Identification Of Small Molecule Melanocortin 2 Receptor Antagonists As Potential Therapeutics For Cushing’s Disease

RAE Forfar1, M Hussain2, P Khurana1, G Genov1, S Lewis1, E McIver1, D Taylor1, A Clark2, L Chan2, J Jerman1, A Kamal1. 1MRC Technology, London, UK, 2Queen Mary University of London, London, UK

The melanocortin 2 (MC2) receptor, a G-protein coupled receptor (GPCR) expressed on the adrenal gland, and its associated protein melanocortin receptor accessory protein (MRAP) are stimulated by adrenocorticotropic hormone (ACTH) produced in the pituitary. Ligand-stimulated Gs-coupling and cyclic adenosine monophosphate (cAMP) generation leads to steroidogenesis with cortisol production regulating a diverse range of physiological processes, including energy metabolism, electrolyte balance and immune function. However, ACTH excess can result from conditions such as pituitary tumours (Cushing’s Disease), small cell lung cancers, and genetic mutations of adrenal enzymes (congenital adrenal hyperplasia) leading to increased morbidity and mortality. Hence, the identification of an MC2/MRAP antagonist would be of clinical benefit to treat these conditions which respond poorly to conventional therapies.

A high-throughput screen of ~200,000 compounds was performed using Chinese hamster ovary (CHO) cells stably expressing human MC2 and MRAP. Using a homogeneous time-resolved fluorescence (HTRF) assay, ~700 unique hits were identified that inhibited ACTH-stimulated cAMP accumulation, with specificity for MC2 established using a cAMP counter-assay against an unrelated Gs-coupled GPCR, the β2 adrenergic receptor. Further pharmacological characterisation of these hits identified four lead compounds that demonstrated at least a log unit rightward-shift of the ACTH concentration response curve (CRC) when incubated with 10μM inhibitor, and Schild analysis consistent with a competitive nature of antagonism (pA2 range: 5.9 to 5.7). A focussed hit-to-lead Medicinal Chemistry programme around these initial hits showed limited structure-activity relationships with related analogues. Significant improvements in affinity were not observed when these and other analogues were profiled against the MC2 receptor stimulated with an EC80 of ACTH (pIC50 range: 5.4 to <4). Similar profiling of the hits and related analogues from another series, series 2, revealed four lead compounds with pIC50 ranging from 5.0 to 4.6, all of which exhibited about half a log unit rightward-shift of the ACTH CRC when incubated with 10μM inhibitor. Hit-to-lead Medicinal Chemistry is currently underway to improve the potency and ADME properties.

The murine adrenal cell line, Y-1, was used to evaluate the effects of representative hit compounds from each series on endogenously expressed MC2 and MRAP using assays that measure ACTH-stimulated cAMP and progesterone release. These studies confirmed antagonist activity in a more physiologically relevant system with endogenous levels of MC2 and MRAP expression. Compound s inhibited a range of concentrations of ACTH-induced progesterone production in a concentration-dependent manner, with higher concentrations of compound inhibiting progesterone release near to basal levels. A number of alternative functional assays including MC2-MRAP proximity assays and dynamic mass redistribution studies are currently ongoing to further pharmacologically characterise preferred compounds, including mechanism of action, before murine in vivo experiments modelling reduction of ACTH-excess are performed.