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.

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.

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.

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.

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.

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.

Evaluation of the role for prolactin-releasing peptide in prolactin secretion induced by ether stress and suckling in the rat: comparison with vasoactive intestinal peptide.

Prolactin (PRL)-releasing peptide (PrRP) is a recently discovered hypothalamic peptide possessing a specific stimulatory action on PRL secretion. In this study, we examined whether PrRP plays a role in mediating ether stress- and suckling-induced PRL secretion in rats through administering anti-PrRP antisera intracerebroventricularly. For comparison, we also tested the effect of anti-vasoactive intestinal peptide (VIP) antisera on the hormonal responses, since VIP is another candidate for a physiological PRL-releasing factor. The immunoneutralization of VIP, but not of PrRP, led to a significant suppression of PRL responses to both ether and suckling. These results suggest that PrRP may not play a significant role, or at least play a much weaker role than VIP, in mediating PRL release induced by ether stress and suckling in the rat.

N-Hydroxyguanidine compound 1-(3,4-dimethoxy- 2-chlorobenzylideneamino)-3-hydroxyguanidine inhibits the xanthine oxidase mediated generation of superoxide radical.

We here show that the novel N-hydroxyguanidine derivative PR5 (1-(3, 4-dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine) is acting as an alternative electron acceptor in xanthine oxidase catalyzed oxidation of xanthine. The reduction product is the corresponding guanidine derivative 1-(3, 4-dimethoxy-2-chlorobenzylideneamino)guanidine (PR9). The reaction occurs under both anaerobic and aerobic conditions. Moreover, EPR measurements show that the action of PR5 is associated with the inhibition of superoxide radical formation seen under aerobic conditions. PR5 also supports xanthine oxidase catalyzed anaerobic oxidation of NADH. Kinetic studies indicate that increasing xanthine concentration significantly increases the apparent K(m) of PR5, but it remains unaltered by changing NADH concentration. Moreover, the molybdenum center inhibitor allopurinol inhibits the PR5-sustained oxidation of xanthine and NADH equally well, whereas the flavin adenine dinucleotide site inhibitor diphenyliodonium (DPI) markedly inhibits only the PR5-sustained oxidation of NADH. We suggest that PR5 binds and becomes reduced at the molybdenum center of the xanthine oxidase. We also found that both PR5 and its reduction product PR9 can inhibit the oxygen-sustained xanthine oxidase reaction. The properties of PR5 suggest that it is a member of a novel class of compounds which we have termed xanthine oxidase electron acceptor-inhibitor drugs. The potential use of xanthine oxidase electron acceptor-inhibitors in the prevention of free radical mediated tissue damage in organ ischemia-reperfusion diseases is discussed.

New highly specific agonistic peptides for human melanocortin MC(1) receptor.

A peptide with very high specificity for the human melanocortin MC(1) receptor identified by phage display was used as a lead for the design of new peptides. Two new peptides, MS05 and MS09, were synthesized and found to bind with sub-nanomolar affinities to the MC(1) receptor. Both these peptides showed strong agonistic activity at the MC(1) receptor. The MS05 was the most MC(1) receptor selective as it showed virtually no binding affinity for the MC(4) and MC(5) receptors and only micromolar affinity for the MC(3) receptor. The selectivity and potency of the new peptides make them potent tools for studies of MC(1) receptors, as well as novel potential candidate drugs for the treatment of inflammatory conditions.

Downregulation of melanocortin receptors in brain areas involved in food intake and reward mechanisms in obese (OLETF) rats.

The melanocortin (MC)4 receptor is important for food intake and weight homeostasis as it mediates the orexigenic and anorexigenic effects of the MCs. OLETF (Otsuka-Long-Evans-Tokushima-Fatty) rats are a selective inbred strain of polygenic variant rats which overeat and develop obesity with elevated leptin levels. We investigated by autoradiography if the expression of MC receptors were altered in ovariectomized estradiol-replaced female OLETF rats compared to their controls (Long-Evans-Tokushima-Otsuka (LETO) rats). We found that OLETF rats show a reduction in total [125I]NDP-MSH MC receptor binding in the ventromedial hypothalamic nucleus, perhaps reflecting an increased release of MC peptides in this region. The levels of MC receptors in the arcuate nucleus and the paraventricular hypothalamic nucleus were not changed. Interestingly, the OLETF rats also showed reduced MC-receptor binding in areas such as the nucleus accumbens shell, and the ventral tegmental area, both of which are believed to be involved in reward systems. Similarities in the changes of MC receptor expression in obese animals and in animals treated with opiates may suggest a neurobiological link between food intake mediated through the MC receptors and reward.

Evidence that melanocortin 4 receptor mediates hemorrhagic shock reversal caused by melanocortin peptides.

Melanocortin peptides are known to be extremely potent in causing the sustained reversal of different shock conditions, both in experimental animals and humans; the mechanism of action includes an essential brain loop. Three melanocortin receptor subtypes are expressed in brain tissue: MC(3), MC(4,) and MC(5) receptors. In a volume-controlled model of hemorrhagic shock in anesthetized rats, invariably causing the death of control animals within 30 min after saline injection, the i.v. bolus administration of the adrenocorticotropin fragment 1-24 (agonist at MC(4) and MC(5) receptors) at a dose of 160 microg/kg i.v. (54 nmol/kg) produced an almost complete and sustained restoration of cardiovascular and respiratory functions. An equimolar dose of gamma(1)-melanocyte stimulating hormone (selective agonist at MC(3) receptors) was completely ineffective. The selective antagonist at MC(4) receptors, HS014, although having no influence on cardiovascular and respiratory functions per se, dose-dependently prevented the antishock activity of adrenocorticotropin fragment 1-24, with the effect being complete either at the i.v. dose of 200 microg/kg or at the i.c.v. dose of 5 microg/rat (17-20 microg/kg). We concluded that the effect of melanocortin peptides in hemorrhagic shock is mediated by the MC(4) receptors in the brain.

Thyrotropin releasing hormone (TRH) selectively binds and activates the melanocortin 1 receptor.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.

Loss of function mutations of the human melanocortin 1 receptor are common and are associated with red hair.

The melanocortin 1 receptor is a G-protein-coupled receptor that acts as a control point for control of the eumelanin/phaeomelanin ratio in mouse hair. MC1 receptor loss of function mutations lead to an increase in the ratio of phaeomelanin/eumelanin in many mammals resulting in yellow or red coat colours. We have previously shown that several common point mutations in the human MC1 receptor are overrepresented in North European redheads and in individuals with pale skin. In order to determine the functional significance of these changes we have carried out transfection and binding studies. Expression of the Val60Leu, Arg142His, Arg151Cys, Arg160Trp, and Asp294His receptors in COS 1 cells revealed that these receptors were unable to stimulate cAMP production as strongly as the wild type receptor in response to alpha-melanocyte-stimulating hormone stimulation. None of the mutant receptors displayed complete loss of alphaMSH binding, with only the Arg142His and Asp294His displaying a slight reduction in binding affinity.

Long-term administration of MC4 receptor antagonist HS014 causes hyperphagia and obesity in rats.

In this study we investigated the long term effects of a potent and selective melanocortin 4 (MC4) receptor antagonist (HS014) on food intake, body weight, body composition and blood glucose levels in adult rats. HS014 was injected i.c.v. either by twice-daily injections (2 x 1 nmol) for 6 days or administered by continuous infusion with osmotic minipumps (0.16 nmol/h) for 2 weeks. The results show a considerable increase in food intake and body weight after both of the treatments without any signs of tachyphylaxis. After 2 weeks of treatment with osmotic pumps, the HS014-treated rats (average weight 425g) had 20% higher body weight than the controls rats (average 360 g). When i.c.v. injections were terminated, the body weight of the twice-daily HS014-treated rats returned to the levels of control group, whereas the rats treated with continuous infusion of HS014 remained hyperphagic and still gained weight. Blood glucose levels in the rats treated with HS014 infusion were significantly increased. Analysis of body composition in HS014-infused rats indicated that body weight was increased due to fat deposits. These data show for the first time that chronic administration of exogenous MC4 receptor antagonist causes hyperphagia and severe obesity in rats. These data also indicate that the melanocortic control of food intake is very robust and suggest that changes induced by such treatment overcome negative feedback signals.

The melanocortin 4 receptor mediates leptin stimulation of luteinizing hormone and prolactin surges in steroid-primed ovariectomized rats.

We have previously reported that leptin, the product of the obese (ob) gene, may play a physiologically relevant role in the generation of estradiol/progesterone-induced luteinizing hormone (LH) and prolactin (PRL) surges in female rats. In the present study, we examined whether the stimulatory effect of leptin on the hormonal surges is mediated through the melanocortin (MC) 4 receptor in the brain, as is leptin's effect on feeding behavior. We also explored whether the MC4 receptor participates in tonic stimulation of steroid-induced LH and PRL surges. Experiments were performed on both normally fed and 3-day starved rats, which were ovariectomized and primed with estradiol and progesterone. At 11:00 h on the day of the experiments, the normally fed rats received an intracerebroventricular administration of artificial cerebrospinal fluid (vehicle), SHU 9119 (a nonselective MC3/MC4 receptor antagonist, 1.0 nmol), or HS014 (a selective MC4 receptor antagonist, 1.0 nmol). The 3-day starved rats were given vehicle, recombinant mouse leptin (0.3 nmol), leptin (0.3 nmol) + SHU9119 (1.0 nmol), or leptin (0.3 nmol) + HS014 (1.0 nmol). From 11:00 to 18:00 h, blood was collected every 30 min to measure LH and PRL. The 3-day starvation completely abolished both LH and PRL surges, but leptin significantly reinstated these hormonal surges. Both SHU9119 and HS014 significantly decreased the magnitude of LH and PRL surges in normally fed rats and also significantly blocked the leptin stimulation of the hormonal surges in starved rats. These results suggest that the MC4 receptor may be the pivotal subtype of MC receptors mediating the leptin stimulation of LH and PRL surges. The data also suggest that endogenous MC(s) may tonically stimulate the hormonal surges in normally fed rats via the MC4 receptor. This is the first report describing a physiological role of a specific MC receptor in regulating the reproductive axis.

Long term orexigenic effect of a novel melanocortin 4 receptor selective antagonist.

1. We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors. 2. One of the substances HS028 (cyclic [AcCys11, dichloro-D-phenylalanine14, Cys18, Asp-NH2(22)]-beta-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. 3. HS028 antagonised a alpha-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. 4. 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. 5. 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.

Further pharmacological characterization of the selective melanocortin 4 receptor antagonist HS014: comparison with SHU9119.

SHU9119 and HS014 are cyclic MSH analogues which are widely used to elucidate the physiology behind the various effects of the MSH peptides and their receptors. We carefully compared the potency of SHU9119 and HS014 in cells expressing the MC receptor clones. We found that both the peptides are partial agonists for the MC1 and MC5 receptors while they are potent antagonists for the MC3 and MC4 receptors. In agreement with earlier binding data, we found that SHU9119 has equal potency for the MC3 and MC4 receptor whereas HS014 has at least 10-fold higher potency for the MC4 receptor than the MC3 receptor in cAMP assay. Moreover, we synthesized analogues of HS014 where the C-terminal was truncated. We found that this C-terminal fragment of HS014, in particular the Lys(14), has a major influence on the affinity for the MC4 receptor without any particular influence on the affinity for the other MC receptors.

Discovery of a novel superpotent and selective melanocortin-4 receptor antagonist (HS024): evaluation in vitro and in vivo.

Several novel cyclic MSH analogs were synthesized, and their binding properties were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. We discovered a novel substance (HS024) that showed about 20-fold selectivity and very high affinity (Ki = 0.29 nM) for the MC4 receptor. HS024 (cyclic [AcCys3,Nle4,Arg5,D-Nal7,Cys-NH2(11)]alpha-MSH-(3-11)) has a 29-membered atom ring structure that includes an Arg in position 5. HS024 was found to antagonize an alphaMSH-induced cAMP response in cells expressing the human MC1, MC3, MC4, and MC5 receptor DNAs. HS024 also caused a dose-dependent increase in food intake, with a maximum response (4-fold increase) at a 1-nmol dose injected intracerebroventricularly in free feeding rats. We also tested SHU9119, a previously described nonselective MC receptor antagonist, and found HS024 and SHU9119 to have similar potencies for increasing food intake, although SHU9119 appeared to induce more serious side-effects. HS024 increased the food intake of free feeding rats to levels comparable to those in food-deprived rats, indicating that blockade of the MC4 receptor is a highly effective way to increase feeding. Moreover, we tested the effects of intracerebroventricular injections of HS024 in elevated plus-maze and open-field experiments on rats. In these tests, HS024 did not appear to affect emotionality or locomotor activity, suggesting that the MC4 receptor does not mediate the anxiogenic-like and locomotor effects related to the melanocortic peptides.