Jarl E. S. Wikberg

Melanocortin receptors: perspectives for novel drugs.

The cloning of five different subtypes of melanocortin receptor subtypes have recently opened up new possibilities for the development of drugs. The physiological roles of the five melanocortin receptors have started to become understood, and compounds with selective actions on some of the five subtypes have become available. Presently, most clinically promising application for drugs active on melanocortin receptors are for control of feeding homeostasis and body weight and for treatment of inflammatory diseases. I review here the cloning, localisation, function and structure of the melanocortin receptors, in relation to the possibilities to develop selective drugs for these receptors.

Major pharmacological distinction of the ACTH receptor from other melanocortin receptors

The mouse adrenocortical cell line Y1, that expresses ACTH receptors (MC2R), was used to probe the binding of ACTH and MSH peptides by using radio-labelled ACTH (1-39). The Y1 cells were found to bind [125I]-labelled ACTH (1-39) with high affinity (Kd approximately 130 pM). However, none of the melanocortin peptides NDP-MSH, alpha-MSH, beta-MSH or gamma 1-MSH could compete with the binding of the labelled ACTH(1-39). When other MC receptor subtype DNAs (MC1, MC3 and MC4) were transfected into the Y1 cells, characteristic binding of the [125I]NDP-MSH appeared for each of the receptor subtype, but no specific binding was present in non-transfected cells. This is the first report clearly demonstrating that the ACTH receptor binds only ACTH, but not other melanocortin peptides.

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.

Characterisation of melanocortin receptor subtypes by radioligand binding analysis

The DNAs encoding three melanocortin receptor subtypes (melanocortin MC1 receptor, melanocortin MC3 receptor and melanocortin MC5 receptor) were expressed individually in COS (CV-1 Origin, SV40) cells to characterise their ligand binding properties. The results indicated that [125I][Nle4, D-Phe7]alpha-MSH (melanocyte stimulating hormone) bound to a single saturable site with Kd values of 85.1 +/- 8.0 pmol/l (mean +/- S.E.M), 396 +/- 65 pmol/l and 5.05 +/- 1.00 nmol/l for melanocortin MC1 receptor, melanocortin MC3 receptor and melanocortin MC3 receptor, respectively. The melanocortin MC1 receptor and the melanocortin MC5 receptor showed a similar potency order to the melanocortic peptides examined which was markedly different from the potency order of the melanocortin MC3 receptor. The melanocortin MC1 receptor and melanocortin MC5 receptor had a relatively higher affinity for alpha-MSH than gamma-MSH and beta-MSH, whereas the melanocortin MC3 receptor had higher affinity for desacetyl-alpha-MSH, gamma-MSH and beta-MSH compared to alpha-MSH. The inclusion of the endopeptidase inhibitor phosphoramidon to prevent the breakdown of ACTH-(1-39) (adrenocorticotrophic hormone) to alpha-MSH, decreased ACTH-(1-39) binding affinity showing that ACTH-(1-39) had a much lower affinity for melanocortin MC1 receptor than reported earlier.

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.

Classification of G-protein coupled receptors by alignment-independent extraction of principal chemical properties of primary amino acid sequences

We have developed an alignment‐independent method for classification of G‐protein coupled receptors (GPCRs) according to the principal chemical properties of their amino acid sequences. The method relies on a multivariate approach where the primary amino acid sequences are translated into vectors based on the principal physicochemical properties of the amino acids and transformation of the data into a uniform matrix by applying a modified autocross‐covariance transform. The application of principal component analysis to a data set of 929 class A GPCRs showed a clear separation of the major classes of GPCRs. The application of partial least squares projection to latent structures created a highly valid model (cross‐validated correlation coefficient, Q2 = 0.895) that gave unambiguous classification of the GPCRs in the training set according to their ligand binding class. The model was further validated by external prediction of 535 novel GPCRs not included in the training set. Of the latter, only 14 sequences, confined in rapidly expanding GPCR classes, were mispredicted. Moreover, 90 orphan GPCRs out of 165 were tentatively identified to GPCR ligand binding class. The alignment‐independent method could be used to assess the importance of the principal chemical properties of every single amino acid in the protein sequences for their contributions in explaining GPCR family membership. It was then revealed that all amino acids in the unaligned sequences contributed to the classifications, albeit to varying extent; the most important amino acids being those that could also be determined to be conserved by using traditional alignment‐based methods.

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.

Bioclipse: an open source workbench for chemo- and bioinformatics

BackgroundThere is a need for software applications that provide users with a complete and extensible toolkit for chemo- and bioinformatics accessible from a single workbench. Commercial packages are expensive and closed source, hence they do not allow end users to modify algorithms and add custom functionality. Existing open source projects are more focused on providing a framework for integrating existing, separately installed bioinformatics packages, rather than providing user-friendly interfaces. No open source chemoinformatics workbench has previously been published, and no sucessful attempts have been made to integrate chemo- and bioinformatics into a single framework.ResultsBioclipse is an advanced workbench for resources in chemo- and bioinformatics, such as molecules, proteins, sequences, spectra, and scripts. It provides 2D-editing, 3D-visualization, file format conversion, calculation of chemical properties, and much more; all fully integrated into a user-friendly desktop application. Editing supports standard functions such as cut and paste, drag and drop, and undo/redo. Bioclipse is written in Java and based on the Eclipse Rich Client Platform with a state-of-the-art plugin architecture. This gives Bioclipse an advantage over other systems as it can easily be extended with functionality in any desired direction.ConclusionBioclipse is a powerful workbench for bio- and chemoinformatics as well as an advanced integration platform. The rich functionality, intuitive user interface, and powerful plugin architecture make Bioclipse the most advanced and user-friendly open source workbench for chemo- and bioinformatics. Bioclipse is released under Eclipse Public License (EPL), an open source license which sets no constraints on external plugin licensing; it is totally open for both open source plugins as well as commercial ones. Bioclipse is freely available at http://www.bioclipse.net.

Evidence for involvement of the melanocortin MC4 receptor in the effects of leptin on food intake and body weight

The hypothesis that the melanocortin MC4 receptor mediates the homeostatic effects of leptin was tested. Leptin (0.3 nmol, i.c.v.) lowered food intake at 4 and 24 h and body weight at 24 h. This effect was inhibited by pretreatment with an analogue of melanocyte stimulating hormone (MSH), the selective melanocortin MC4 receptor antagonist HS014 (cyclic [AcCys11,D-Nal14,Cys18,Asp-NH2(22)]-beta-MSH11-2 2, 0.3 nmol, i.c.v.). HS014 alone at this dose did not modify food intake or body weight. At a higher dose (1.0 nmol, i.c.v.) HS014 stimulated food intake and this orexigenic effect of HS014 was attenuated by leptin pretreatment (0.3 nmol, i.c.v.). These results confirm earlier findings that leptin inhibits food intake and lowers body weight via the melanocortin system and suggest that leptin affects signalling at the melanocortin MC4 receptor.