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.

Interplay between glucose and leptin signalling determines the strength of GABAergic synapses at POMC neurons.

Regulation of GABAergic inhibitory inputs and alterations in POMC neuron activity by nutrients and adiposity signals regulate energy and glucose homeostasis. Thus, understanding how POMC neurons integrate these two signal molecules at the synaptic level is important. Here we show that leptin's action on GABA release to POMC neurons is influenced by glucose levels. Leptin stimulates the JAK2-PI3K pathway in both presynaptic GABAergic terminals and postsynaptic POMC neurons. Inhibition of AMPK activity in presynaptic terminals decreases GABA release at 10 mM glucose. However, postsynaptic TRPC channel opening by the PI3K-PLC signalling pathway in POMC neurons enhances spontaneous GABA release via activation of presynaptic MC3/4 and mGlu receptors at 2.5 mM glucose. High-fat feeding blunts AMPK-dependent presynaptic inhibition, whereas PLC-mediated GABAergic feedback inhibition remains responsive to leptin. Our data indicate that the interplay between glucose and leptin signalling in glutamatergic POMC neurons is critical for determining the strength of inhibitory tone towards POMC neurons.

Activation of the central melanocortin system contributes to the increased arterial pressure in obese Zucker rats.

We have previously demonstrated that leptin-mediated activation of the central nervous system (CNS) melanocortin system reduces appetite and increases sympathetic activity and blood pressure (BP). In the present study we examined whether endogenous melanocortin system activation, independent of leptin's actions, contributes to the regulation of BP and metabolic functions in obese Zucker rats, which have mutated leptin receptors. The long-term cardiovascular and metabolic effects of central melanocortin-3/4 receptor (MC3/4R) antagonism with SHU-9119 were assessed in lean (n = 6) and obese (n = 8) Zucker rats. BP and heart rate (HR) were measured 24-h/day by telemetry and an intracerebroventricular cannula was placed in the brain lateral ventricle. After stable control measurements, SHU-9119 was infused intracerebroventricularlly (1 nmol/h) for 10 days followed by a 10-day recovery period. Chronic CNS MC3/4R antagonism significantly increased food intake and body weight in lean (20 ± 1 to 45 ± 2 g and 373 ± 11 to 432 ± 14 g) and obese (25 ± 2 to 35 ± 2 g and 547 ± 10 to 604 ± 11 g) rats. No significant changes were observed in plasma glucose levels in lean or obese rats, whereas plasma leptin and insulin levels markedly increased in lean Zucker rats during CNS MC3/4R antagonism. Chronic SHU-9119 infusion in obese Zucker rats reduced mean arterial pressure (MAP) and HR by 6 ± 1 mmHg and 24 ± 5 beats/min, whereas in lean rats SHU-9119 infusion reduced HR by 31 ± 9 beats/min while causing only a transient decrease in MAP. These results suggest that in obese Zucker rats the CNS melanocortin system contributes to elevated BP independent of leptin receptor activation.

Memory impairment induced by IL-1beta is reversed by alpha-MSH through central melanocortin-4 receptors.

Interleukin-1beta (IL-1beta) significantly influences memory consolidation. Treatments that raise the level of IL-1beta in the brain, given after training, impair contextual fear conditioning. The melanocortin alpha-MSH exerts potent anti-inflammatory actions by physiologically antagonizing the effect of pro-inflammatory cytokines. Five subtypes of melanocortin receptors (MC1R-MC5R) have been identified, with MC3R and MC4R predominating in the central nervous system. The present experiments show that injection of IL-1beta (5 ng/0.25 microl) in dorsal hippocampus up to 15 min after training decreased freezing during the contextual fear test. The treatment with IL-1beta (5 ng/0.25 microl) 12h after conditioning cause amnesia when animals were tested 7 days post training. Thus, our results also demonstrated that IL-1beta can influence persistence of long-term memory. We determined that animals previously injected with IL-1beta can acquire a new contextual fear memory, demonstrating that the hippocampus was not damaged. Treatment with alpha-MSH (0.05 microg/0.25 microl) blocked the effect of IL-1beta on contextual fear memory. Administration of the MC4 receptor antagonist HS014 (0.5 microg/0.25 microl) reversed the effect of alpha-MSH. However, treatment with gamma-MSH (0.5 microg/0.25 microl), an MC3 agonist, did not affect IL-1beta-induced impairment of memory consolidation. These results suggest that alpha-MSH, through central MC4R can inhibit the effect of IL-1beta on memory consolidation.

Amygdalar opioids modulate hypothalamic melanocortin-induced anorexia.

We wanted to assess the possibility that opioid activity in the central amygdala (CeA) could modulate the feeding inhibition of melanocortin stimulation of the paraventricular hypothalamus (PVN). The melanocortin system is important in both the acute regulation of satiety and feeding behavior and in the integration of long-term appetite signals. Melanotan II (MTII) is a synthetic MC3R and MC4R agonist which reduces food intake when given intracerebroventricularly (ICV) and into the PVN. Tyr-D-Ala-Gly-(me) Phe-Gly-ol (DAMGO), a micro-opioid receptor agonist, increases food intake, while opioid antagonists, like naltrexone (NTX), inhibit food intake after injection into many brain sites involved in appetite regulation, including the CeA. In food-deprived male Sprague-Dawley rats, co-injected intra-PVN MTII partially blocked the orexigenic effect of co-injected intra-CeA DAMGO. Intra-CeA NTX co-injected with intra-PVN MTII reduced food intake significantly more than either alone. NTX administered intra-CeA reduced c-Fos-immunoreactivity (IR) in nucleus accumbens neurons significantly compared to the intra-PVN MTII treated animals, animals co-injected intra-PVN with MTII and intra-CeA with NTX animals, and control animals. Intra-PVN MTII induced c-Fos-IR in significantly more PVN neurons than observed in control animals. Intra-CeA NTX co-injected with intra-PVN MTII induced c-Fos-IR significantly in PVN neurons relative to control and intra-CeA NTX animals. Such data support the significance of opioid action within the CeA as a modulator of the feeding regulation action of melanocortins within the PVN, occurring within the context of a larger appetitive network.

Further structure-activity studies of lactam derivatives of MT-II and SHU-9119: their activity and selectivity at human melanocortin receptors 3, 4, and 5.

Recently we have demonstrated that replacing His(6) by constrained amino acids(2) in the well-known antagonist SHU-9119 resulted in potent and selective antagonist ligands especially at the hMC3R and hMC5 receptors. With the aim to further explore position 6 in the sequence of SHU-9119 and MT-II, we have designed, synthesized, and pharmacologically characterized a series of peptide analogues of MT-II and SHU-9119 at the human melanocortin receptors subtypes MC3R, MC4R and MC5R. All these peptides were modified at position 6 with constrained amino acids which are commercially available. In this study, we have identified new selective ligands for the hMC4R, and an antagonist for the hMC3/hMC4 receptors. Additionally, we have discovered an interesting new selective antagonist at the hMC3R, Ac-Nle-c[Asp-betaAla-DNal(2')-Arg-Trp-Lys]-NH(2) (2, PG-106) which represents an important tool in further biological investigations of the hMC3R. PG-106 will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.

Hypothalamic pro-opiomelanocortin gene delivery ameliorates obesity and glucose intolerance in aged rats.

AIMS/HYPOTHESIS: Age-related obesity is associated with impaired hypothalamic pro-opiomelanocortin (Pomc) gene expression. We assessed whether overproduction of POMC in the hypothalamus ameliorates age-related obesity in rats. METHODS: Recombinant adeno-associated virus (rAAV) encoding Pomc (rAAV-Pomc) or control vector was delivered bilaterally into the basomedial hypothalamus of aged obese rats with coordinates targeting the arcuate nucleus. Energy balance, glucose metabolism, brown adipose tissue thermogenesis and mRNA levels of hypothalamic neuropeptides and melanocortin receptors were assessed. RESULTS: Forty-two days after Pomc gene delivery, hypothalamic Pomc expression increased 12-fold while agouti-related protein and neuropeptide Y mRNA levels remained unchanged. Using a punch technique, we detected the highest Pomc RNA level in the arcuate nucleus. Pomc overexpression reduced food consumption from day 10 after vector injection, but this anorexic effect abated by day 30. In contrast, there was a steady decrease in body weight without apparent attenuation. Pomc gene delivery decreased visceral adiposity and induced uncoupling protein 1 in brown adipose tissue in aged rats. Serum NEFA and triglyceride levels were also diminished by rAAV-Pomc treatment. Improved glucose metabolism and insulin sensitivity were observed on day 36 but not day 20 after Pomc gene delivery. The expression of hypothalamic melanocortin 3 and 4 receptor decreased by 17% and 25%, respectively in rAAV-Pomc rats. CONCLUSIONS/INTERPRETATION: This study demonstrates that targeted Pomc gene therapy in the hypothalamus reduces body weight and visceral adiposity, and improves glucose and fat metabolism in aged obese rats. Thus long-term activation of the central melanocortin system may be a viable strategy to combat age-related obesity and diabetes.

Design and pharmacology of peptoids and peptide-peptoid hybrids based on the melanocortin agonists core tetrapeptide sequence.

A series of N-substituted glycine oligomers (peptoids) and peptide-peptoid hybrids were synthesized based on the Ac-His-Phe-Arg-Trp-NH(2) tetrapeptide template. The compounds were pharmacologically characterized at the mouse melanocortin receptors (MC1R, MC3R-MC5R) for agonist activity.

Structure-activity relationships of gamma-MSH analogues at the human melanocortin MC3, MC4, and MC5 receptors. Discovery of highly selective hMC3R, hMC4R, and hMC5R analogues.

It has been shown by extensive studies that melanotropin bioactivities are critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, and in alpha-MSH it has been demonstrated further that a reverse-turn type conformation exists at this pharmacophore. To probe the receptor active conformation of the pharmacophore His-Phe-Arg-Trp in gamma-MSH, two different series of gamma-MSH analogues have been designed and synthesized and their biological activities determined at hMC3R, hMC4R, and hMC5R. The 1st series consists of a cyclic scan using different disulfides or lactam bridges. It was found that cyclization of the native gamma-MSH around the highly conserved sequence can lead to shifts in affinity and selectivity for hMC4R instead of the hMC3R as seen in the native peptide. Furthermore, a 23-membered ring is desirable for potency (e.g., analogues 6 and 10) whereas a 26-membered ring (analogue 1, H-Tyr-Val-c[Cys-Gly-His-Phe-Arg-Trp-Cys]-Arg-Phe-Gly-NH(2) with Gly(4)) is more important for selectivity. The 2nd series is made of d-2-naphthylalanine (d-Nal(2')) scan of the gamma-MSH sequence at position 6 and 8 and the replacement of His(5) with Pro (analogue 13). Analogue 12, H-Tyr-Val-Nle-Gly-His-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a potent and selective antagonist at the hMC4R, and analogue 15, H-Tyr-Val-Nle-Gly-Aib-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a highly selective and potent agonist of the hMC5R. A most promising analogue is 13, H-Tyr-Val-Nle-Gly-Pro-d-Nal(2')-Arg-Trp-Asp-Arg-Phe-Gly-NH(2), which is a very potent agonist of the hMC4R, and this analogue can be further evaluated for feeding behavior and the regulation of fat stores.

Extensive structure-activity studies of lactam derivatives of MT-II and SHU-9119: their activity and selectivity at human melanocortin receptors 3, 4, and 5.

The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nm) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-II and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nm, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.

Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors.

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC(3) receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH(2) (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC(3) receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC(3)/MC(4) receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(1-24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC(3) receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC(3) receptors, the C-terminal Arg-Phe structure being relevant.

Brainstem melanocortin 3/4 receptor stimulation increases uncoupling protein gene expression in brown fat.

Central administration of melanocortin 3 and 4 receptor (MC3/4-R) agonists increases energy expenditure, with the hypothalamus commonly held as the primary site of action. It is also clear, however, that MC4-R are expressed in caudal brainstem structures of relevance to the control of energy expenditure. Three experiments investigated whether hindbrain MC-R contribute to the energy expenditure effects of central MC3/4-R agonist treatments; in each, we examined the effect of fourth intracerebroventricular (i.c.v.) administration of a MC3/4-R agonist, MTII (three injections, each separated by 12 h), on uncoupling protein 1 (UCP-1) gene expression in brown adipose tissue (BAT). First, we compared the effects of fourth and third i.c.v. administration of MTII and found that the hindbrain and forebrain treatments were equally effective at elevating UCP-1 mRNA expression in BAT compared with the respective vehicle-treated group results. A second experiment demonstrated that the fourth i.c.v. MTII-induced rise in UCP-1 expression was mediated by sympathetic outflow to BAT by showing that this response was abolished by surgical denervation of BAT. In the third experiment, we showed that chronic decerebrate rats, like their neurologically intact controls, elevated UCP-1 mRNA expression in response to fourth i.c.v. MTII administration. Taken together, the results indicate that: 1) there is an independent caudal brainstem MC3/4-R trigger for a sympathetically stimulated elevation in BAT UCP-1 gene expression, and 2) the MTII-induced rise in UCP-1 expression can be mediated by circuitry intrinsic to the caudal brainstem and spinal cord.