Felikss Mutulis

Discovery of novel melanocortin4 receptor selective MSH analogues.

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, MC4 and MC5 receptors. We discovered that compounds with 26 membered rings of [Cys4,D‐Nal7,Cys11]α‐MSH(4–11) displayed specific MC4 receptor 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 MC4 receptor with high and the MC1 receptor with low relative affinities. HS964 and HS014 have 12 and 17 fold MC4/MC3 receptor 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 MC5 receptor than the MC1 receptor. HS014, which was the most potent and selective MC4 receptor ligand (Ki 3.2 nM, which is ∼300 fold higher affinity than for α‐MSH), was also demonstrated to antagonize α‐MSH stimulation of cyclic AMP in MC4 receptor 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 MC3 receptor. This is the first study to describe ligands that are truly MC4 selective and a ligand having a high affinity for the MC3 receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4 and MC5 receptors.

Targeting melanocortin receptors: an approach to treat weight disorders and sexual dysfunction

The melanocortin system has multifaceted roles in the control of body weight homeostasis, sexual behaviour and autonomic functions, and so targeting this pathway has immense promise for drug discovery across multiple therapeutic areas. In this Review, we first outline the physiological roles of the melanocortin system, then discuss the potential of targeting melanocortin receptors by using MC3 and MC4 agonists for treating weight disorders and sexual dysfunction, and MC4 antagonists to treat anorectic and cachectic conditions. Given the complexity of the melanocortin system, we also highlight the challenges and opportunities for future drug discovery in this area.

Differential influence of a selective melanocortin MC4 receptor antagonist (HS014) on melanocortin-induced behavioral effects in rats

We injected i.c.v. the natural agonist alpha-MSH (melanocyte-stimulating hormone) and the first selective melanocortin MC4 receptor antagonist HS014 (cyclic [AcCys11, D-Nal14, Cys18, Asp-NH(2)22]-beta-MSH(11-22) in rats and scored a number of behavioral effects which have been related to the melanocortic peptides. The results showed that HS014 (5 microg/rat) completely blocked alpha-MSH (3 and 5 microg/rat)-induced grooming, yawning and stretching. Penile erections induced by alpha-MSH were, however, only partially blocked by HS014. Injections of alpha-MSH decreased food intake in food-deprived rats, whereas HS014 increased food intake. When the peptides were given together, the food intake was similar to that of saline treated controls. Locomotion/exploration and resting were not influenced by either peptide. Our data show that exogenous beta-MSH decreases food intake, and that an endogenous central melanocortinergic inhibitory tone on feeding prevails which can be blocked with HS014, leading to an increase in food intake. Our data also provide evidence that grooming, stretching and yawning in rats may be mediated by the melanocortin MC4 receptor, whereas penile erections might perhaps be mediated by some other melanocortin receptor.

PHAGE DISPLAY SELECTION ON WHOLE CELLS YIELDS A PEPTIDE SPECIFIC FOR MELANOCORTIN RECEPTOR 1

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 α-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 μm. This is the first time phage display has been used successfully with G-protein-coupled receptors lacking an extracellular binding domain.

Anorexigenic cocaine- and amphetamine-regulated transcript peptide intensifies fear reactions in rats

An increasing number of appetite-regulating peptides are being discovered. The list of regulators inhibiting food intake is considerably longer than that of appetite stimulators. In many cases, the peptides inhibiting food intake facilitate fear reactions, whereas the majority of the agents reducing anxiety responses stimulate appetite. Cocaine- and amphetamine-regulated transcript (CART) cDNA was isolated from hypothalamic libraries and CART was reported to inhibit food intake and to mediate the anorectic effects of leptin. Here, we show that the active core fragment of CART (CART(89-103), 0.04-5.0 nmol) injected into lateral cerebral ventricle not only inhibits food intake, but also causes a dose-dependent increase in anxiety-like reactions in elevated plus-maze test. Intracerebroventricular administration of CART(82-103) (0.04-5.0 nmol) did not inhibit water intake and did not affect spontaneous locomotor activity in the open field test ruling out unspecific effects of the peptide. Our results suggest that CART could be an endogenous factor in the brain mediating the effects of stress on appetite.

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

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.

Binding of cyclic and linear MSH core peptides to the melanocortin receptor subtypes.

We report here the binding of 5-, 6- and 7-amino-acid-long linear and cyclic core peptides of MSH (melanocyte-stimulating hormone) to cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that, in contrast to the natural peptides, the core peptides did not differentiate between the melanocortin MC3 and MC4 receptors. All tested cyclic peptides had much lower affinities than their corresponding linear homologues. Interestingly, the relative loss of binding due to the cyclisation did not change as the ring size decreased. Therefore, decreasing the ring size does not seem to force the peptide into a more unfavourable conformation.

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.

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.

Selective properties of C- and N-terminals and core residues of the melanocyte-stimulating hormone on binding to the human melanocortin receptor subtypes

We synthesised nine analogues of [Nle4,D-Phe7]alpha-MSH (melanocyte-stimulating hormone) (NDP) where (1) the N- or C-terminals were deleted or exchanged by those of beta- or gamma-MSH and (2) the core residues His6, Phe7, Arg8 and Trp9 were individually substituted by Glu6, beta-(2-naphthyl)-D-alanine (D-Nal7), Lys8 and His9, respectively. We tested these analogues in ligand binding assays with cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that the N-terminal segment (Ser1-Tyr2-Ser3) of NDP was not important for binding to melanocortin MC1 and MC4 receptors whereas it affects binding to melanocortin MC3 and MC5 receptors. The C-terminal segment (Gly10-Lys11-Pro12-Val13) of NDP was clearly important for binding to all the four melanocortin receptor subtypes. The data indicate that the low affinity of gamma-MSH for the melanocortin MC4 receptor is due to its C-terminal (Asp10)-Arg11-Phe12). Substitution of D-Phe7 by D-Nal7 increased the affinity for the melanocortin MC4 receptor but not for the other melanocortin receptor subtypes. The other core residue substitutions lowered the affinity in a differentiated manner for each of the melanocortin receptors. These results are valuable for the molecular modelling and design of selective drugs for the melanocortin receptors.