The discovery of the endogenous melanocortin agonists in the 1950s have resulted in sixty years of melanocortin ligand research. Early efforts involved truncations or select modifications of the naturally occurring agonists leading to the development of many potent and selective ligands. With the identification and cloning of the five known melanocortin receptors, many ligands were improved upon through bench-top in vitro assays. Optimization of select properties resulted in ligands adopted as clinical candidates. A summary of every melanocortin ligand is outside the scope of this review. Instead, this review will focus on the following topics: classic melanocortin ligands, selective ligands, small molecule (non-peptide) ligands, ligands with sex-specific effects, bivalent and multivalent ligands, and ligands advanced to clinical trials. Each topic area will be summarized with current references to update the melanocortin field on recent progress.

Aldehyde dehydrogenases (ALDHs) catalyze the NAD(P)-dependent oxidation of aldehydes to carboxylic acids and are important for metabolism and detoxification. Although the ALDH superfamily fold is well established, some ALDHs contain an uncharacterized domain of unknown function (DUF) near the C terminus of the polypeptide chain. Herein, we report the first structure of a protein containing the ALDH superfamily DUF. Proline utilization A from Sinorhizobium meliloti (SmPutA) is a 1233-residue bifunctional enzyme that contains the DUF in addition to proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase catalytic modules. Structures of SmPutA with a proline analog bound to the proline dehydrogenase site and NAD bound to the ALDH site were determined in two space groups at 1.7-1.9 Å resolution. The DUF consists of a Rossmann dinucleotide-binding fold fused to a three-stranded β-flap. The Rossmann domain resembles the classic ALDH superfamily NAD-binding domain, whereas the flap is strikingly similar to the ALDH superfamily dimerization domain. Paradoxically, neither structural element performs its implied function. Electron density maps show that NAD does not bind to the DUF Rossmann fold, and small-angle X-ray scattering reveals a novel dimer that has never been seen in the ALDH superfamily. The structure suggests that the DUF is an adapter domain that stabilizes the aldehyde substrate binding loop and seals the substrate-channeling tunnel via tertiary structural interactions that mimic the quaternary structural interactions found in non-DUF PutAs. Kinetic data for SmPutA indicate a substrate-channeling mechanism, in agreement with previous studies of other PutAs.

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The five melanocortin receptors (MC1R–MC5R) are involved in numerous biological pathways, including steroidogenesis, pigmentation, and food intake. In particular, MC3R and MC4R knockout mice suggest that the MC3R and MC4R regulate energy homeostasis in a non-redundant manner. While MC4R-selective agonists have been utilized as appetite modulating agents, the lack of MC3R-selective agonists has impeded progress in modulating this receptor in vivo. In this study, the (pI)DPhe position of the tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 (an MC3R agonist/MC4R antagonist ligand) was investigated with a library of 12 compounds. The compounds in this library were found to have higher agonist efficacy and potency at the mouse (m) MC3R compared to the MC4R, indicating that the Arg-DPhe motif preferentially activates the mMC3R over the mMC4R. This observation may be used in the design of new MC3R-selective ligands, leading to novel probe and therapeutic lead compounds that will be useful for treating metabolic disorders.

Methylenetetrahydrofolate reductase (MTHFR) is a flavoprotein that utilizes two substrates, NADH and 5,10‐methylenetetrahydrofolate (CH2‐H4folate), in a ping pong bi‐bi mechanism. In the reaction, bound FAD is reduced by NADH and then oxidized by CH2‐H4folate. The pH dependence of the reductive half‐reaction was studied with steady‐state kinetics using the NADH‐menadione oxidoreductase assay. In this assay the reductive half‐reaction is rate‐limiting, and menadione is used to reoxidize the flavin. The wild‐type MTHFR reaction was found to be pH‐dependent with an inflection point (pKa) at 4.9. We have hypothesized that this pKa belongs to the active‐site amino acid glutamate 28 (Glu28). When Glu28 is deprotonated, the negatively‐charged side chain can stabilize the positively‐charged nicotinamide ring of NADH during the reductive half‐reaction. To determine whether Glu28 is responsible for the pKa of 4.9, we have examined a mutant enzyme with Glu28 replaced by a glutamine. Kinetic parameters were obtained for the NADH‐menadione reaction catalyzed by the Glu28Gln mutant at pH values 4.5 to 8. Our results suggest that a pKa ~5 still exists in the pH rate profile of the Glu28Gln mutant. However, instability and denaturation have plagued our studies. In current work, we are testing alternate buffers conditions to stabilize the mutant enzyme.