MC4R
October 2024
The melanocortin receptor 4 (MC4R) is a class A peptide G-protein coupled receptor (GPCR) of the melanocortin receptor family. It is involved in regulating hunger and satiety and, thus, body weight. MC4R mutations significantly affect food intake regulation, with loss-of-function mutations leading to severe early-onset obesity, while gain-of-function mutations increase satiety, resulting in lower body weight.
Therapeutic interventions with drugs that stimulate MC4R signaling (agonists) have shown promise in controlling satiety and food consumption, notably with the approval of the peptide MC4R agonist setmelanotide for treating rare genetic obesity disorders. (1) However, its lack of specificity for MC4R caused off-target effects on other melanocortin receptor subtypes (MC3R, MC1R, and with lower potency MC5R), (2,3) highlighting the need for more selective MC4R agonists.
Antibody therapeutics could offer a safer, more targeted approach. However, the discovery of GPCR antibodies, especially agonistic ones, is particularly challenging due to the high conformational complexity of these receptors.
Confo Therapeutics recently identified a panel of potent, highly specific MC4R agonistic nanobodies and solved the 3D structure of the MC4R complex with the most potent nanobody, pN162.4 This work elucidates its binding mode and provides a structural foundation for developing safer anti-obesity therapies (4).
Nanobodies
Nanobodies are small, single-domain antibody fragments derived from heavy-chain-only antibodies found in camelids. Despite lacking a light chain, they retain unimpaired binding capabilities comparable to other antibody types. Their small size, simple structure, high stability, solubility, and strong antigen-binding affinity make nanobodies potentially overcome many limitations of conventional monoclonal antibodies.
The scientists produced MC4R in a stabilized active conformation to identify full agonist MCR4 antibodies. They achieved this by using an active state-stabilized version of MC4R (MC4R-β2AR hybrid) and genetically fusing it with a ConfoBody (Cb), a single-domain antibody specifically designed to stabilize a particular conformational state of a dynamic protein like GPCRs. Using this active state MC4R-Cb chimera, they successfully identified and produced potent, MC4R-specific agonistic nanobodies, with the most potent being nanobody pN162.
To elucidate the interaction mode between pN162 and MC4R, the researchers determined the cryo-EM structure of the complex, revealing that pN162 binds deeply in the orthosteric pocket (Image 1). The complementary determining region 3 (CDR3) of pN162 occupies the same site as the endogenous ligand α-melanocyte-stimulating hormone (α-MSH, zoomed panel Image 1).
Image 1. 3D structure of the MC4R complex with nanobody pN162 (PDB: 8QJ2). MC4R is colored in light orange, pN162 in white, ConfoBody Cb35 in light green, and Gαs, Gβ, and Gγ in purple, red, and grey, respectively. The panel zooms in on the orthosteric pocket and pN162 CDR3 binding. The binding pose of the endogenous agonist α-MSH is represented in red and is obtained by overlay of the MC4R:α-MSH complex structure (PDB: 7F53; the cartoon representation of MC4R is omitted for clarity). The pictures were produced using the 3decision® software and its Annotation browser feature.
Superimposing the pN162:MC4R complex with previously published active (Image 2A) and inactive (Image 2B) state structures of MC4R shows a high similarity between the pN162-stabilized conformation and the active state MC4R. This includes key activation features, such as the large outward movement of transmembrane helix 6 (TM6) compared to the inactive state (Image 2B), which is characteristic of receptor activation.
Image 2. Comparison between active and inactive MC4R. A) Overlay of MC4R complexes with nanobody pN162 (PDB: 8QJ2, white), α-MSH (PDB: 7F53, pink), and setmelanotide (PDB: 7PIU, blue). In all three structures it is shown how the binders stabilize the active form of the receptor. B) Overlay of MC4R complexes with nanobody pN162 (PDB: 8QJ2, white), and the antagonist SHU9119 (PDB: 6W25, orange). The antagonist SHU9119 stabilizes the receptor in an inactive conformation, as indicated by the position of TM6 in the inward conformation, which is highlighted in the light blue circle. The pictures were produced using the 3decision® software.
Analysis of the interactions stabilizing pN162 binding showed that the nanobody exhibits a distinct set of interactions compared to other known agonists. Previous studies reported that a calcium ion in the orthosteric site coordinates agonist ligands and mediates MC4R binding, acting as a cofactor for initiating the MC4R signaling pathway (Image 3A). In contrast, the pN162 nanobody structure lacks the calcium ion in the orthosteric pocket. Instead, the residue pN162Arg101 forms a salt bridge with MC4RGlu100 and MC4RAsp126, effectively replacing the role of the metal ion (Image 3B). Mutagenesis screening confirmed that pN162Arg101 is critical for activating the MC4R signaling pathway.
Image 3. Analysis of the binding modes of agonist ligands to MC4R. Dashed lines represent the interaction networks: polar interactions are in yellow, hydrogen bonds in blue, and metal-mediated interactions in purple. A) Setmelanotide interaction with MC4R (PDB: 7PIU, MC4R in yellow, setmelanotide in pink, Ca2+ ion in light grey) shows calcium ion coordination of MC4RGlu100 and MC4RAsp126 residues (labeled in yellow). B) pN162 binding with MC4R (PDB: 8QJ2, MC4R in orange, pN162 in green) does not require the presence of Ca2+: the pN162Arg101 residue forms salt bridges with the residues MC4RGlu100 and MC4RAsp126 instead of the calcium coordination. The pictures were produced with the 3decision® software.
This study presented a rare example of the development and characterization of a full-agonistic, highly specific GPCR antibody. The solved pN162-MC4R structure offered structural insights that helped rationalize the observed therapeutic effect. Additionally, it serves as the basis for structure-based strategies aimed to further enhance potency and advance innovative anti-obesity therapies.
References:
Clément K, van den Akker E, Argente J, et al. Efficacy and safety of setmelanotide, an MC4R agonist, in individuals with severe obesity due to LEPR or POMC deficiency: single-arm, open-label, multicentre, phase 3 trials. Lancet Diabetes Endocrinol. 2020;8(12):960-970. doi:10.1016/S2213-8587(20)30364-8
Collet TH, Dubern B, Mokrosinski J, et al. Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency. Mol Metab. 2017;6(10):1321-1329. doi:10.1016/j.molmet.2017.06.015
Yeo GSH, Chao DHM, Siegert AM, et al. The melanocortin pathway and energy homeostasis: From discovery to obesity therapy. Mol Metab. 2021;48:101206. doi:10.1016/j.molmet.2021.101206
Fontaine T, Busch A, Laeremans T, et al. Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist. Nat Commun. 2024;15(1):7029. Published 2024 Oct 1. doi:10.1038/s41467-024-50827-7
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