Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling.

Thermal proteome profiling with label-free quantitation using ion-mobility-enhanced LC-MS offers versatile data sets, providing information on protein differential expression, thermal stability, and the activities of transcription factors. We developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments that incorporates the aspects of gene set enrichment analysis, differential protein expression analysis, and inference of transcription factor activities from LC-MS data. We applied it to study the signaling processes downstream of melanocortin 3 receptor (MC3R) activation by endogenous agonists derived from the proopiomelanocortin prohormone: ACTH, α-MSH, and γ-MSH. The obtained information was used to map signaling pathways downstream of MC3R and to deduce transcription factors responsible for cellular response to ligand treatment. Using our workflow, we identified differentially expressed proteins and investigated their thermal stability. We found in total 298 proteins with altered thermal stability, resulting from MC3R activation. Out of these, several proteins were transcription factors, indicating them as being downstream target regulators that take part in the MC3R signaling cascade. We found transcription factors CCAR2, DDX21, HMGB2, SRSF7, and TET2 to have altered thermal stability. These apparent target transcription factors within the MC3R signaling cascade play important roles in immune responses. Additionally, we inferred the activities of the transcription factors identified in our data set. This was done with Bayesian statistics using the differential expression data we obtained with label-free quantitative LC-MS. The inferred transcription factor activities were validated in our bioinformatic pipeline by the phosphorylated peptide abundances that we observed, highlighting the importance of post-translational modifications in transcription factor regulation. Our multidimensional data analysis workflow allows for a comprehensive characterization of the signaling processes downstream of MC3R activation. It provides insights into protein differential expression, thermal stability, and activities of key transcription factors. All proteomic data generated in this study are publicly available at DOI: 10.6019/PXD039945.

Effects of Polyethylene Glycol and 8-Aminooctanoic Acid Linkers on Melanoma Uptake of [99mTc]Tc-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized α-MSH Peptides.

The purpose of this study was to evaluate the effect of linkers on tumor targeting and biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex {[99mTc]Tc(CO)3-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-d-Phe-Arg-Trp-Lys]-CONH2} and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex {[99mTc]Tc(CO)3-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on B16/F10 melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and radiolabeled with [99mTc]Tc via the {[99mTc]Tc(CO)3(OH2)3}+ intermediate. The biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiochemical yields and exhibited MC1R-specific binding on B16/F10 melanoma cells. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex exhibited a higher tumor uptake than [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2, 4, and 24 h postinjection. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 13.63 ± 1.13, 31.93 ± 2.57, 20.31 ± 3.23, and 1.33 ± 0.15% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 1.6 and 3.4 times the tumor uptake of [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2 and 4 h postinjection, respectively. Meanwhile, the normal organ uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was lower than 1.8% ID/g at 2 h postinjection. The renal uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was only 1.73 ± 0.37, 0.73 ± 0.14, and 0.03 ± 0.01% ID/g at 2, 4, and 24 h postinjection, respectively. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios at 2 h postinjection. Single-photon emission computed tomography imaging revealed that the B16/F10 melanoma lesions could be clearly visualized by [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex at 2 h postinjection. Overall, the high tumor uptake and low kidney uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex highlighted its potential for melanoma imaging and warranted the future evaluation of [188Re]Re(CO)3-NOTA-PEG2Nle-CycMSHhex for melanoma therapy.

Setmelanotide optimization through fragment-growing, molecular docking in-silico method targeting MC4 receptor.

Obesity has emerged as a global issue, but with the complex structures of multiple related important targets and their agonists or antagonists determined, the mechanism of ligand-protein interaction may offer new chances for developing new generation agonists anti-obesity. Based on the molecule surface of the cryo-EM protein structure 7AUE, we tried to replace D-Ala3 with D-Met in setmelanotide as the linker site for fragment-growing with De novo evolution. The simulation results indicate that the derivatives could improve the binding abilities with the melanocortin 4 receptor and the selectivity over the melanocortin 1 receptor. The improved selectivity of the newly designed derivatives is mainly due to the shape difference of the molecular surface at the orthosteric peptide-binding pocket between melanocortin 4 receptor and melanocortin 1 receptor. The new extended fragments could not only enhance the binding affinities but also function as a gripper to seize the pore, making it easier to balance and stabilize the other component of the new derivatives. Although it is challenging to synthesize the compounds designed in silico, this study may perhaps serve as a trigger for additional anti-obesity research.Communicated by Ramaswamy H. Sarma.

61Cu-Labelled radiodiagnostics of melanoma with NAPamide-targeted radiopharmaceutical.

Malignant melanoma is a major public health problem with an increasing incidence and mortality in the Caucasian population due to its significant metastatic potential. The early detection of this cancer type by imaging techniques like positron emission tomography acts as an important contributor to the long-term survival. Based on literature data, the radio labelled alpha-MSH analog NAPamide molecule is an appropriate diagnostic tool for the detection of melanoma tumors. Inspired by these facts, a new radiotracer, the [61Cu]Cu-KFTG-NAPamide has been synthesized to exploit the beneficial features of the positron emitter 61Cu and the melanoma specificity of the NAPamide molecule. In this work, we report a new member of the CB-15aneN5 ligand family (KFTG) as the chelator for 61Cu(II) complexation. On the basis of the thorough physico-chemical characterization, the rigid [Cu(KFTG)]+ complex exhibits fast complex formation (t1/2 = 155 s at pH 5.0 and 25 °C) and high inertness (t1/2 = 2.0 h in 5.0 M HCl at 50 °C) as well as moderate superoxide dismutase activity (IC50 = 2.3 µM). Furthermore, the [61Cu]Cu-KFTG-NAPamide possesses outstanding features in the diagnostics of B16-F10 melanoma tumors by PET imaging: (T/M(SUVs) (in vivo): appr. 14, %ID/g: 7 ± 1 and T/M (ex vivo): 315 ± 24 at 180 min).

The c.612A>G mutation of MC4R affects constitutive activity and signaling in domestic goats.

As a key gene for balancing energy and regulating feeding behavior, MC4R is relevant to the growth of ruminants. In this presentation, a highly conserved c.612A>G site in the coding sequence (CDS) of MC4R has been selected during a selective sweep analysis of 35 Yiling goats and 20 other wild goats. This site mutation results in an amino acid change from Ile to Met. The genotyping analysis of the c.612A>G site revealed that the A allele was the dominant allele in the domestic goat populations, while the wild goat individuals only had the G allele. For a better understanding of the biological significance of this site, we examined the protein localization and signal detection to explain the function of the two MC4R receptors. The results showed that both the M204 and I204 receptors can normally localize on the membrane. When stimulating the M204 type without α-MSH, it was defective at the level of basal cAMP and decreased significantly against the I204 type. In contrast, the signaling capacity of the M204 receptor was also lower than that of I204 under the stimulation of α-MSH. In the ERK1/2 pathway, stimulating MC4R with NDP-α-MSH, both the M204 and I204 receptors had normal pERK1/2 levels. These results indicate that the p.I204M mutation may change the function by damaging the constitutive activity and signaling, and thus may regulate goats' appetite. This study has potential application for rearing domestic goats.

Novel Al18F-NOTA-Conjugated Lactam-Cyclized α-Melanocyte-Stimulating Hormone Peptides with Enhanced Melanoma Uptake.

The purpose of this study was to evaluate the effect of linker on tumor targeting and biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex {Al18F-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-poly(ethylene glycol)-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and Al18F-NOTA-AocNle-CycMSHhex {Al18F-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of Al18F-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake and lower renal uptake than that of Al18F-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2/AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex were readily prepared with more than 55% of radiolabeling yields and displayed melanocortin-1 receptor (MC1R)-specific binding on B16/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake and lower kidney and liver uptake than Al18F-NOTA-AocNle-CycMSHhex at 1 and 2 h post injection. The tumor and renal uptakes of Al18F-NOTA-PEG2Nle-CycMSHhex were 17.44 ± 0.76 and 2.07 ± 0.43% ID/g at 1 h post injection, respectively. Al18F-NOTA-PEG2Nle-CycMSHhex showed the high tumor to normal organ uptake ratios after 1 h post injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using Al18F-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 1 and 2 h post injection. Overall, high tumor uptake, low kidney and liver uptake, and fast urinary clearance of Al18F-NOTA-PEG2Nle-CycMSHhex highlighted its potential as an MC1R-targeted imaging probe for melanoma detection.

Novel 64Cu-Labeled NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptides with Enhanced Tumor to Kidney Uptake Ratios.

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of 64Cu-NOTA-PEG2Nle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 64Cu-NOTA-AocNle-CycMSHhex {64Cu-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and purified by HPLC. The melanocortin-1 (MC1) receptor binding affinities of the peptides were examined on B16/F10 melanoma cells. The biodistributions of 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of 64Cu-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake than 64Cu-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 melanoma cells. 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiolabeling yields and showed MC1R-specific binding on B16/F10 cells. 64Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake than 64Cu-NOTA-AocNle-CycMSHhex at 0.5, 2, 4, and 24 h post-injection. The tumor uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 16.23 ± 0.42, 19.59 ± 1.48, 12.83 ± 1.69, and 8.78 ± 2.29% ID/g at 0.5, 2, 4, and 24 h post-injection, respectively. Normal organ uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2% ID/g at 2 h post-injection except for kidney uptake. The renal uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 3.66 ± 0.52, 3.27 ± 0.52, and 1.47 ± 0.56 ID/g at 2, 4, and 24 h post-injection, respectively. 64Cu-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using 64Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 2 h post-injection. High tumor uptake and low kidney uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted theranostic peptide for melanoma imaging and therapy.

[213Bi]Bi3+/[111In]In3+-neunpa-cycMSH: Theranostic Radiopharmaceutical Targeting Melanoma─Structural, Radiochemical, and Biological Evaluation.

With the emergence of [225Ac]Ac3+ as a therapeutic radionuclide for targeted α therapy (TAT), access to clinical quantities of the potent, short-lived α-emitter [213Bi]Bi3+ (t1/2 = 45.6 min) will increase over the next decade. With this in mind, the nonadentate chelator, H4neunpa-NH2, has been investigated as a ligand for chelation of [213Bi]Bi3+ in combination with [111In]In3+ as a suitable radionuclidic pair for TAT and single photon emission computed tomography (SPECT) diagnostics. Nuclear magnetic resonance (NMR) spectroscopy was utilized to assess the coordination characteristics of H4neunpa-NH2 on complexation of [natBi]Bi3+, while the solid-state structure of [natBi][Bi(neunpa-NH3)] was characterized via X-ray diffraction (XRD) studies, and density functional theory (DFT) calculations were performed to elucidate the conformational geometries of the metal complex in solution. H4neunpa-NH2 exhibited fast complexation kinetics with [213Bi]Bi3+ at RT achieving quantitative radiolabeling within 5 min at 10-8 M ligand concentration, which was accompanied by the formation of a kinetically inert complex. Two bioconjugates incorporating the melanocortin 1 receptor (MC1R) targeting peptide Nle-CycMSHhex were synthesized featuring two different covalent linkers for in vivo evaluation with [213Bi]Bi3+ and [111In]In3+. High molar activities of 7.47 and 21.0 GBq/µmol were achieved for each of the bioconjugates with [213Bi]Bi3+. SPECT/CT scans of the [111In]In3+-labeled tracer showed accumulation in the tumor over time, which was accompanied by high liver uptake and clearance via the hepatic pathway due to the high lipophilicity of the covalent linker. In vivo biodistribution studies in C57Bl/6J mice bearing B16-F10 tumor xenografts showed good tumor uptake (5.91% ID/g) at 1 h post-administration with [213Bi][Bi(neunpa-Ph-Pip-Nle-CycMSHhex)]. This study demonstrates H4neunpa-NH2 to be an effective chelating ligand for [213Bi]Bi3+ and [111In]In3+, with promising characteristics for further development toward theranostic applications.

CLIPSing Melanotan-II to Discover Multiple Functionally Selective hMCR Agonists.

The pleiotropic role played by melanocortin receptors (MCRs) in both physiological and pathological processes has stimulated medicinal chemists to develop synthetic agonists/antagonists with improved potency and selectivity. Here, by deploying the Chemical Linkage of Peptide onto Scaffolds strategy, we replaced the lactam cyclization of melanotan II (MT-II), a potent and unselective agonist of human MCRs (hMCRs), with different xylene-derived thioethers. The newly designed peptides displayed binding affinities toward MCRs ranging from the low nanomolar to the sub-micromolar range, highlighting a correlation between the explored linkers and the affinity toward hMCRs. In contrast to the parent peptide (MT-II), compound 5 displayed a remarkable functional selectivity toward the hMC1R. Enhanced sampling molecular dynamics simulations were found to be instrumental in outlining how the employed cyclization strategy affects the peptides' conformational behavior and, as a consequence, the detected hMC1R affinity. Additionally, a model of the peptide 5/hMC1R complex employing the very recently reported cryogenic electron microscopy receptor structure was provided.

Binding of cationic analogues of α-MSH to lipopolysaccharide and disruption of the cytoplasmic membranes caused bactericidal action against Escherichia coli.

In earlier reports, we have shown the antimicrobial activity of a host neuropeptide, alpha-melanocyte stimulating hormone (α-MSH) and its cationic analogues against Staphylococcus aureus. These analogues of α-MSH showed enhanced staphylocidal activity without any significant mammalian cell toxicity. Therefore, here, we explored the antimicrobial activity of α-MSH and its cationic analogues against Escherichia coli. Though the presence of lipopolysaccharide (LPS) in Gram-negative bacteria enables them to resist most conventional antibiotics, encouragingly α-MSH and its four analogues showed killing of both logarithmic and stationary phase E. coli cells in a time, dose and cationicity-dependent manner. In fact, the most cationic analogue, KKK-MSH with a + 5 charge, demonstrated successful eradication of 105 CFU/mL of E. coli cells within 15 min at a concentration as low as 1 µM. BC displacement experiment revealed that cationicity of the peptides was directly related to the killing efficacy of these α-MSH analogues against E. coli cells via initial LPS-binding, leading to rapid disruption of the LPS-outer membrane complex followed by inner bacterial membrane damage and eventual cell death. Here, we propose α-MSH based cationic peptides as promising future agents with broad-spectrum antibacterial efficacy against both Gram-negative and Gram-positive pathogens.

The Effect of Albumin-Binding Moiety on Tumor Targeting and Biodistribution Properties of 67Ga-Labeled Albumin Binder-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

Background: The purpose of this study was to examine the effect of 4-p-(tolyl)butyric acid as an albumin-binding (ALB) moiety on tumor targeting and biodistribution properties of 67Ga-labeled albumin binder-conjugated alpha-melanocyte-stimulating hormone peptides. Materials and Methods: DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(ALB)-Gly/GlyGly/GlyGlyGly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} were synthesized with 4-p-(tolyl)butyric acid serving as an ALB moiety. The melanocortin-1 receptor (MC1R)-binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 67Ga-DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex was examined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 67Ga-DOTA-Lys(ALB)-GGNle-CycMSHhex {67Ga-ALB-G2} were determined on B16/F10 melanoma-bearing C57 mice. Results: The IC50 value of DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {ALB-G1, ALB-G2, ALB-G3} was 0.67 ± 0.07, 0.5 ± 0.09 and 0.51 ± 0.03 nM on B16/F10 cells, respectively. 67Ga-ALB-G2 was further evaluated as a lead peptide because of its higher tumor uptake (30.25 ± 3.24%ID/g) and lower kidney uptake (7.09 ± 2.22%ID/g) than 67Ga-ALB-G1 and 67Ga-ALB-G3 at 2 h postinjection. The B16/F10 melanoma uptake of 67Ga-ALB-G2 was 15.64 ± 4.55, 30.25 ± 3.24, 26.76 ± 3.23, and 10.71 ± 1.21%ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 67Ga-ALB-G2 as an imaging probe at 2 h postinjection. Conclusions: The introduction of 4-p-(tolyl)butyric acid as an ALB moiety increased the blood retention, and resulted in higher tumor/kidney ratio of 67Ga-ALB-G2 as compared with its counterpart without an albumin binder. However, the resulting high uptake of 67Ga-ALB-G2 in blood and liver need to be further reduced to facilitate its therapeutic application when replacing 67Ga with therapeutic radionuclides.

Zebrafish Bioassay for Screening Therapeutic Candidates Based on Melanotrophic Activity.

In this study, we used the zebrafish animal model to establish a bioassay by which physiological efficacy differential of alpha-melanocyte-stimulating hormone (α-MSH) analogues could be measured by melanosome dispersion in zebrafish larvae. Brain-skin connection research has purported the interconnectedness between the nervous system and skin physiology. Accordingly, the neuropeptide α-MSH is a key regulator in several physiological processes, such as skin pigmentation in fish. In mammals, α-MSH has been found to regulate motivated behavior, appetite, and emotion, including stimulation of satiety and anxiety. Several clinical and animal model studies of autism spectrum disorder (ASD) have already demonstrated the effectiveness of α-MSH in restoring the social deficits of autism. Therefore, we sought to analyze the effect of synthetic and naturally-occurring α-MSH variants amongst different species. Our results showed that unique α-MSH derivatives from several fish species produced differential effects on the degree of melanophore dispersion. Using α-MSH human form as a standard, we could identify derivatives that induced greater physiological effects; particularly, the synthetic analogue melanotan-II (MT-II) exhibited a higher capacity for melanophore dispersion than human α-MSH. This was consistent with previous findings in an ASD mouse model demonstrating the effectiveness of MT-II in improving ASD behavioral symptoms. Thus, the melanophore assay may serve as a useful screening tool for therapeutic candidates for novel drug discovery.

Structure reveals the activation mechanism of the MC4 receptor to initiate satiation signaling.

Obesity is a global epidemic that causes morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human MC4R-Gs signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. In addition, our findings indicate that calcium (Ca2+) is required for agonist, but not antagonist, efficacy. These results fill a gap in the understanding of MC4R activation and could guide the design of future weight-management drugs.

Melanocortin receptor activation alleviates amyloid pathology and glial reactivity in an Alzheimer's disease transgenic mouse model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder with no disease-modifying treatment. AD progression is characterized by cognitive decline, neuroinflammation, and accumulation of amyloid-beta (Aß) and neurofibrillary tangles in the brain, leading to neuronal and glial dysfunctions. Neuropeptides govern diverse pathophysiological processes and represent key players in AD pathogenesis, regulating synaptic plasticity, glial cell functions and amyloid pathology. Activation of the pro-opiomelanocortin (POMC)-derived neuropeptide and its receptor from the melanocortin receptor (MCR) family have previously been shown to rescue the impairment in hippocampus-dependent synaptic plasticity in the APP/PS1 mouse model of AD. However, the functional roles of MCR signaling in AD conditions, particularly in glial functions, are largely unknown. In this study, we investigated the potential benefits of MCR activation in AD. In APP/PS1 transgenic mice, we demonstrate that MCR activation mediated by the central administration of its agonist D-Tyr MTII substantially reduces Aß accumulation, while alleviating global inflammation and astrocytic activation, particularly in the hippocampus. MCR activation prominently reduces the A1 subtype of reactive astrocytes, which is considered a key source of astrocytic neurotoxicity in AD. Concordantly, MCR activation suppresses microglial activation, while enhancing their association with amyloid plaques. The blunted activation of microglia may contribute to the reduction in the neurotoxic phenotypes of astrocytes. Importantly, transcriptome analysis reveals that MCR activation restores the impaired homeostatic processes and microglial reactivity in the hippocampus in APP/PS1 mice. Collectively, our findings demonstrate the potential of MCR signaling as therapeutic target for AD.

Setmelanotide: First Approval.

Setmelanotide (IMCIVREE™, Rhythm Pharmaceuticals) is a melanocortin-4 (MC4) receptor agonist developed for the treatment of obesity arising from proopiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency. The drug has received its first approval in the USA for chronic weight management in patients 6 years and older with obesity caused by POMC, PCSK1 and LEPR deficiency and has been granted PRIority MEdicines (PRIME) designation by the European Medicines Agency for the treatment of obesity and the control of hunger associated with deficiency disorders of the MC4 receptor pathway. Setmelanotide is also being developed in other rare genetic disorders associated with obesity including Bardet-Biedl Syndrome, Alström Syndrome, POMC and other MC4R pathway heterozygous deficiency obesities, and POMC epigenetic disorders. This article summarizes the milestones in the development of setmelanotide leading to this first approval for obesity caused by POMC, PCSK1 and LEPR deficiency.

Facile preparation of a novel Ga-67-labeled NODAGA-conjugated lactam-cyclized alpha-MSH peptide at room temperature for melanoma targeting.

In this study, the melanoma targeting property of 67Ga-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-D-Phe-Arg-Trp-Lys]-CONH2} was determined on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NODAGA as a radiometal chelator for facile room temperature radiolabeling of NODAGA-GGNle-CycMSHhex. The IC50 value of NODAGA-GGNle-CycMSHhex was 0.87 ± 0.12 nM on B16/F10 melanoma cells. 67Ga-NODAGA-GGNle-CycMSHhex was readily prepared at room temperature with greater than 98% radiolabeling yield and displayed MC1R-specific binding on B16/F10 melanoma cells. The B16/F10 melanoma uptake of 67Ga-NODAGA-GGNle-CycMSHhex was 10.31 ± 0.78, 14.96 ± 1.34, 13.7 ± 3.33 and 10.4 ± 2.2% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 85% of the injected dose was cleared out the body via urinary system at 2 h post-injection. 67Ga-NODAGA-GGNle-CycMSHhex showed high tumor/blood, tumor/muscle and tumor/skin uptake ratios after 2 h post-injection. Overall, 67Ga-NODAGA-GGNle-CycMSHhex could be easily prepared at room temperature and exhibited favorable melanoma targeting property, suggesting the potential use of NODAGA as a radiometal chelator for facile room temperature radiolabeling of α-MSH peptides.

Structural Complexity and Plasticity of Signaling Regulation at the Melanocortin-4 Receptor.

The melanocortin-4 receptor (MC4R) is a class A G protein-coupled receptor (GPCR), essential for regulation of appetite and metabolism. Pathogenic inactivating MC4R mutations are the most frequent cause of monogenic obesity, a growing medical and socioeconomic problem worldwide. The MC4R mediates either ligand-independent or ligand-dependent signaling. Agonists such as α-melanocyte-stimulating hormone (α-MSH) induce anorexigenic effects, in contrast to the endogenous inverse agonist agouti-related peptide (AgRP), which causes orexigenic effects by suppressing high basal signaling activity. Agonist action triggers the binding of different subtypes of G proteins and arrestins, leading to concomitant induction of diverse intracellular signaling cascades. An increasing number of experimental studies have unraveled molecular properties and mechanisms of MC4R signal transduction related to physiological and pathophysiological aspects. In addition, the MC4R crystal structure was recently determined at 2.75 Å resolution in an inactive state bound with a peptide antagonist. Underpinned by structural homology models of MC4R complexes simulating a presumably active-state conformation compared to the structure of the inactive state, we here briefly summarize the current understanding and key players involved in the MC4R switching process between different activity states. Finally, these perspectives highlight the complexity and plasticity in MC4R signaling regulation and identify gaps in our current knowledge.

Alpha-MSH Targeted Liposomal Nanoparticle for Imaging in Inflammatory Bowel Disease (IBD).

BACKGROUND: The purpose of our study was to find a novel targeted imaging and drug delivery vehicle for inflammatory bowel disease (IBD). IBD is a common and troublesome disease that still lacks effective therapy and imaging options. As an attempt to improve the disease treatment, we tested αMSH for the targeting of nanoliposomes to IBD sites. αMSH, an endogenous tridecapeptide, binds to the melanocortin-1 receptor (MC1-R) and has anti-inflammatory and immunomodulating effects. MC1-R is found on macrophages, neutrophils and the renal tubule system. We formulated and tested a liposomal nanoparticle involving αMSH in order to achieve a specific targeting to the inflamed intestines. METHODS: NDP-αMSH peptide conjugated to Alexa Fluor™ 680 was linked to the liposomal membrane via NSuccinyl PE and additionally loaded into the lumen of the liposomes. Liposomes without the αMSH-conjugate and free NDP-αMSH were used as a control. The liposomes were also loaded with ICG to track them. The liposomes were tested in DSS treated mice, which had received DSS via drinking water order to develop a model IBD. Inflammation severity was assessed by the Disease Activity Index (DAI) score and ex vivo histological CD68 staining of samples taken from different parts of the intestine. The liposome targeting was analyzed by analyzing the ICG and ALEXA 680 fluorescence in the intestine compared to the biodistribution. RESULTS: NPD-αMSH was successfully labeled with Alexa and retained its biological activity. Liposomes were identified in expected regions in the inflamed bowel regions and in the kidneys, where MC1-R is abundant. In vivo liposome targeting correlated with the macrophage concentration at the site of the inflammation supporting the active targeting of the liposomes through αMSH. The liposomal αMSH was well tolerated by animals. CONCLUSION: This study opens up the possibility to further develop an αMSH targeted theranostic delivery to different clinically relevant applications in IBD inflammation but also opens possibilities for use in other inflammations like lung inflammation in Covid 19.

Novel [99mTc]-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized Alpha-MSH Peptide with Enhanced Melanoma Uptake and Reduced Renal Uptake.

The purpose of this study was to examine the melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex {1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NOTA/NODAGA as metal chelators for 99mTc(CO)3+ radiolabeling. NOTA/NODAGA-GGNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The IC50 values of NOTA/NODAGA-GGNle-CycMSHhex were 0.8 ± 0.1 and 0.9 ± 0.1 nM on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were readily prepared via the [99mTc(CO)3(OH2)3]+ intermediate and displayed MC1R-specific binding on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex was further evaluated as a lead peptide because of its higher tumor uptake (19.76 ± 3.62% ID/g) and lower kidney uptake (1.59 ± 0.52% ID/g) at 2 h postinjection than 99mTc(CO)3-NODAGA-GGNle-CycMSHhex. The B16/F10 melanoma uptake of 99mTc(CO)3-NOTA-GGNle-CycMSHhex was 16.07 ± 4.47, 19.76 ± 3.62, 11.30 ± 2.81, and 3.16 ± 2.28% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. 99mTc(CO)3-NOTA-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h postinjection. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 99mTc(CO)3-NOTA-GGNle-CycMSHhex as an imaging probe at 2 h postinjection. High tumor uptake, low kidney uptake, and fast urinary clearance of 99mTc(CO)3-NOTA-GGNle-CycMSHhex highlighted its potential for melanoma imaging and facilitated the evaluation of 188Re(CO)3-NOTA-GGNle-CycMSHhex for melanoma therapy.

Molecular determinants of ACTH receptor for ligand selectivity.

The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. ACTH receptor is a subtype of the melanocortin receptor family which is a member of the G-protein coupled receptor (GPCR) superfamily. ACTH receptor has unique characteristics among MCRs. α-MSH, ß-MSH, γ-MSH and ACTH are agonists for MCRs but only ACTH is the agonist for ACTH receptor. In addition, the melanocortin receptor accessory protein (MRAP) is required for ACTH receptor expression at cell surface and function. In this review, we summarized the information available on the relationship between ACTH and ACTH receptor and provide the latest understanding of the molecular basis of the ACTH receptor responsible for ligand selectivity and function.