Identification And Profiling Of Novel Melanocortin 2 Receptor (MC2) Antagonists.

Mashal Hussain1, Ahmad Kamal2, Jeff Jerman2, Ed McIver2, Debra Taylor2, Jenny Cook2, Puneet Khurana2, Peter King1, Adrian Clark1, Li Chan1, Rachel Forfar2. 1Queen Mary University of London, London, UK, 2MRC Technology Centre for Therapeutics Discovery, London, UK

Abstract:

The pituitary hormone adrenocorticotropin hormone (ACTH) stimulates production of glucocorticoids from the adrenal gland by acting on a specific receptor complex, the ACTH receptor or melanocortin 2 receptor (MC2) and its accessory protein MRAP (melanocortin receptor-associated protein) (1). Excessive ACTH production in conditions such as pituitary tumours (Cushing’s disease), small cell lung cancers and inherited adrenal enzyme defects lead to adrenal hormone overproduction and increased morbidity and mortality (2). Such diseases respond poorly to conventional treatment and hence the blockade of ACTH action with a MC2-specific antagonist would have a valuable clinical role.

With the aim of identifying MC2-specific antagonists, ~200,000 small molecules comprising a collection of diverse chemical structures (approximately 75%), specific target class collections (kinases, ion channels), natural products and known bioactive compounds were screened, in Chinese hamster ovary cells stably co-expressing human MC2 and MRAP, using a high throughput homogeneous time-resolved fluorescence (HTRF) detection assay to measure cyclic adenosine monophosphate (cAMP) accumulation following ACTH stimulation. A significant number of hits which demonstrated MC2 antagonism were identified and these were subsequently counter-screened against the β2 adrenergic receptor, a related Gs-coupled receptor to confirm that compounds were selective for MC2. Hit confirmation of these MC2 selective compounds is currently on-going, and to date has identified four novel compounds that antagonised the effects of ACTH on cAMP accumulation (Table 1).

Further characterisation of these compounds, through Schild plot analysis and determination of antagonist affinity (pA2), suggests that they are competitive MC2 antagonists with an estimated affinity of XμμM (Figure 1).

The effect of these compounds at the murine orthologue receptor on cAMP accumulation and steroidogenesis was evaluated in the murine adrenal cell line, Y-1. Confirmation of the pharmacological and physiological effects of these putative antagonists would represent the first description of a small molecule antagonist for MC2, with the potential to reverse endocrine and oncological disorders characterised by overproduction of ACTH.

Table 1. Four identified antagonists, tested at XμM produced a log shift in the ACTH half maximal effective concentration (EC50) values..

Figure 1. Rightward shift in the ACTH concentration response curve with increasing concentrations of compound MRCT-x (a), which was used to estimate the pA2 value, by Schild regression (b)

(1) Metherell LA, Chapple JP, Cooray S, David A, Becker C, Rüschendorf F, Naville D, Begeot M, Khoo B, Nürnberg P, Huebner A, Cheetham ME & Clark AJL (2005) Nat Genet 37:166–170

(2) Shepherd FA, Laskey J, Evans WK, Goss PE, Johansen E & Khamsi F (1992) J Clin Oncol. 10:21-7

Shepherd FA, Laskey J, Evans WK, Goss PE, Johansen E & Khamsi F (1992) J Clin Oncol. 10:21-7