1)- and gamma(2)-MSH induced moderate catalepsy, but could at the same time attenuate haloperidol induced catalepsia. We conclude that gamma(2)-MSH mediates a central analgesic effect via GABA-receptor dependent pathway that is distinct from melanocortic- and opioid-receptors. Moreover, the mechanism for gamma(2)-MSH's analgesic effect appears to be distinct from that causing moderate catalepsia by gamma-MSH's.

Detection of regions in the MC1 receptor of importance for the selectivity of the MC1 receptor super-selective MS04/MS05 peptides.

We have investigated the ability of our earlier identified MS04-MS05 MSH-peptide analogues to bind to chimeric MC1-MC3 receptors. While the MS04 and MS05 peptides bind with nanomolar and sub-nanomolar affinities to the wild type MC1 receptor, they bind only with micromolar affinities for the wild type MC3 receptor, thus being the hitherto most MC1 receptor selective ligands. Upon exchanging portions involving transmembrane regions TM1, TM2-3, and TM6-7 of the MC1 receptor with corresponding portions of the MC3 receptor both of these peptides showed major losses of affinities. By contrast exchanges involving TM4-5 did not appreciably affect the affinity of either MS04 or MS05. Our data suggest that the binding pocket for the MS04-MS05 MSH-peptides is located between TM1-3 and TM6-7 of the melanocortin receptors.

PLS modeling of chimeric MS04/MSH-peptide and MC1/MC3-receptor interactions reveals a novel method for the analysis of ligand-receptor interactions.

A novel method has been developed for the analysis of ligand-receptor interactions. The method utilizes binding data generated from the analysis of chimeric proteins with chimeric peptides. To each chimeric part of the peptide and receptor are assigned descriptors, thus creating a matrix of X descriptors. These descriptors are then correlated with the experimentally determined interaction binding affinities for each chimeric receptor/peptide pair by use of partial least-squares projection to latent structures (PLS). The method was applied to analyze the interactions of chimeric MSH-peptides with wild-type MC1 and MC3 receptors, and MC1/MC3 receptor chimeras (in total 40 peptide-receptor combinations). Two types of PLS models could be created, one that revealed the relationships between receptor and peptide structure and peptide binding pK(i) values (i.e., affinity) (R2 and Q2 being 0.71 and 0.62, respectively), and another that revealed the relationships between peptide and receptor structure and peptide-receptor selectivity (R2 and Q2 being 0.64 and 0.57, respectively). After addition of cross-terms these models improved significantly; the R2 and Q2 being 0.93 and 0.75 for affinity, and 0.92 and 0.72 for selectivity, respectively. The analysis shows that the high affinity of the MSH-peptides is primarily achieved by interactions of the peptides' C-terminal amino acids with TM2 and TM3 of the receptor, and, to a lesser extent, by the interaction of the N-terminus with TM1, TM2 and TM3 of the receptor. However, in contrast, the MC1 receptor selectivity is primarily determined by an interaction of the peptides' N-termini with TM2/3 of the receptor. Moreover, the cross-terms of the PLS model revealed the existence of a strong interaction between TM6/7 and TM2/3 of the receptors.

Development of proteo-chemometrics: a novel technology for the analysis of drug-receptor interactions.

A novel method for the analysis of drug receptor interactions has been developed and used to explore mechanisms involved in the binding of 4-piperidyl oxazole antagonists to alpha1a-, alpha1b- and alpha1d-adrenoceptors. The method exploits affinity data for a series of organic chemical compounds binding to wild-type and artificially mutated receptors. The receptor sequences and compounds are assigned predictor variables that are correlated to the measured pharmacological activities using partial least-squares projections to latent structures. The predictor variables consist of one descriptor block derived from the chemical properties of the receptors' primary amino acid sequences and another descriptor block derived from the chemical properties of the organic compounds. The cross-terms generated from the two descriptor blocks are also derived. Using this approach, very sturdy models were generated describing the interactions of the chemical compounds with the receptors. Models are useful to predict binding affinity and receptor subtype selectivity of compounds prior to their synthesis, and may find use in rational drug design. Moreover, models also give quantitative information about the interactions of the amino acids of the receptors with the ligands, thereby giving an insight into the molecular mechanisms involved in ligand binding.

Design of new small cyclic melanocortin receptor-binding peptides using molecular modelling: Role of the His residue in the melanocortin peptide core.

The conserved core of melanocyte stimulating hormones (MSH), His-Phe-Arg-Trp, was probed by comparing a cyclic pentapeptide containing His-DPhe-Arg-Trp, with three structurally similar cyclic peptides, that lacked the His residue. All three peptides bound to the MC(1), MC(3), MC(4) and MC(5) receptors with similar affinities. Molecular modelling indicated that the 3D structure of the DPhe-Arg-Trp of all three peptides were closely similar. The data indicate that the His residue of the small rigid cyclic MSH core peptides does not participate in binding with the melanocortin receptors.

Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells: evidence for dual mechanisms of action.

The pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) mediates broad anti-inflammatory and immunomodulatory effects, which include inhibition of the production and release of proinflammatory cytokines and nitric oxide (NO) from macrophages. We investigated the effects of alpha-MSH, alpha-MSH(1-10), and alpha-MSH(11-13) on NO production and nuclear factor-kappaB (NF-kappaB) translocation in RAW 264.7 macrophages. After stimulation of the cells with bacterial lipopolysaccharide/interferon-gamma (LPS/IFN-gamma), all three peptides inhibited NO production with an order of potency alpha-MSH > or = alpha-MSH(11-13) > alpha-MSH(1-10). All three MSH peptides inhibited NF-kappaB nuclear translocation with the maximal effect of alpha-MSH and alpha-MSH(11-13) being seen in the range 1 nM-1 microM, and that of alpha-MSH(1-10) at 1 microM. By use of (125)I-(Nle(4),D-Phe(7))alpha-MSH(NDP-MSH) radioligand binding, MC(1) receptor-binding sites were demonstrated on RAW 264.7 cells. alpha-MSH and alpha-MSH(1-10) competed with the (125)I-NDP-MSH binding at these MC(1) receptor-binding sites, but alpha-MSH(11-13) even in concentrations up to 1 mM did not. Moreover, alpha-MSH and alpha-MSH(1-10) caused powerful stimulation of cyclic 3',5'-adenosine monophosphate (cAMP) in the RAW 264.7 cell, whereas alpha-MSH(11-13) was ineffective. Forskolin stimulated cAMP and inhibited NO production to the same extent as alpha-MSH and alpha-MSH(1-10), but did not modify the translocation of NF-kappaB. Whereas the protein kinase A inhibitor H89 did not modify the effect of alpha-MSH on NF-kappaB translocation, H89 caused a partial inhibition of the inhibitory effect of alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin on NO production. In addition alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin also inhibited the activity of an NF-kappaB-dependent luciferase reporter and these effects were partially counteracted by H89. We suggest that melanocortin peptides act via dual mechanisms of action: one cAMP-independent and causing inhibition of NF-kappaB translocation and the other dependent on MC(1) receptor/cAMP activation.

Neuropeptide Y Y(5) receptor antagonist CGP71683A: the effects on food intake and anxiety-related behavior in the rat.

The effects of neuropeptide Y Y(5) receptor antagonist (trans-naphtalene-1-sulphonic acid [4-[(4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethyl]-amide hydrochloride; CGP71683A), on food intake, anxiety and locomotor activity were studied. CGP71683A (1-10 mg/kg, i.p.) dose-dependently decreased nocturnal and fasting-induced food intake. CGP71683A did not have an anxiogenic-like effect in the rat social interaction test. In the elevated plus-maze test, where novel neuropeptide Y Y(1) receptor antagonist (2R)-5-([amino(imino)methyl)amino)-2-[(2.2-diphenylacetyl)-amino]-N-[(1R)-1-(4-hydroxyphenyl)ethyl-pentanamide (H 409/22) had anxiogenic-like effect, CGP71683A was inactive. In the open-field test, carried out immediately after the elevated plus-maze test, CGP71683A inhibited horizontal and vertical activity. CGP71683A did modify the habituation of locomotor response in novel environment. These data show that the inhibition of food intake induced by CGP71683A could not be explained by increased fearfulness, a state that is induced by neuropeptide Y Y(1) receptor antagonists. Thus, our data, obtained with first neuropeptide Y Y(5) receptor antagonist CGP71683A, suggest that in contrast to the neuropeptide Y Y(1) receptor, Y(5) receptor is not involved in tonic neuropeptide Y-induced anxiolysis.

The anabolic-androgenic steroid nandrolone decanoate affects the density of dopamine receptors in the male rat brain.

In recent years a male group of anabolic-androgenic steroid misusers has been identified to share socio-demographic and personality related background factors with misusers of psychotropic substances, as well as being involved in habits of multiple drug use. The present study aimed to assess whether anabolic-androgenic steroids (AAS) would affect the density of the dopamine receptors in areas implicated in reward and behaviour in the male rat brain. The effects of 2 weeks of treatment with i.m. injections of nandrolone decanoate (15 mg/kg/day) on the expression of the D(1)-like and D(2)-like receptors were evaluated by autoradiography. Specific binding of D(1)-like receptors was significantly down regulated in the caudate putamen, the nucleus accumbens core and shell. D(2)-like receptor densities were down regulated in the nucleus accumbens shell, but up regulated in the caudate putamen, the nucleus accumbens core and the ventral tegmental area. These results are compatible with nandrolone induced neuroadaptive alterations in dopamine circuits associated with motor functions and behavioural paradigms known to be affected following AAS misuse.

New aspects on the melanocortins and their receptors.

Knowledge of melanocortins and their receptors has increased tremendously over the last few years. The cloning of five melanocortin receptors, and the discovery of two endogenous antagonists for these receptors, agouti and agouti-related peptide, have sparked intense interest in the field. Here we give a comprehensive review of the pharmacology, physiology and molecular biology of the melanocortins and their receptors. In particular, we review the roles of the melanocortins in the immune system, behaviour, feeding, the cardiovascular system and melanoma. Moreover, evidence is discussed suggesting that while many of the actions of the melanocortins are mediated via melanocortin receptors, some appear to be mediated via mechanisms distinct from melanocortin receptors.