Targeting human melanocortin type 2 receptor (MC2R) with small molecules and designed peptides for the treatment of congenital adrenal hyperplasia

Abstract

<b>Abstract ID 24264</b> <b>Poster Board 321</b> <b>Background:</b> The adrenocorticotropic hormone (ACTH) is a 39 amino acid polypeptide secreted by the anterior pituitary and regulates cortisol secretion from the adrenal cortex. Cortisol has negative feedback and regulates the synthesis and secretion of ACTH. Excess ACTH is associated with a wide range of diseases including congenital adrenal hyperplasia (CAH). Classic CAH due to the 21-hydroxylase (CYP21A2) deficiency causes a reduction or loss of cortisol synthesis. Here the negative feedback is removed, causing an excess of ACTH which then leads to an increase in the production of adrenal androgens. This high level of androgens compromises both the growth and the fertility in CAH patients and remains a challenging side effect even under steroid treatment. Blocking the ACTH action by targeting MC2R may be a potential treatment option in patients with CAH. <b>Methods:</b> We modeled the interaction of ACTH with MC2R and designed peptides to understand ACTH binding and activation of MC2R. A model of MC2R was made using the recently described structure of the MC4R protein. We docked the ACTH into the MC2R structure using AUTODOCK-VINA and performed Molecular dynamics simulations of ACTH binding to MC2R that provided hints for the design of peptides to block ACTH-MC2R interaction. <i>In-vitro</i> assays were performed to test the potency of designed peptides which were then optimized further to produce a second batch of antagonists. For assays, the OS3 cells transfected with MC receptor constructs were used and cyclic AMP (cAMP) was measured by luciferase assay. The potential to shift the ACTH concentration-response curve (CRC) was evaluated to characterize the antagonist activity of the designed peptides. Several natural compounds were also screened by computational docking and activity assays to screen for potential ACTH antagonism. <b>Results:</b> We have analyzed the MC receptors and aligned the sequences of MC1R, MC2R, MC3R, MC4R, and MC5R to study the similarities and differences between these receptors. Sequence analysis identified two specific regions that are distinct for MC2R compared to other MC receptors and are also critical for the binding of ACTH to MC2R. After three rounds of peptide design, we made 4, 6, and 8 residue linear as well as circular peptides for targeting MC2R. A sequence optimization of peptides and ACTH residues was performed to improve the peptide binding to MC2R and to improve peptide stability. Mutation in the core sequence (M4, R8) of ACTH abolished MC2R activation as predicted. One lead peptide inhibitor was identified which shifted the ACTH CRC towards the right by half log, indicating antagonism. We identified several chemical leads for the further development of ACTH antagonists. <b>Conclusion:</b> We have modeled the binding of ACTH to MC2R and designed peptides that could target the ACTH-MC2R interaction and signaling. Three rounds of peptide designs were made which provided information about key residues involved in ACTH-MC2R interaction and created more specific and tighter binding peptides to MC2R that can be used to block ACTH-MC2R interaction. Further optimization of our designed peptides as well as modification of small molecules identified in our screening could be used for developing specific inhibitors of ACTH-MC2R interaction to suppress the action of ACTH and elevated androgen production in CAH and other disorders of steroid biosynthesis. This study was funded in part by a Swiss Government Excellence Scholarship and from a grant by IFCAH Foundation, France.