Development of potent selective competitive-antagonists of the melanocortin type 2 receptor Elise Bouw, Martin Huisman, Sebastian J.C.M.M. Neggers, Axel P.N. Themmen, A.J. van der Lely, Patric J.D. Delhanty ⇑ Department of Internal Medicine, Erasmus MC, 3000 CA Rotterdam, The Netherlands article info Article history: Received 11 March 2014 Received in revised form 16 June 2014 Accepted 3 July 2014 Available online 11 July 2014 Keywords: Melanocortin 2 receptor (MC2R), Adrenocorticotropic hormone (ACTH) Antagonist Cushing’s disease abstract Cushing’s disease, a hypercortisolemic state induced by an ACTH overexpressing pituitary adenoma, causes increased morbidity and mortality. Selective antagonism of the melanocortin type 2 receptor (MC2R) may be a novel treatment modality. Five structurally related peptides with modified HFRW sites but intact putative MC2R binding sites were tested for antagonistic activity at MC1R, MC2R/MRAP, MC3R, MC4R and MC5R. Two of these peptides (GPS1573 and GPS1574) dose-dependently antagonized ACTH- stimulated MC2R activity (IC 50 s of 66 ± 23 nM and 260 ± 1 nM, respectively). GPS1573 and 1574 sup- pressed the R max but not EC 50 of ACTH on MC2R, indicating non-competitive antagonism. These peptides did not antagonize a-MSH stimulation of MC1R and antagonized MC3, 4 and 5R at markedly lower potency. GP1573 and GPS1574 antagonize MC4R with IC 50 s of 950 nM and 3.7 lM, respectively. In con- clusion, two peptide antagonists were developed with selectivity for MC2R, forming a platform for devel- opment of a medical treatment for Cushing’s disease. Ó 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cushing’s syndrome is an endocrine disease caused by exposure to excessive glucocorticoids from endogenous or exogenous (iatrogenic) origin. There are several aetiologies of endogenous hypercortisolism. The subtypes are broadly divided into adrenocor- ticotropic hormone (ACTH)-dependent and ACTH-independent hypercortisolism. Pituitary ACTH-dependent hypercortisolism is the most common cause of endogenous hypercortisolism and is also known as Cushing’ s disease (Guaraldi and Salvatori, 2012). Untreated or inadequately treated Cushing’s disease is associated with cardiovascular disease, central obesity, skeletal fractures, insu- lin-resistant hyperglycaemia, arterial hypertension, limiting proxi- mal muscle weakness, persistent cognitive defects and increased mortality (Lindholm et al., 2001; Schteingart, 2009; Arnaldi et al., 2003; Newell-Price et al., 2006; Plotz et al., 1952). However, when eucortisolemia is achieved, the observed mortality rate returns to normal over a 10–20 year follow-up period (Clayton, 2010). Surgery is still the first choice of treatment for ACTH-dependent Cushing’s syndrome (Biller et al., 2008; Fleseriu and Petersenn, 2012). Transsphenoidal resection of a pituitary adenoma is success- ful in 69–98% of the patients (Kelly, 2007). However, in 5–11.5% of patients persistent and recurrent disease occurs, as has been docu- mented by long-term surveillance (Kelly, 2007). If surgery is not curative or contra-indicated, hypercortisolism has to be resolved by continued treatment. Second-line therapeutic options include repeated transsphenoidal surgery, radiation therapy and bilateral adrenalectomy (Kelly, 2007; Liu et al., 2007; Hofmann et al., 2006). Medical therapy can offer temporary relief, while more definitive therapy becomes effective (Biller et al., 2008). Medical therapies can be divided into three groups: cortisol secretion inhib- itors, neuromodulatory drugs and glucocorticoid receptor antago- nists (Schteingart, 2009; Morris and Grossman, 2002). Currently, an effective, minimally toxic, medical monotherapy is not available for the treatment of Cushing’ s disease (Feelders et al., 2010; Mancini et al., 2010). Given the limitations of surgery and radiation therapy, new medical treatment options are needed for patients with Cushing’s disease (Mancini et al., 2010). A recent study found that Pasireotide, a somatostatin analogue, leads to a significant decrease in cortisol levels in patients with Cushing’s disease. A major disadvantage of this treatment is the high incidence of hyper- glycaemia-related adverse events (Colao et al., 2012). The ACTH receptor, also known as the melanocortin 2 receptor (MC2R), is a member of a family of five highly homologous G pro- tein-coupled receptors (MC1–5R) (Cone et al., 1993; Cooray and http://dx.doi.org/10.1016/j.mce.2014.07.003 0303-7207/Ó 2014 Elsevier Ireland Ltd. All rights reserved. ⇑ Corresponding author. Address: Room Ee532, Department of Internal Medicine, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Tel.: +31 10 704 35 77; fax: +31 10 703 54 30. E-mail address: p.delhanty@erasmusmc.nl (P.J.D. Delhanty). Molecular and Cellular Endocrinology 394 (2014) 99–104 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce