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

Abstract

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 β2 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.

Keywords: GPCR antagonism; MC2R; MRAP; adrenal; drug discovery.

Figure 1

Hit compound confirmation and inhibition curves of compounds. Hit compounds from the high-throughput screen were confirmed if they were able to inhibit the MC2 receptor stimulated with an EC80 of ACTH in a concentration-dependent manner. Exemplar compounds shown are MCR2-7084, MCR2-7301, and MCR2-4788. Of note is MCR2-4788 which gave a pIC50 value of 6.2 and thus showed the greatest potential as a small-molecule antagonist of MC2R and MRAP.

Figure 2

Antagonist characterisation. Distinct shifts were observed in the ACTH CRCs upon addition of the candidate compounds. Three compounds caused at least unit log shift in the values for half maximal effective concentration (EC50). The assay was performed on three separate days, and each condition was tested in triplicate (n  = 3). The nine replicates obtained confirmed reproducibility. s.d. between the replicates is represented by the error bars. Statistical significances between the LogEC₅₀ levels are denoted graphically. **P < 0.01, ***P < 0.01 and ****P < 0.001.

Figure 3

Antagonist characterisation. Functional activity of MCR2-4788 in cAMP accumulation assay. A, concentration-dependent stimulation of cAMP accumulation response in CHO-K1 cells expressing human MC2R and MRAP by ACTH in the presence of increasing concentrations of MCR2-4788. The data were derived from HTRF measurement and normalised to percentage of activity based on the effect of 100 nM ACTH. Each data point is mean ± s.d. (n  = 4). B, Schild plot of the data from A. Dose ratio is the ratio of the ACTH EC50 value in the presence of MCR2-4788 divided by the EC50 value in the absence of the antagonist. Calculated pA₂ = 5.7.

Figure 4

Antagonism of ACTH-induced cAMP generation and progesterone production in adrenal cells. (A) MCR2-4788 demonstrated antagonism of ACTH-induced cAMP accumulation in murine Y-1 adrenal cells, while MCR2-7301 and MCR2-7084 did not (negative data not shown). (B) MCR2-4788 inhibited ACTH-induced progesterone release in murine Y-1 adrenal cells. Progesterone release in Y-1 cells was induced by ACTH at 100 nM, 10 nM, 1 nM, and 100 pM concentrations, each in the presence of no candidate MCR2-4788, and 1 μM, 10 μM, and 30 μM of the MCR2-4788. The mean unstimulated baseline level of progesterone release is indicated by the dashed line (142.81 pg/mL +/–6.309). The assay was performed on 3 days, each condition being tested in triplicate (n  = 3). Error bars represent s.d. Statistical significance between each bar is shown graphically. *P < 0.05, **P < 0.01.

Figure 5

MCR selectivity against α-MSH and ACTH. (A) The α-MSH dose responses for cells expressing MC1R, MC3R, MC4R, and MC5R were not affected by MCR2-4788, whereas the ACTH dose responses for MC2R-expressing cells underwent a rightward shift. (B) Dose–response curves for ACTH at MC1R, MC2R, MC3R, MC4R, and MC5R, with ACTH alone and with the three concentrations of lead compound 4 indicated (n  = 3). MCR2-4788 did not antagonise MC1R, MC3R, MC4R or MC5R when stimulated with α-MSH or ACTH at the concentrations tested.