ESPE Abstracts (2018) 89 FC1.3

Targetting the Binding of ACTH to the Melanocortin Receptor by Structure Modeling and Design of Peptide antagonists to Block Excess Androgens in 21-hydroxylase Deficiency

Shaheena Parweena,b, Christa E Flücka,b & Amit V Pandeya,b

aUniversity Children’s Hospital Bern, Bern, Switzerland; bUniversity of Bern, Bern, Switzerland

Background: 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 the 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 the CAH patients.

Aim: To model the interaction of ACTH with MC2R and design ACTH antagonists for reducing the abnormal adrenal androgen production by blocking the binding of ACTH to the MC2R.

Methods: Three-dimensional protein structure models of both the MC2R and the ACTH were built by homology modeling and optimized by molecular dynamics (MD) simulations. A computational docking of ACTH to MC2R was performed and contact points of ACTH and MC2R were generated. This interaction information was then used to design peptide-based inhibitors. In-vitro 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 antagonist activity of the designed peptides.

Results and conclusion: Activation and inhibition of MC2R by designed peptides were tested and the assay results confirmed the structural bioinformatics predictions. 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. This study could be useful in ACTH/MC2R antagonist development and inhibiting the binding of ACTH to the MC2R could be a novel approach for CAH management.

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