JKC 363

Recent studies have suggested that melanocortins contribute to the generation and/or maintenance of pathological pain. Experimental evidence indicates a primary role for melanocortin 4 (MC4) receptors in pathological pain. In a previous study, we described the presence of MC4 receptor transcripts in the dorsal root ganglia (DRG). This finding prompted us to investigate the peripheral antinociceptive effects of MC4 receptor antagonists. In addition, we assess the expression of MC4 receptors in the spinal cord and the DRG of rats subjected to neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Injection of the MC4 receptor antagonists Asp3-Lys8- Ac-Nle-Asp-His-D-Nal(2')-Arg-Trp-Lys-NH(2) (SHU9119) and Mpr1-Cys8-Mpr-Glu-His-(D-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH(2) (JKC-363) into the ipsilateral paw resulted in a significant and dose-dependent alleviation of mechanical allodynia (assayed by the von Frey test) and thermal hyperalgesia (assayed by the Hargreaves test). Compared to naive control animals, immunohistochemistry revealed a 40% and 22% increase in MC4 receptor-immunoreactivity (IR) in the dorsal horn of the spinal cord ipsilateral to the injury at 3 and 14 days after CCI, respectively. Similarly, in the ipsilateral L4-L5 DRG, a 21.1% enhancement in MC4 receptor-IR was seen 3 days after CCI, as well as a 40.5% increase 14 days after CCI. Together, painful neuropathy resulted in the up-regulation of MC4 receptors in the spinal and peripheral nociceptive pathways. This up-regulation of MC4 receptors promotes the pronociceptive action of their endogenous ligands. Therefore, a block of the MC4 receptors results in the antagonism of neuropathic pain and such treatment could be beneficial therapeutically for individuals with chronic neuropathic pain.

To study the peripheral effects of melanocortin on fuel homeostasis in skeletal muscle, we assessed palmitate oxidation and AMP kinase activity in alpha-melanocyte-stimulating hormone (alpha-MSH)-treated muscle cells. After alpha-MSH treatment, carnitine palmitoyltransferase-1 and fatty acid oxidation (FAO) increased in a dose-dependent manner. A strong melanocortin agonist, NDP-MSH, also stimulated FAO in primary culture muscle cells and C2C12 cells. However, [Glu6]alpha-MSH-ND, which has ample MC4R and MC3R agonistic activity, stimulated FAO only at high concentrations (10(-5) M). JKC-363, a selective MC4R antagonist, did not suppress alpha-MSH-induced FAO. Meanwhile, SHU9119, which has both antagonistic activity on MC3R and MC4R and agonistic activity on both MC1R and MC5R, increased the effect of alpha-MSH on FAO in both C2C12 and primary muscle cells. Small interference RNA against MC5R suppressed the alpha-MSH-induced FAO effectively. cAMP analogues mimicked the effect of alpha-MSH on FAO, and the effects of both alpha-MSH and cAMP analogue-mediated FAO were antagonized by a protein kinase A inhibitor (H89) and a cAMP antagonist ((Rp)-cAMP). Acetyl-CoA carboxylase activity was suppressed by alpha-MSH and cAMP analogues by phosphorylation through AMP-activated protein kinase activation in C2C12 cells. Taken together, these results suggest that alpha-MSH increases FAO in skeletal muscle, in which MC5R may play a major role. Furthermore, these results suggest that alpha-MSH-induced FAO involves cAMP-protein kinase A-mediated AMP-activated protein kinase activation.

We previously identified melanocortin receptor 4 (MC4R) in a search for genes associated with hypoglossal nerve regeneration. As melanocortins promote nerve regeneration after axonal injury, we investigated whether MC4R functions as a key receptor for peripheral nerve regeneration. In situ hybridization revealed that MC4R mRNA is induced in mouse hypoglossal motor neurons after axonal injury, whereas mRNAs for MC1R, MC2R, MC3R, and MC5R are not expressed either before or after nerve injury. This result was confirmed by RT-PCR. The level of MC4R mRNA expression increased significantly from day 3 after axotomy, reached a peak on day 5, and decreased to the control level on day 14. Similar induction of MC4R was observed in axotomized mouse dorsal root ganglia (DRGs). MC4R mRNA expression was induced exclusively among the MCR family in the L4-6 DRG after sciatic nerve injury. We further examined whether alpha-melanocortin stimulating hormone (alpha-MSH) promotes neurite elongation via MC4R. In mouse DRG neuron culture, alpha-MSH significantly promoted neurite outgrowth at a concentration of 10(-8) mol/L. This neurite-elongation effect was entirely inhibited by the addition of a selective MC4R blocker, JKC-363. Therefore, it is concluded that alpha-MSH could stimulate neurite elongation via MC4R in DRG neurons. The present results suggest that induction of MC4R is crucial for motor and sensory neurons to regenerate after axonal injury.

The solution structures and inter-molecular interaction of the cyclic melanocortin antagonists SHU9119, JKC363, HS014, and HS024 with receptor molecules have been determined by NMR spectroscopy and molecular modeling. While SHU9119 is known as a nonselective antagonist, JKC363, HS014, and HS024 are selective for the melanocortin subtype-4 receptor (MC4R) involved in modulation of food intake. Data from NMR and molecular dynamics suggest that the conformation of the Trp9 sidechain in the three MC4R-selective antagonists is quite different from that of SHU9119. This result strongly supports the concept that the spatial orientation of the hydrophobic aromatic residue is more important for determining selectivity than the presence of a basic, "arginine-like" moiety responsible for biological activity. We propose that the conformation of hydrophobic residues of MCR antagonists is critical for receptor-specific selectivity.