Identification of a novel specific small-molecule melanocortin-2-receptor antagonist.

The overproduction of adrenocorticotropic hormone (ACTH), in conditions such as Cushing's disease and congenital adrenal hyperplasia (CAH), leads to significant morbidity. Current treatment with glucocorticoids does not adequately suppress plasma ACTH, resulting in excess adrenal androgen production. At present, there is no effective medical treatment in clinical use that would directly block the action of ACTH. Such a therapy would be of great clinical value. ACTH acts via a highly selective receptor, the melanocortin-2 receptor (MC2R) associated with its accessory protein MRAP. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and the high degree of ligand specificity suggest that antagonism of this receptor could provide a useful therapeutic strategy in the treatment of conditions of ACTH excess. To this end, we screened an extensive library of low-molecular-weight drug-like compounds for MC2R antagonist activity using a high-throughput homogeneous time-resolved fluorescence cAMP assay in Chinese hamster ovary cells stably co-expressing human MC2R and MRAP. Hits that demonstrated MC2R antagonist properties were counter-screened against the ß2 adrenergic receptor and dose-response analysis undertaken. This led to the identification of a highly specific MC2R antagonist capable of antagonising ACTH-induced progesterone release in murine Y-1 adrenal cells and having selectivity for MC2R amongst the human melanocortin receptors. This work provides a foundation for the clinical investigation of small-molecule ACTH antagonists as therapeutic agents and proof of concept for the screening and discovery of such compounds.

Old drugs with new skills: fenoprofen as an allosteric enhancer at melanocortin receptor 3.

The efficiency of drug research and development has paradoxically declined over the last decades despite major scientific and technological advances, promoting new cost-effective strategies such as drug repositioning by systematic screening for new actions of known drugs. Here, we performed a screening for positive allosteric modulators (PAMs) at melanocortin (MC) receptors. The non-steroidal anti-inflammatory drug fenoprofen, but not the similar compound ibuprofen, presented PAM activity at MC3, MC4, and MC5 receptors. In a model of inflammatory arthritis, fenoprofen afforded potent inhibition while ibuprofen was nearly inactive. Fenoprofen presented anti-arthritic actions on cartilage integrity and synovitis, effects markedly attenuated in Mc3r-/- mice. Fenoprofen displayed pro-resolving properties promoting macrophage phagocytosis and efferocytosis, independently of cyclooxygenase inhibition. In conclusion, combining repositioning with advances in G-protein coupled receptor biology (allosterism) may lead to potential new therapeutics. In addition, MC3 PAMs emerged as a viable approach to the development of innovative therapeutics for joint diseases.

ACTH Antagonists.

Adrenocorticotropin (ACTH) acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R), is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP) for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1) Cushing's disease and ectopic ACTH syndrome - especially while preparing for definitive treatment of a causative tumor, or in refractory cases, or (2) congenital adrenal hyperplasia - as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article, we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role.

One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix-helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs.

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection.

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Combined use of two transcriptional reporters improves signalling assays for G protein-coupled receptors in fission yeast.

The biochemical and genetic tractability of yeasts make them ideal hosts for the analysis of signalling from G protein-coupled receptors (GPCRs). Selected modifications to the strains allow the introduction of non-yeast components, while signal-dependent expression of reporter genes provides growth selection or enzyme read-out as assays for signalling. One issue with such systems is reporter expression in the absence of stimulation, usually because of spontaneous activation of intracellular signalling components and/or incomplete repression of the signal-dependent promoter. This limits the difference between reporter activity in the presence and absence of stimulation, often referred to as the signal:background ratio. In an effort to extend the applicability of the yeast system, we generated a Schizosaccharomyces pombe strain containing pheromone-dependent reporters for both growth selection and beta-galactosidase production. Simultaneous use of the two reporters provided several advantages over strains expressing only one reporter, particularly when coupled to the use of a competitive inhibitor of the nutritional reporter. For example, the beta-galactosidase signal:background ratio following stimulation with 10(-6) M P-factor increased from 35 for a strain containing a single lacZ reporter to almost 2500 for the double reporter. The sensitivity of the system was also improved, with higher signal:background ratios allowing detection of lower concentrations of P-factor. Although we have used Sz. pombe and focused on GPCR-based induction of beta-galactosidase, the principles described can be applied to other yeasts, different signalling pathways and alternative reporters.

Identification of Gnr1p, a negative regulator of G alpha signalling in Schizosaccharomyces pombe, and its complementation by human G beta subunits.

G protein-coupled receptors (GPCRs) are involved in the response of eukaryotic cells to a wide variety of stimuli, traditionally mediating their effects through heterotrimeric G proteins comprised of G alpha, G beta and G gamma subunits. The fission yeast Schizosaccharomyces pombe is an established tool for GPCR research, possessing two G alpha-dependent signalling cascades. A complete G alpha beta gamma complex has been characterised for the glucose-sensing pathway, but only the G alpha subunit, Gpa1p, has been identified in the pheromone-response pathway. Here, we report the use of the yeast two-hybrid system to identify a novel protein, Gnr1p, which interacts with Gpa1p. Gnr1p is predicted to contain seven WD repeats and to adopt a structure similar to typical G beta subunits. Disruption and overexpression studies reveal that Gnr1p negatively regulates the pheromone-response pathway but is not required for signalling. Human G beta subunits complement the loss of Gnr1p, functioning as negative regulators of G alpha signalling in fission yeast.