Identification of Tetrapeptides from a Mixture Based Positional Scanning Library That Can Restore nM Full Agonist Function of the L106P, I69T, I102S, A219V, C271Y, and C271R Human Melanocortin-4 Polymorphic Receptors (hMC4Rs)

Human obesity has been linked to genetic factors and single nucleotide polymorphisms (SNPs). Melanocortin-4 receptor (MC4R) SNPs have been associated with up to 6% frequency in morbidly obese children and adults. A potential therapy for individuals possessing such genetic modifications is the identification of molecules that can restore proper receptor signaling and function. These compounds could serve as personalized medications improving quality of life issues as well as alleviating diseases symptoms associated with obesity including type 2 diabetes. Several hMC4 SNP receptors have been pharmacologically characterized in vitro to have a decreased, or a lack of response, to endogenous agonists such as α-, β-, and γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin hormone (ACTH). Herein we report the use of a mixture based positional scanning combinatorial tetrapeptide library to discover molecules with nM full agonist potency and efficacy to the L106P, I69T, I102S, A219V, C271Y, and C271R hMC4Rs. The most potent compounds at all these hMC4R SNPs include Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2, and Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, revealing new ligand pharmacophore models for melanocortin receptor drug design strategies

Discovery of Molecular Interactions of the Human Melanocortin-4 Receptor (hMC4R) Asp189 (D189) Amino Acid with the Endogenous G-Protein-Coupled Receptor (GPCR) Antagonist Agouti-Related Protein (AGRP) Provides Insights to AGRP\u27s Inverse Agonist Pharmacology at the hMC4R

The melanocortin receptors (MCRs) are important for numerous biological pathways, including feeding behavior and energy homeostasis. In addition to endogenous peptide agonists, this receptor family has two naturally occurring endogenous antagonists, agouti and agouti-related protein (AGRP). At the melanocortin-4 receptor (MC4R), the AGRP ligand functions as an endogenous inverse agonist in the absence of agonist and as a competitive antagonist in the presence of agonist. At the melanocortin-3 receptor (MC3R), AGRP functions solely as a competitive antagonist in the presence of agonist. The molecular interactions that differentiate AGRP\u27s inverse agonist activity at the MC4R have remained elusive until the findings reported herein. Upon the basis of homology molecular modeling approaches, we previously postulated a unique interaction between the D189 position of the hMC4R and Asn114 of AGRP. To further test this hypothesis, six D189 mutant hMC4Rs (D189A, D189E, D189N, D189Q, D189S, and D189K) were generated and pharmacologically characterized resulting in the discovery of differences in inverse agonist activity of AGRP and an 11 macrocyclic compound library. These data support the hypothesized interaction between the hMC4R D189 position and Asn114 residue of AGRP and define critical ligand-receptor molecular interactions responsible for the inverse agonist activity of AGRP at the hMC4R

Discovery of Mixed Pharmacology Melanocortin-3 Agonists and Melanocortin-4 Receptor Tetrapeptide Antagonist Compounds (TACOs) Based on the Sequence Ac-Xaa

The centrally expressed melanocortin-3 and -4 receptors (MC3R/MC4R) have been studied as possible targets for weight management therapies, with a preponderance of studies focusing on the MC4R. Herein, a novel tetrapeptide scaffold [Ac-Xaa1-Arg-(pI)DPhe-Xaa4-NH2] is reported. The scaffold was derived from results obtained from a MC3R mixture-based positional scanning campaign. From these results, a set of 48 tetrapeptides were designed and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. This resulted in the serendipitous discovery of nine compounds that were MC3R agonists (EC50 \u3c 1000 nM) and MC4R antagonists (5.7 \u3c pA2 \u3c 7.8). The three most potent MC3R agonists, 18 [Ac-Arg-Arg-(pI)DPhe-Tic-NH2], 1 [Ac-His-Arg-(pI)DPhe-Tic-NH2], and 41 [Ac-Arg-Arg-(pI)DPhe-DNal(2\u27)-NH2] were more potent (EC50 \u3c 73 nM) than the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2. This template contains a sequentially reversed Arg-(pI)DPhe motif with respect to the classical Phe-Arg melanocortin signaling motif, which results in pharmacology that is first-in-class for the central melanocortin receptors

Discovery of Mixed Pharmacology Melanocortin-3 Agonists and Melanocortin-4 Receptor Tetrapeptide Antagonist Compounds (TACOs) Based on the Sequence Ac-Xaa1-Arg-(pI)DPhe-Xaa4-NH2

The centrally expressed melanocortin-3 and -4 receptors (MC3R/MC4R) have been studied as possible targets for weight management therapies, with a preponderance of studies focusing on the MC4R. Herein, a novel tetrapeptide scaffold [Ac-Xaa1-Arg-(pI)DPhe-Xaa4-NH2] is reported. The scaffold was derived from results obtained from a MC3R mixture-based positional scanning campaign. From these results, a set of 48 tetrapeptides were designed and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. This resulted in the serendipitous discovery of nine compounds that were MC3R agonists (EC50 \u3c 1000 nM) and MC4R antagonists (5.7 \u3c pA2 \u3c 7.8). The three most potent MC3R agonists, 18 [Ac-Arg-Arg-(pI)DPhe-Tic-NH2], 1 [Ac-His-Arg-(pI)DPhe-Tic-NH2], and 41 [Ac-Arg-Arg-(pI)DPhe-DNal(2′)-NH2] were more potent (EC50 \u3c 73 nM) than the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2. This template contains a sequentially reversed “Arg-(pI)DPhe” motif with respect to the classical “Phe-Arg” melanocortin signaling motif, which results in pharmacology that is first-in-class for the central melanocortin receptors

Human β‑Defensin 1 and β‑Defensin 3 (Mouse Ortholog mBD14) Function as Full Endogenous Agonists at Select Melanocortin Receptors

β-Defensin 3 (BD3) was identified as a ligand for the melanocortin receptors (MCRs) in 2007, although the pharmacology activity of BD3 has not been clearly elucidated. Herein, it is demonstrated that human BD3 and mouse BD3 are full micromolar agonists at the MCRs. Furthermore, mouse β-defensin 1 (BD1) and human BD1 are also MCR micromolar agonists. This work identifies BD1 as an endogenous MCR ligand and clarifies the controversial role of BD3 as a micromolar agonist

Discovery of Mixed Pharmacology Melanocortin‑3 Agonists and Melanocortin‑4 Receptor Tetrapeptide Antagonist Compounds (TACOs) Based on the Sequence Ac-Xaa¹‑Arg-(pI)DPhe-Xaa⁴‑NH₂

The centrally expressed melanocortin-3 and -4 receptors (MC3R/MC4R) have been studied as possible targets for weight management therapies, with a preponderance of studies focusing on the MC4R. Herein, a novel tetrapeptide scaffold [Ac-Xaa¹-Arg-(pI)­DPhe-Xaa⁴-NH₂] is reported. The scaffold was derived from results obtained from a MC3R mixture-based positional scanning campaign. From these results, a set of 48 tetrapeptides were designed and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. This resulted in the serendipitous discovery of nine compounds that were MC3R agonists (EC₅₀ < 1000 nM) and MC4R antagonists (5.7 < pA₂ < 7.8). The three most potent MC3R agonists, <b>18</b> [Ac-Arg-Arg-(pI)­DPhe-Tic-NH₂], <b>1</b> [Ac-His-Arg-(pI)­DPhe-Tic-NH₂], and <b>41</b> [Ac-Arg-Arg-(pI)­DPhe-DNal(2′)-NH₂] were more potent (EC₅₀ < 73 nM) than the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH₂. This template contains a sequentially reversed “Arg-(pI)­DPhe” motif with respect to the classical “Phe-Arg” melanocortin signaling motif, which results in pharmacology that is first-in-class for the central melanocortin receptors