Incorporation of Three Extracyclic Arginine Residues into a Melanocortin Macrocyclic Agonist (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Arg-Arg-Arg-Ac)-DPro]) Decreases Food Intake When Administered Intrathecally or Subcutaneously Compared to a Macrocyclic Ligand Lacking Extracyclic Arginine Residues (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro)].

Of the three Food and Drug Administration-approved melanocortin peptide drugs, two possess a cyclic scaffold, demonstrating that cyclized melanocortin peptides have therapeutic relevance. An extracyclic Arg residue, critical for pharmacological activity in the approved melanocortin cyclic drug setmelanotide, has also been demonstrated to increase the signal when fluorescently labeled cell-penetrating cyclic peptides are incubated with HeLa cells, with the maximal signal observed with three extracyclic Arg amino acids. Herein, a branching Lys residue was substituted into two macrocyclic melanocortin peptide agonists to incorporate 0-3 extracyclic Arg amino acids. Incorporation of the Arg residues resulted in equipotent or increased agonist potency at the mouse melanocortin receptors in vitro, suggesting that these substitutions were tolerated in the macrocyclic scaffolds. Further in vivo evaluation of one parent ligand (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-Pro]) and the three Arg derivative (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Ac-Arg-Arg-Arg)-Pro)] demonstrated that the three Arg derivative further decreased food intake compared to the parent macrocycle when the compounds were administered either via intrathecal injection or subcutaneous dosing. This suggests that three extracyclic Arg amino acids may be beneficial in the design of cyclic melanocortin ligands and that in vitro pharmacological profiling may not predict the in vivo efficacy of melanocortin ligands.

Functional Mixture-Based Positional Scan Identifies a Library of Antagonist Tetrapeptide Sequences (LAtTeS) with Nanomolar Potency for the Melanocortin-4 Receptor and Equipotent with the Endogenous AGRP(86-132) Antagonist.

The melanocortin-4 receptor (MC4R) plays an important role in appetite. Agonist ligands that stimulate the MC4R decrease appetite, while antagonist compounds increase food consumption. Herein, a functional mixture-based positional scan identified novel MC4R antagonist sequences. Mixtures comprising a library of 12,960,000 tetrapeptides were screened in the presence and absence of the NDP-MSH agonist. These results led to the synthesis of 48 individual tetrapeptides, of which 40 were screened for functional activity at the melanocortin receptors. Thirteen compounds were found to possess nanomolar antagonist potency at the MC4R, with the general tetrapeptide sequence Ac-Aromatic-Basic-Aromatic-Basic-NH2. The most notable results include the identification of tetrapeptide 48 [COR1-25, Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2], an equipotent MC4R antagonist to agouti-related protein [AGRP(86-132)], more potent than miniAGRP(87-120), and possessing 15-fold selectivity for the MC4R versus the MC3R. These tetrapeptides may serve as leads for novel appetite-inducing therapies to treat states of negative energy balance, such as cachexia and anorexia.

The melanocortin pathway and energy homeostasis: From discovery to obesity therapy.

BACKGROUND: Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency. SCOPE OF REVIEW: Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target. MAJOR CONCLUSIONS: Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug.

Journeying through the Field of Medicinal Chemistry: Perspectives from Graduate Researchers.

The training of new medicinal chemists is vital to the future of the field, and as graduate students at this critical stage, we are uniquely positioned to comment on our training. Herein, we discuss the perspectives from graduate researchers before, during, and after graduate school by utilizing survey data obtained from five medicinal chemistry programs in the Midwest and recent alumni of the University of Minnesota. We also reflect on the female perspective within the field of medicinal chemistry. Finally, we offer recommendations to both students and faculty in the hopes of helping future generations succeed in the field.

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.

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 ß-hairpin 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[Pro1-Arg2-Phe3-Phe4-Xaa5-Ala6-Phe7-dPro8], where Xaa was Dap5 or Asn5, 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. Compounds MDE 6-82-3c, ZMK 2-82, MDE 6-82-1c, ZMK 2-85, and ZMK 2-112 are also the first AgRP-based chemotypes that antagonize the MC5R.

Maintenance DNA Methyltransferase Activity in the Presence of Oxidized Forms of 5-Methylcytosine: Structural Basis for Ten Eleven Translocation-Mediated DNA Demethylation.

A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten eleven translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. In this study, we investigated the catalytic activity of human DNMT1 in the presence of oxidized forms of mC. A mass spectrometry-based assay was employed to study the kinetics of DNMT1-mediated cytosine methylation in CG dinucleotides containing C, mC, hmC, fC, or caC across from the target cytosine. Homology modeling, coupled with molecular dynamics simulations, was used to explore the structural consequences of mC oxidation with regard to the geometry of protein-DNA complexes. The DNMT1 enzymatic activity was strongly affected by the oxidation status of mC, with the catalytic efficiency decreasing in the following order: mC > hmC > fC > caC. Molecular dynamics simulations revealed that DNMT1 forms an unproductive complex with DNA duplexes containing oxidized forms of mC as a consequence of altered interactions of the target recognition domain of the protein with the C-5 substituent on cytosine. Our results provide new structural and mechanistic insight into TET-mediated DNA demethylation.

Arg-Phe-Phe d-Amino Acid Stereochemistry Scan in the Macrocyclic Agouti-Related Protein Antagonist Scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro] Results in Unanticipated Melanocortin-1 Receptor Agonist Profiles.

The melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), endogenous agonists derived from the proopiomelanocortin gene transcript, and naturally occurring antagonists agouti and agouti-related protein (AGRP) have been linked to biological pathways associated with energy homeostasis. The active tripeptide sequence of AGRP, Arg111-Phe112-Phe113, is located on a hypothesized ß-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was Asn or diaminopropionic acid (Dap). Macrocyclic peptides were synthesized with one, two, or three residues of the Arg-Phe-Phe sequence substituted with the corresponding d-isomer(s), generating a 14 compound library. While l-to-d inversions of the Arg-Phe-Phe sequence in a 20-residue AGRP-derived ligand previously resulted in agonist activity at the MC1R, MC3R, MC4R, and MC5R, only the MC1R was consistently stimulated by the macrocyclic ligands in the present study, with varying ligand potencies and efficacies observed at the MC1R. A general trend of increased MC4R antagonist potency was observed for Dap-containing compounds, while MC5R inverse agonist activity was observed for select ligands. It was observed that stereochemical modification of the Arg-Phe-Phe active tripeptide sequence was insufficient to convert melanocortin antagonist into agonists. Overall, these observations are important in the design of melanocortin ligands possessing potent and selective agonist and antagonist activities.