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An unusual conformation of ?-melanocyte-stimulating hormone analogues leads to a selective human melanocortin 1 receptor antagonist for targeting melanoma cells.

ABSTRACT: ?-MSH (?-melanocyte-stimulating hormone, H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH), with its exquisite specificity and potency, has recently created much excitement as a drug lead. However, this peptide is like most peptides susceptible to proteolysis in vivo, which potentially decreases its beneficial activities. In our continued effort to design a proteolytically stable ligand with specific receptor binding, we have engineered peptides by cyclizing ?-MSH using a thioether bridge. A number of novel cyclic truncated ?-MSH analogues were designed and synthesized, in which a thioether bridge was incorporated between a cysteine side chain and an N-terminal bromoacyl group. One of these peptides, cyclo-[(CH(2))(3)CO-Gly(1)-His(2)-D-Phe(3)-Arg(4)-D-Trp(5)-Cys(S-)(6)]-Asp(7)-Arg(8)-Phe(9)-Gly(10)-NH(2), demonstrated potent antagonist activity and receptor selectivity for the human melanocortin 1 receptor (hMC1R) (IC(50) = 17 nM). This novel peptide is the most selective antagonist for the hMC1R to date. Further pharmacological studies have shown that this peptide can specifically target melanoma cells. The nuclear magnetic resonance analysis of this peptide in a membrane-like environment revealed a new turn structure, specific to the hMC1R antagonist, at the C-terminus, where the side chain and backbone conformation of D-Trp(5) and Phe(9) of the peptide contribute to hMC1R selectivity. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

SUBMITTER: Cai M

PROVIDER: S-EPMC3641192 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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An unusual conformation of γ-melanocyte-stimulating hormone analogues leads to a selective human melanocortin 1 receptor antagonist for targeting melanoma cells.

Cai Minying M Stankova Magda M Muthu Dhanasekaran D Mayorov Alexander A Yang Zhehui Z Trivedi Devendra D Cabello Christopher C Hruby Victor J VJ

Biochemistry 20130115 4

γ-MSH (γ-melanocyte-stimulating hormone, H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH), with its exquisite specificity and potency, has recently created much excitement as a drug lead. However, this peptide is like most peptides susceptible to proteolysis in vivo, which potentially decreases its beneficial activities. In our continued effort to design a proteolytically stable ligand with specific receptor binding, we have engineered peptides by cyclizing γ-MSH using a thioether bridge. A ...[more]

PMID: 23276279

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Project description:BackgroundAlthough a 3-arm DOTA construct, which has three carboxylic acids, h has been applied for conjugation to many peptides, we investigated if a 4-arm DOTA construct conjugated to peptides improves chemical properties for melanoma imaging of the melanocortin 1 receptor compared to 3-arm DOTA-conjugated peptides.MethodsSpecific activities, radiolabeling efficiencies, and partition coefficients were evaluated using 111In-labeled 3-arm and 4-arm DOTA-α-melanocyte-stimulating hormone (MSH). For assessment of MC1-R affinity and accumulation in tumor cells in vitro, B16-F1 melanoma and/or 4T1 breast cancer cells were incubated with 111In-labeled 3-arm and 4-arm DOTA-α-MSH with and without α-MSH as a substrate. The stability was evaluated using mouse liver homogenates and plasma. Biological distribution and whole-body single photon emission computed tomography imaging of 111In-labeled 3-arm and 4-arm DOTA-α-MSH were obtained using B16-F1 melanoma-bearing mice.ResultsSpecific activities and radiolabeling efficiencies of both radiotracers were about 1.2 MBq/nM and 90-95%, respectively. The partition coefficients were -0.28 ± 0.03 for 111In-labeled 3-arm DOTA-α-MSH and -0.13 ± 0.04 for 111In-labeled 4-arm DOTA-α-MSH. Although accumulation was significantly inhibited by α-MSH in B16-F1 cells, the inhibition rate of 111In-labeled 4-arm DOTA-α-MSH was lower than that of 111In-labeled 3-arm DOTA-α-MSH. 111In-labeled 4-arm DOTA-α-MSH was taken up early into B16-F1 cells and showed higher accumulation than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation. Although these stabilities were relatively high, the stability of 111In-labeled 4-arm DOTA-α-MSH was higher than that of 111In-labeled 3-arm DOTA-α-MSH. Regarding biological distribution, 111In-labeled 4-arm DOTA-α-MSH showed significantly lower average renal accumulation (1.38-fold) and significantly higher average melanoma accumulation (1.32-fold) than 111In-labeled 3-arm DOTA-α-MSH at all acquisition times. 111In-labeled 4-arm DOTA-α-MSH showed significantly higher melanoma-to-kidney, melanoma-to-blood, and melanoma-to-muscle ratios than 111In-labeled 3-arm DOTA-α-MSH.ConclusionsThe 4-arm DOTA construct has better chemical properties for peptide radiotracers than the 3-arm DOTA construct.

Project description:Key pointsAlpha-melanocyte stimulating hormone (α-MSH) is an anorexigenic peptide. Injection of the α-MSH analog MTII into the ventral tegmental area (VTA) decreases food and sucrose intake and food reward. Melanocortin-3 receptors (MC3R) are highly expressed in the VTA, suggesting that the effects of intra-VTA α-MSH may be mediated by α-MSH changing the activity of MC3R-expressing VTA neurons. α-MSH increased the firing rate of MC3R VTA neurons in acute brain slices from mice, although it did not affect the firing rate of non-MC3R VTA neurons. The α-MSH induced increase in MC3R neuron firing rate is probably activity-dependent, and was independent of fast synaptic transmission and intracellular Ca2+ levels. These results help us to better understand how α-MSH acts in the VTA to affect feeding and other dopamine-dependent behaviours.AbstractThe mesocorticolimbic dopamine system, the brain's reward system, regulates multiple behaviours, including food intake and food reward. There is substantial evidence that the melanocortin system of the hypothalamus, an important neural circuit controlling feeding and body weight, interacts with the mesocorticolimbic dopamine system to affect feeding, food reward and body weight. For example, melanocortin-3 receptors (MC3Rs) are expressed in the ventral tegmental area (VTA) and our laboratory previously showed that intra-VTA injection of the MC3R agonist, MTII, decreases home-cage food intake and operant responding for sucrose pellets. However, the cellular mechanisms underlying the effects of intra-VTA alpha-melanocyte stimulating hormone (α-MSH) on feeding and food reward are unknown. To determine how α-MSH acts in the VTA to affect feeding, we performed electrophysiological recordings in acute brain slices from mice expressing enhanced yellow fluorescent protein in MC3R neurons to test how α-MSH affects the activity of VTA MC3R neurons. α-MSH significantly increased the firing rate of VTA MC3R neurons without altering the activity of non-MC3R expressing VTA neurons. In addition, the α-MSH-induced increase in MC3R neuron activity was independent of fast synaptic transmission and intracellular Ca2+ levels. Finally, we show that the effect of α-MSH on MC3R neuron firing rate is probably activity-dependent. Overall, these studies provide an important advancement in the understanding of how α-MSH acts in the VTA to affect feeding and food reward.

Project description:It is estimated that melanoma accounted for 76,380 new cases and 10,130 deaths in the United States in 2016. The melanocortin 1 receptor (MC1R) is highly expressed in the vast majority of melanomas, which makes it an attractive target for molecular imaging and radionuclide therapy. Lactam bridge-cyclized ?-melanocyte-stimulating hormone (Ac-Nle4-cyclo[Asp5-His-D-Phe7-Arg-Trp-Lys10]-NH2, or Nle-CycMSHhex) analogues have been successfully developed and studied for MC1R-targeted imaging, predominantly with single-photon emission computed tomography (SPECT). The goal of this study was to design and evaluate novel peptides for melanoma imaging with positron emission tomography (PET). We designed and synthesized three peptides, DOTA-PEG2-Nle-CycMSHhex (CCZ01047), DOTA-4-amino-(1-carboxymethyl) piperidine (Pip)-Nle-CycMSHhex (CCZ01048), and DOTA-Pip-Pip-Nle-CycMSHhex (CCZ01056). All three peptides exhibited high binding affinity to MC1R with sub-nanomolar Ki values, rapid internalization into B16F10 melanoma cells and high in vivo stability with more than 93% remaining intact at 15 min post-injection (p.i.) in blood plasma. All three 68Ga-labeled tracers produced high contrast PET images in C57BL/6J mice bearing B16F10 tumors, and their respective tumor uptakes were 8.0 ± 3.0, 12.3 ± 3.3, and 6.5 ± 1.4 %ID/g at 1 h p.i. Minimal normal organ activity was observed at 1 h p.i., except for kidneys (5.1 ± 1.4, 4.7 ± 0.5, and 6.2 ± 2.0 %ID/g, respectively), and thyroid (4.1 ± 0.6 %ID/g for CCZ01047 and 2.4 ± 0.6 %ID/g for CCZ01048). Due to high accumulation at tumor sites and rapid background clearance of 68Ga-CCZ01048, we further evaluated it at 2 h p.i., and a tumor uptake of 21.9 ± 4.6 %ID/g was observed, with background activity further decreased. Exceptional image contrast was also achieved, i.e. tumor-to-blood, tumor-to-muscle, tumor-to-bone and tumor-to-kidney ratios were 96.4 ± 13.9, 210.9 ± 20.9, 39.6 ± 11.9 and 4.0 ± 0.9, respectively. A blocking study was also performed by co-injection of excess amount of non-radioactive Ga-coupled of CCZ01048, which confirmed that the tumor uptake was MC1R mediated. In conclusion, the introduction of a cationic Pip linker to Nle-CycMSHhex, CCZ01048, not only improved tumor uptake, but also generated high tumor-to-normal tissue contrast with PET imaging in a preclinical melanoma model. Therefore, CCZ01048 is a promising candidate for PET imaging of melanoma, and potentially as a theranostic agent for radionuclide therapy of melanoma when labeled with ? or ? emitters.

Project description:BackgroundThe ?-Melanocyte Stimulating Hormone (?MSH)/Melanocortin-1 receptor (MC1R) interaction promotes melanogenesis through the cAMP/PKA pathway. The direct induction of this pathway by Forskolin (FSK) is also known to enhance melanocyte proliferation. ?MSH acts as a mitogenic agent in melanocytes and its effect on proliferation of melanoma cells is less known. We previously identified the ?MSH/Peroxisome Proliferator Activated Receptor (PPAR?) pathway as a new pathway on the B16-F10 mouse melanoma cell line. ?MSH induced the translocation of PPAR? into the nucleus as an active transcription factor. This effect was independent of the cAMP/PKA pathway and was mediated by the activation of the PI(4,5)P2/PLC pathway, a pathway which we have described to be triggered by the ?MSH-dependent MC1R stimulation. Moreover, in the same study, preliminary experiments showed that mouse melanoma cells responded to ?MSH by reducing proliferation and that PPAR? was involved in this effect. Due to its key role in the control of cell proliferation, PPAR? agonists are used in therapeutic models for different forms of cancer, including melanoma. The purpose of this study was: (a) to confirm the different proliferative behavior in response to ?MSH in healthy and in melanoma condition; (b) to verify whether the cAMP/PKA pathway and the PLC/PPAR? pathway could exert an antagonistic function in the control of proliferation; (c) to deepen the knowledge of the molecular basis responsible for the down-proliferative response of melanoma cells after exposure to ?MSH.MethodsWe employed B16-F10 cell line, a human melanoma cell line (Mel 13) and two primary cultures of human melanocytes (NHM 1 and NHM 2, respectively), all expressing a wild type MC1R and responding to the ?MSH in terms of pigmentation. We evaluated cell proliferation through: a) cell counting, b) cell cycle analysis c) protein expression of proliferation modulators (p27, p21, cyclin D1 and cyclin E).ResultsThe ?MSH acted as a mitogenic agent in primary cultures of human melanocytes, whereas it determined a slow down of proliferation in melanoma cell lines. FSK, as an inducer of the cAMP/PKA pathway, reproduced the ?MSH mediated effect on proliferation in NHMs but it did not mimic the ?MSH effect on proliferation in B16-F10 and Mel 13 melanoma cell lines. Meanwhile, 3 M3-FBS (3 M3), as an inducer of PI(4,5)P2/PLC pathway, reproduced the ?MSH proliferative effect. Further experiments, treating melanoma cell lines with ?MSH in the presence/absence of GW9662, as an inhibitor of PPAR?, confirmed the key role of this transcription factor in decreasing cell proliferation in response to the hormone exposure.ConclusionsIn both melanoma cell lines, ?MSH determined the reduction of proliferation through the PI(4,5)P2/PLC pathway, employing PPAR? as an effector element. These evidence could offer perspectives for new therapeutic approaches for melanoma.

Dataset Information

An unusual conformation of ?-melanocyte-stimulating hormone analogues leads to a selective human melanocortin 1 receptor antagonist for targeting melanoma cells.

Publications

An unusual conformation of γ-melanocyte-stimulating hormone analogues leads to a selective human melanocortin 1 receptor antagonist for targeting melanoma cells.

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