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.
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.
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.
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 β-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 cPro
-Arg
-Phe
-Phe
-Xaa
-Ala
-Phe
-dPro
, where Xaa was Dap
or Asn
, 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.
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-NH
. The most notable results include the identification of tetrapeptide
, Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH
, 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-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 β-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold cPro-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.
The melanocortin receptors are a family of rhodopsin-like (family A) G protein-coupled receptors (GPCRs) that are known to have a variety of conserved physiological functions. The following chapters ...of this dissertation explore four different projects designed to examine this receptor family through: 1) synthesis of novel ligands using traditional structure activity relationships (SAR), 2) use of a novel mixture based positional scanning approach for ligand discovery, 3) synthesis of endogenous ligand mimetics with established scaffolds, and 4) use of various functional, binding affinity, and proximity assays for in vivo evaluation. Chapter 1 serves as an introduction to the melanocortin receptor (MCR) family as well as ligands (endogenous and synthetic) that are well known in the field. This chapter also introduces the concept of selectivity between the five melanocortin receptors, particularly the centrally expressed melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R). Chapter 2 describes all the materials and methods used in Chapters 3-6 for peptide synthesis, characterization, and pharmacological evaluation. Chapter 3 describes the exploration of single nucleotide polymorphisms in the antagonist signaling molecule Agouti-related Protein (AgRP) by incorporating them into a modified AgRP octapeptide macrocyclic scaffold. Chapter 4 investigates the roles of ligands in protein:protein interactions, specifically those of the MC4R and the kappa opioid receptor (KOR). Chapter 5 demonstrates the complexity involved in designing a selective ligand through focused tetrapeptide SAR and functional assessment in a cAMP accumulation assay. Chapter 6 outlines the discovery of a completely novel MC3R antagonist scaffold using a mixture based positional scanning approach. Lastly, Chapter 7 broadly summarizes the dissertation presented and reflects upon knowledge and experienced gained, advancements to the field, and potential future directions for the work presented in this thesis.
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.
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