Incorporation of Agouti-Related Protein (AgRP) Human Single Nucleotide Polymorphisms (SNPs) in the AgRP-Derived Macrocyclic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-dPro] Decreases Melanocortin-4 Receptor Antagonist Potency and Results in the Discovery of Melanocortin-5 Receptor Antagonists

Abstract

While the melanocortin receptors (MCRs) are known to be involved in numerous biological pathways, the potential roles of the MC5R have not been clearly elucidated in humans. Agouti-related protein (AgRP), an MC3R/MC4R antagonist and MC4R inverse agonist, contains an exposed βhairpin loop composed of six residues (Arg-Phe-Phe-Asn-Ala-Phe) that is imperative for binding and function. Within this active loop of AgRP, four human missense polymorphisms were deposited into the NIH Variation Viewer database. These polymorphisms, Arg111Cys, Arg111His, Phe112Tyr, and Ala115Val (AgRP full-length numbering), were incorporated into the peptide macrocycles c[Pro 1 -Arg 2 -Phe 3 -Phe 4 -Xaa 5 -Ala 6 -Phe 7 -DPro 8 ], where Xaa was Dap 5 or Asn 5 , to explore the functional effects of these naturally occurring substitutions in a simplified AgRP scaffold. All peptides lowered potency at least 10-fold in a cAMP accumulation assay compared to the parent sequences at the MC4Rs. are also the first AgRP-based chemotypes that antagonize the MC5R.

Peptoid NPhe4 in AGRP-Based c[Pro1-Arg2-Phe3-Phe4-Xxx5-Ala6-Phe7-DPro8] Scaffolds Maintain Mouse MC4R Antagonist Potency

“…Similar to prior reports, inverse agonist activity was observed for select ligands at the MC5R. ,, Due to the increased MC4R antagonist potency, three compounds were examined for antagonist potency using a Schild experimental design at the MC5R, the parent 1 and the NPhe 4 containing 4 and 11 . The parent 1 was previously reported to possess nanomolar MC5R antagonist potency (pA 2 = 7.3), similar to the value observed in the present study (pA 2 = 7.5). The NPhe 4 , Dap 5 4 resulted in an equipotent MC5R antagonist (pA 2 = 7.4) to 1 , indicating that peptoid substitution at this position was not detrimental to MC5R antagonism activity.…”

“…As a comparison to the NPhe 4 , Dap 5 4 peptide, the antagonist potency of NPhe 4 , Asn 5 11 was measure at the MC5R (Table ). This compound possessed submicromolar antagonist potency at the MC5R (pA 2 = 6.1), 20-fold decreased potency compared to 4 but similar to the reported value for 8 (pA 2 = 6.4) …”

“…The use of <5.5 indicates that no antagonist potency was observed in the highest concentration ranged assayed (10,000, 5,000, 1,000, and 500 nM). Statistical analysis was performed using a student t test compared to the respective controls 1 (Dap 5 ) and 8 (Asn 5 ) receptor values with * p < 0.05. b The pharmacology for AGRP and macrocyclic peptide 8 has previously been reported and is included for reference. , …”

“… b The pharmacology for AGRP and macrocyclic peptide 8 has previously been reported and is included for reference. , …”

The central melanocortin system and human obesity

“…Most recently, these SNPs have been tested in vitro for potential impacts on cellular signaling and functions of the MC4R. All the SNPs tested result in at least a 10-fold decreased potency in inhibiting the MC4R, suggesting that SNPs may impact AGRP functions ( Koerperich et al., 2020 ).…”