Melanocortin Receptor Research Articles

A novel pathogenic variant in MRAP2 in an obese patient with successful outcome of bariatric surgery.

Mutations in genes encoding proteins located in the leptin/melanocortin pathway have been identified in the rare cases of genetic obesities. Heterozygous variants of MRAP2, encoding a G coupled-protein receptor accessory protein implicated in energy control notably via the melanocortin-4 receptor, have been recently identified. A 24-year-old patient with early-onset severe obesity (body mass index [BMI]: 64 kg/m2) associated with hypertension, respiratory complications, nonalcoholic fatty liver disease, and type 2 diabetes was referred to our department. Sleeve gastrectomy was successful. A new heterozygous variant in MRAP2 (NM_138409.4: c.154G>C/p.G52R) variant was identified in the patient DNA. Functional assessment confirmed that this new variant was pathogenic. We report a new pathogenic loss-of-function mutation in MRAP2 in a patient suffering from a severe multicomplicated obesity. This confirms the metabolic phenotype in patients with this monogenic form of obesity. Longer follow-up will be necessary. Our finding will allow a personalized medicine.

Novel Melanocortin-3 and -4 Receptor Functional Variants in Asian Children With Severe Obesity.

Genetic variants in melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) genes are strongly associated with childhood obesity. This study aims to identify and functionally characterize MC3R and MC4R variants in an Asian cohort of children with severe early-onset obesity. Whole-exome sequencing was performed to screen for MC3R and MC4R coding variants in 488 Asian children with severe early-onset obesity (body mass index for age ≥97th percentile). Functionality of the identified variants were determined via measurement of intracellular cyclic adenosine monophosphate (cAMP) concentrations and luciferase activity. Four MC3R and 2 MC4R heterozygous nonsynonymous rare variants were detected. There were 3 novel variants: MC3R c.151G > C (p.Val51Leu), MC4R c.127C > A (p.Gln43Lys), and MC4R c.272T > G (p.Met91Arg), and 3 previously reported variants: MC3R c.127G > A (p.Glu43Lys), MC3R c.97G > A (p.Ala33Thr), and MC3R c.437T > A (p.Ile146Asn). Both MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants demonstrated defective downstream cAMP signaling activity. The MC4R c.127C > A (p.Gln43Lys) variant showed reduced cAMP signaling activity at low substrate concentration but the signaling activity was restored at high substrate concentration. The MC3R c.151G > C (p.Val51Leu) variant did not show a significant reduction in cAMP signaling activity compared to wild-type (WT) MC3R. Coexpression studies of the WT and variant MC3R/MC4R showed that the heterozygous variants did not exhibit dominant negative effect. Our functional assays demonstrated that MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants might predispose individuals to early-onset obesity, and further studies are needed to establish the causative effect of these variants in the pathogenesis of obesity.

Computer screening of peptidomimetics and small-molecule ligands of B-cell membrane proteins for therapy of Burkitt lymphoma

The capabilities of molecular modelling and docking allow for the discovery of new potential drug agents to improve the treatment of diseases, which is a current concern. The objective of this study was to conduct in silico screening for antibody mimetics to B-cell membrane proteins for the treatment of Burkitt lymphoma through virtual screening. In this work, a standard protocol for structure-based virtual screening was employed, with the distinction that pharmacophores for screening were built not based on small-molecule ligands but on selected amino acid residues of antibodies. Based on literature data and the presence of a mechanism of direct cytotoxic action, as well as the availability of 3D structures of complexes, three monoclonal antibodies were selected: obinutuzumab, epratuzumab, and atezolizumab. The identification of biological targets was carried out by searching for 3D structures of selected complexes with target proteins in the Protein Data Bank. For virtual screening, the web service Pharmit was chosen. Using the Molecular Operating Environment program, pharmacophore models were constructed for three complexes: CD20 and obinutuzumab, CD22 and epratuzumab, and PD-L1 and atezolizumab. Docking with the CD20, CD22, and PD-L1 proteins was conducted at the binding sites recognised by the original antibody. Through in silico virtual screening using the Molecular Operating Environment software, a search for antibody mimetics to B-cell membrane proteins for Burkitt lymphoma treatment was conducted, resulting in the selection of 5 potential anti-lymphoma agents: CHEMBL505179 for the CD20 receptor, an antagonist of the melanocortin receptor for CD20 (PubChem-44406884), an inhibitor of blood clotting Factor Xa for CD22 (PubChem-136510605), and a blocker of epithelial Na+ channels for CD22 (PubChem-126761430), and an agonist of the melanocortin receptor for PD-L1 (PubChem-25078192). The obtained results can be applied in the pharmaceutical industry and oncological practice to enhance therapeutic outcomes in the treatment of patients with Burkitt lymphoma

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.

SAT654 Regulation Of The Melanocortin-4 Receptor (mc4r) By Attractin Like Protein 1 (alp1)

Abstract Disclosure: P. Buscaglia: None. J. Sebag: None. The Melanocortin-4 Receptor (MC4R) plays a central role in the regulation of energy homeostasis. Mutations in MC4R are responsible for up to 6% of early onset obesity in humans and deletion of MC4R in mice results in severe obesity due to hyperphagia. MC4R is a GPCR expressed in different region of the brains including the paraventricular nucleus of the hypothalamus. Stimulation of MC4R by its agonist αMSH and inhibition by its endogenous inverse agonist AGRP result in inhibition or stimulation of food intake respectively. MC4R is known to interact with two single transmembrane proteins, the Melanocortin Receptor Accessory Protein 2 (MRAP2) and Attractin Like Protein 1 (ALP1). Whereas MRAP2 has been shown to promote MC4R signaling in vitro and in vivo, the pharmacological and physiological relevance of ALP1 for MC4R signaling and actions is not known. To determine if ALP1 alters MC4R signaling, we measured αMSH-stimulated cAMP production in cells transfected with MC4R alone or with either ALP1 or MRAP2. We found that ALP1 increases MC4R signaling to a larger extent than MRAP2. We also showed that ALP1 inhibits β-arrestin recruitment to MC4R, thus likely preventing desensitization of the receptor. To assess the physiological importance of ALP1, we generated an ALP1Flox mouse and bred it to MC4RCRE mice. Deletion of ALP1 in MC4R neurons resulted in increased body weight compared to MC4RCRE control on standard diet. This increase in body weight was further exacerbated when animals were fed a high fat diet. In conclusion our results suggest that ALP1 regulates MC4R signaling and that deletion of ALP1 from MC4R neurons causes weight gain that may be due to decreased MC4R activity. Presentation: Saturday, June 17, 2023

SAT027 Novel Obesity-Associated Melanocortin 4 Receptor Variants Modulate Signaling Differently

Abstract Disclosure: A.V. Rodriguez Rondon: None. M.S. Welling: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus regulating appetite and energy expenditure. Extensive evidence from genetic and biological studies show that MC4R is a key player in the homeostatic regulation of body weight. MC4R loss of function (LoF) variants are the most common cause of monogenic obesity. We have identified 1 homozygous and 16 heterozygous MC4R variants in patients with obesity at our academic Obesity Center CGG, Erasmus MC. The impact of these variants on MC4R signaling and the association with obesity are unknown. Here, we functionally characterized these variants by analyzing four different aspects of MC4R signaling. HEK293 cells were transiently transfected with expression plasmids encoding wild type (WT) or variant MC4R and stimulated with melanocyte stimulating hormone. Cyclic AMP response (cAMP) was measured using GloSensor cAMP bioluminescence assay, and β-arrestin-2 recruitment (βarr2) was measured using NanoBiT complementation luminescence assay. Furthermore, HiBiT Detection System was used to measure cell surface expression, and serum response element (SRE) reporter assay was used to measure extracellular signal-regulated kinase (ERK) activation. Variants were classified according to the percent of maximum response (Emax) relative to WT: complete LoF (cLoF): 0-20%; partial LoF (pLoF): 20-80%; WT: 80-120%; gain of function (GoF): >120%. The MC4R variants were identified by sequencing the MC4R gene of adult and pediatric patients with obesity using an obesity gene panel. Eleven of these variants have not been functionally studied previously. The median age of onset obesity for all patients is 3 years (range 0 – 6). Furthermore, the median BMI of the adult patients was 48.8 kg/m2 (range 36 - 58.8) and the median BMI-SDS of the pediatric patients was +4.2 SDS (range +3 – 7). Twelve out of 17 patients presented with hyperphagia. MC4R variants had differential effects on signaling. In 7 out of 17 variants, cLoF for cAMP was observed. This could be explained in 6 out of 7 of these variants due to lack of cell surface expression. Intriguingly, five out of 17 variants had a normal or GoF cAMP response and normal ERK activation. However, three out of these 5 variants showed cLoF or pLoF for βarr2, indicating a disrupted desensitization mechanism. In addition, the remaining five variants displayed pLoF for most studied pathways. We show that these MC4R variants identified in our patients with obesity affect signaling differently, through modulation of cAMP, βarr2, cell surface expression, and ERK pathways. This demonstrates the value of examining different aspects of MC4R signaling to understand possible biased effects of variants on these pathways. Overall, our results show the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity. Presentation: Saturday, June 17, 2023

FRI048 Cardiometabolic Risk Markers In Children With Obesity And Variants In MC4R Pathway Related Genes

Abstract Disclosure: M. Salama: None. S. Kumar: None. F. Pinto e Vairo: None. R. Hentz: None. Objective: The association between disrupted melanocortin 4 receptor (MC4R) pathway function and childhood obesity is well established. Several variants in MC4R pathway related genes have been implicated to influence cardiometabolic parameters including lipid metabolism and glucose homeostasis. We compared the cardiometabolic risk markers in children with obesity and MC4R related clinically reported genetic variants relative to children with obesity and no MC4R related genetic variants. Methods: This was a retrospective case-control study conducted by chart review of children (age <18 years) with obesity who underwent a multi-gene panel testing for monogenic obesity between February 2020 and November 2022. Children were divided into two groups according to their genetic test results: The first group was of children with clinically reported variants (pathogenic, likely pathogenic, risk allele or variant of uncertain significance) within the MC4R pathway), and the second group was of children with no reported variants in the MC4R pathway (negative genetic testing or variants in non-MC4R related genes). Data on systolic and diastolic blood pressure percentiles, fasting lipid panel, fasting blood glucose, hemoglobin A1c and ALT were extracted. Tests for group differences were performed using the independent two-sample t-test, Wilcoxon rank-sum test, Chi-Square test, or Fisher’s exact test as appropriate for each variable type and test assumptions. Results: Data on a total of 91 children were reviewed. Mean age at testing was 11 years (SD: 4) and mean peak BMI% of the 95th percentile was 158 (SD: 28). 36 patients had clinically reported variants in the MC4R pathway, and 55 patients had no reported MC4R related variants. The blood pressure percentiles, fasting glucose, hemoglobin A1C, total cholesterol, high density lipoprotein cholesterol and low-density lipoprotein cholesterol were not different between both groups. Conclusion: In this pilot study, among children with obesity, variants in the MC4R pathway related genes were not associated with cardiometabolic risk markers. Larger studies are warranted to assess the correlation between MC4R pathway genes and cardiometabolic risk. Presentation: Friday, June 16, 2023

OR0302 Regulation Of Human Melanocortin-5 Receptor By Isoforms Of Melanocortin-2 Receptor Accessory Proteins

Abstract Disclosure: S. Jiang: None. Y. Tao: None. Melanocortin-5 receptor (MC5R) is ubiquitously expressed in central nervous system and peripheral tissues, has unique pharmacological properties, and physiological functions. Melanocortin-2 receptor accessory proteins (MRAPs) are regulators of melanocortin receptor family. To investigate the effects of MRAPs on trafficking, ligand binding and signaling properties of human (h) MC5R, HEK293T cells were transiently co-transfected with plasmids encoding MC5R and different isoforms of MRAPs (hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c). The results showed that hMRAP1a and hMRAP2a increased the cell surface expression of hMC5R. All MRAPs have no effect on affinity to the superpotent analog of α-melanocyte stimulating hormone (α-MSH), NDP-MSH, while hMRAP2c significantly increased maximal binding. Additionally, α-MSH induced ERK1/2 activation. A higher basal level of ERK1/2 phosphorylation was observed when co-transfected with some MRAPs. All MRAPs had no effect on α-MSH-stimulated cAMP production (Gs-cAMP pathway). In summary, the two MRAP1s and three MRAP2s had differential effects on MC5R trafficking, binding, and signaling. These findings led to a better understanding of the regulation of MC5R by MRAP1s and MRAP2s. Presentation: Thursday, June 15, 2023

SAT026 Melanocortin Receptors In The Earliest Chordates?

Abstract Disclosure: R. Ji: None. S. Jiang: None. G. Kleinau: None. P. Scheerer: None. Y. Tao: None. Melanocortin receptors (MCRs) genes have been identified to be functional in the genome of early chordates, e.g. the cyclostomata, around 360 million years ago. Whether it appears in the most ancient chordates, such as cephalochordate and urochordata (500 million years ago), however, remained unknown until now. Herein, therefore, we studied experimentally four putative, sequence based predicted melanocortin-4 receptor (mc4r) like receptors and one putative MC1R-like receptor from urochordata and cephalochordate (from NCBI database), including Styela clava (sc), Ciona intestinalis (ci), Branchiostoma floridae (fl), and Branchiostoma belcheri (bb). No genes coding the endogenous MCR ligands were observed in these species. We further expressed these putative receptor plasmids in HEK293T cells and stimulated with α-melanocyte stimulating hormone (α-MSH) and synthetic MCR ligands to verify whether these receptors are functional MCRs. Our results showed that four receptors (so far named flMC4R, bbMC4R-1, bbMC4R-2, and ciMC1R) had high cell surface expression in HEK293T cells, whereas scMC4R had low cell surface expression. Four receptors (except scMC4R) showed high basal cAMP signaling, suggesting that these receptors are likely coupled to the stimulatory Gs protein. However, no specific binding to the superpotent α-MSH analog, NDP-MSH, were observed at these five receptors, and in accordance they had no α-MSH-induced cAMP signaling coupled to Gs stimulation. Additionally, all five receptors had low basal and no α-MSH-stimulated ERK1/2 signaling. Four human MC4R inverse agonists, including Agouti-related protein and three small molecular compounds (Ipsen5i, MCL0020, and ML00253764), had no effect on cAMP and ERK1/2 signaling of the four predicted MC4R-like receptors. In summary, our results suggested that the four predicted MCR-like receptors (except scMC4R) in ancient chordates are indeed functional receptors, with high constitutive activity, but they are all not MCR-like receptors based on our experimental data with common endogenous and synthetic MCR ligands. We hypothesize that these receptors might be MCR-like receptors, in that receptors might appear earlier than appearance of endogenous ligands; or these receptors might be other ancient G protein-coupled receptors (GPCRs) with other unidentified ligands. However, further studies are needed to identify the ancient GPCR system, which may include studies on the appearance of ligands or precursors. Presentation: Saturday, June 17, 2023

THU452 Polyostotic Fibrous Dysplasia Complicated By Aneurysmal Bone Cyst In A Patient With McCune-Albright Syndrome

Abstract Disclosure: E. Milazzo: None. K.L. Hermayer: None. Introduction: McCune-Albright Syndrome (MAS) is a rare mosaic disorder with GNAS mutations, encoding the alpha-subunit of the Gs G-coupled protein receptor, activating it, and inappropriately producing cAMP. The hyperfunctioning endocrinopathies associated with MAS are due to signaling through the TSH, LH, FSH, GHRH, and ACTH receptors. MAS clinical presentation occurs with café-au-lait skin macules, precocious puberty, and fibrous dysplasia (FD) of the bone. FD is a benign tumor that leads to increased rates of bone fractures and if it causes facial deformities, can be complicated with orbital neuropathies. An aneurysmal bone cyst (ABC) is a blood-filled cyst that can be a primary or secondary lesion due to co-existing lesions. ABC is a rare complication of FD. We report a rare case of ABC in a MAS patient who presented with acute visual disturbance and enlargement of the forehead and was successfully treated with surgery. Clinical Case: A 20-year-old female diagnosed with MAS at the age of 2 who had precocious puberty by the 3rd year of life and developed polyostotic FD in the right arm, left femur, cranial, and skull bones with multiple long bone fractures by the age of 5-6 and required numerous orthopedic surgeries. She presented with the characteristic café-au-lait skin macules on the back and right arm. In 2018, a slow decline of left eye vision required left cranial decompression of the optic nerve, with pathology showing fibrous tissue compatible with FD. In 2021, she complained of acute right forehead enlargement, compressing her right eye with blurry vision. An MRI showed FD involving the facial and skull bones with a new expansible lesion in the right frontal bone with internal layering fluid, exerting a mild mass effect on the frontal lobe and extending superiorly to the orbit with inferior displacement of the globe. These findings were consistent with an ABC. Right frontal craniectomy and cranioplasty were performed in 12/2021, with pathology showing a fibrous pseudocyst wall with associated lymphoplasmacytic inflammation and evidence of prior hemorrhage with Ki-67 in the cyst wall at 15%. Lab work showed chronic alkaline phosphatase elevation at 567IU/L (42-106IU/L), normal calcium at 9.2mg/dl (8.4-10.3mg/dl), phosphate at 3.3mg/dl (3.3-5.1mg/dl), and PTH at 26pg/ml (15-65mg/dl), with Vitamin D insufficiency at 25.1ng/ml (30-100ng/ml). Endocrinology workup was negative for thyroid, adrenal, gonadotrophic, and GH dysfunction. 5 months post-surgery, the patient demonstrated complete recovery, and MRI showed no recurrence of the ABC. Conclusion: ABC is a benign solitary tumor generally arising from the metaphysis of long bones and rarely involves the cranium. ABC associated with FD has only been reported in a few cases. The treatment is surgical excision, with radiation and cryotherapy used in nonorbital cases. Presentation: Thursday, June 15, 2023

FRI082 Impact Of Setmelanotide On Metabolic Parameters And Vital Signs In Patients With Hypothalamic Obesity

Abstract Disclosure: M.J. Abuzzahab: Advisory Board Member; Self; Pfizer, Inc., Endo Pharmaceuticals, Rhythm Pharmaceuticals, Inc., Consynance. Consulting Fee; Self; Pfizer, Inc., Endo Pharmaceuticals, Consynance, Rhythm Pharmaceuticals, Inc. A. Shoemaker: Advisory Board Member; Self; Rhythm Pharmaceuticals, Saniona. M. Gottschalk: Advisory Board Member; Self; Rhythm Pharmaceuticals, Inc. Consulting Fee; Self; Rhythm Pharmaceuticals, Inc. J. Miller: Other; Self; Study funding from Rhythm Pharmaceuticals, Inc. G. Yuan: Employee; Self; Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Rhythm Pharmaceuticals, Inc. S. Malhotra: Employee; Self; Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Rhythm Pharmaceuticals, Inc. C. Scimia: Employee; Self; Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Rhythm Pharmaceuticals, Inc. C.L. Roth: Other; Self; Study funding from Rhythm Pharmaceuticals, Inc. Background: Hypothalamic obesity (HO) is a severe form of obesity that results from insult to the hypothalamus, primarily from tumors, tumor resections, and radiotherapy treatment, which can impair melanocortin-4 receptor (MC4R) pathway signaling. Treatment with setmelanotide, an MC4R agonist, resulted in weight loss and hunger reduction in a Phase 2 trial of patients with HO. This analysis evaluated the impact of 16 weeks of setmelanotide on metabolic parameters and vital signs in patients with HO. Methods: A Phase 2 open-label trial of setmelanotide enrolled patients aged ≥6 to ≤40 years with obesity resulting from hypothalamic injury who completed tumor-related treatment ≥6 months before enrollment (NCT04725240). The primary endpoint was the proportion of patients with ≥5% reduction in baseline body mass index (BMI) after 16 weeks of setmelanotide. Exploratory endpoints of change from baseline in waist circumference and metabolic parameters, including fasting glucose and glycated hemoglobin, were evaluated. Vital signs, including heart rate and blood pressure, were also assessed. Descriptive statistics including changes at Week 16 from baseline are reported. Results: Eighteen patients were included in the study. At Week 16, most patients met the primary endpoint (88.9% [n/N=16/18]; P<0.0001), and 13 of 18 patients who completed treatment had ≥10% BMI reduction (mean [standard deviation (SD)] percent change, −14.5% [9.5%]). Across all patients, body weight was reduced by a mean (SD) of 12.6% (9.1%), and change in BMI Z score in patients aged ≥6 to <18 years was −1.3 (1.0). Overall, waist circumference was reduced by a mean (SD) of 12.0 (5.8) cm, which was a 10.4% (5.2%) reduction. Weight loss was predominately due to reduction in total fat mass rather than loss of lean mass. Most mean metabolic and vital sign values were within normal ranges at baseline, and given the short duration of the study, no changes in metabolic parameters were noted. Vital signs continued to remain normal during the 16-week study duration. Treatment-related adverse events occurred in all patients. The most frequent adverse events included nausea (61.1%; n=11), vomiting (33.3%; n=6), skin hyperpigmentation (33.3%; n=6), and diarrhea (22.2%; n=4). Conclusions: In a heterogeneous population of patients with HO secondary to treatment of hypothalamic tumors, setmelanotide led to weight loss and reduction in BMI and a favorable change in body composition. Efficacy and safety of setmelanotide will continue to be evaluated in a larger Phase 3 clinical trial. Presentation: Friday, June 16, 2023

SAT656 Weight Reduction After 6 Months Of Setmelanotide Treatment In Patients With Hypothalamic Obesity

Abstract Disclosure: C.L. Roth: Other; Self; Received research support from Rhythm Pharmaceuticals, Inc. A.H. Shoemaker: Advisory Board Member; Self; Received payments from Rhythm Pharmaceuticals, Inc and Saniona for advisory boards and lectures. Other; Self; Received research support from Rhythm Pharmaceuticals, Inc. M. Gottschalk: Advisory Board Member; Self; Received compensation for consulting and participation on advisory boards from Rhythm Pharmaceuticals, Inc. Other; Self; Received funding for clinical trials from Rhythm Pharmaceuticals, Inc. J. Miller: Other; Self; Received research support from Harmony Biosciences, Rhythm Pharmaceuticals, Inc, Soleno Therapeutics, and Tryp Therapeutics. G. Yuan: Employee; Self; Employee of Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Has company-awarded RSU and options. S. Malhotra: Employee; Self; Employee of Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Stockholder in in Rhythm Pharmaceuticals, Inc. C. Scimia: Employee; Self; Employee of Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Has company-awarded RSU and options. M. Abuzzahab: Advisory Board Member; Self; Received compensation for advisory board participation from Consynance, Endo Pharmaceutical, Pfizer, and Rhythm Pharmaceuticals, Inc. Consulting Fee; Self; Received compensation for consulting from Consynance, Endo Pharmaceutical, Pfizer, and Rhythm Pharmaceuticals, Inc. Speaker; Self; Received compensation for speaking engagements from Consynance, Endo Pharmaceutical, Pfizer, and Rhythm Pharmaceuticals, Inc. Other; Self; Received research support from Ascendis, Levo Pharmaceuticals, Lumos, NovoNordisk, Rhythm Pharmaceuticals, Inc, Saniona, and Soleno. Background: Hypothalamic obesity (HO) is an acquired form of severe obesity characterized by rapid and excessive weight gain resulting from insult to the hypothalamus—primarily caused by tumor invasion, resection, or radiotherapy—that can impair melanocortin-4 receptor (MC4R) pathway signaling. Treatment with setmelanotide, an MC4R agonist, resulted in weight loss and hunger reduction at 16 weeks in a Phase 2 trial of patients with HO. Here, we report changes in age-appropriate weight-related parameters after 6 months of treatment with setmelanotide in patients with HO who entered a long-term extension (LTE) trial. Methods: A Phase 2, multicenter, open-label study (NCT04725240) examined the efficacy and safety of 16-week setmelanotide treatment in patients aged ≥6 to ≤40 years with a clinical diagnosis of HO. Patients who met the primary endpoint of ≥5% body mass index (BMI) reduction from baseline at Week 16 or experienced investigator-determined clinically meaningful benefit and demonstrated adequate safety were eligible to enroll in the LTE trial (NCT03651765). We assessed the proportion of patients who achieved ≥10% BMI reduction as well as changes in age-appropriate weight-related parameters (ie, mean percent change in body weight for adults [aged ≥18 years] and mean change in BMI Z score and percent of the 95th BMI percentile [%BMI95] for children [aged <18 years]) from index trial baseline to Month 6 of setmelanotide treatment. Results: Of 18 patients originally enrolled in the index trial, 14 (77.8%) continued into the LTE and 13 (72.2%) had received ≥6 months of setmelanotide treatment at the time of this analysis. HO was diagnosed in these patients secondary to treatment of craniopharyngioma (n=11), hypothalamic hamartoma (n=1), and juvenile pilocytic astrocytoma (n=1). At Month 6, 11 of 13 patients (84.6% [90% confidence interval, 59.0%–97.2%) achieved ≥10% BMI reduction from index trial baseline; the remaining 2 patients achieved ≥5% reduction from baseline at Month 6. In adults (n=2), the mean (standard deviation [SD]) percent change in body weight from baseline to Month 6 was −16.2% (7.2%). In children (n=11), the mean (SD) change from baseline in BMI Z score was −1.7 (1.1) and in %BMI95 was −34.8 (14.9) percentage points at Month 6. The most frequent adverse events were nausea (n=9 [69.2%]), skin hyperpigmentation (n=5 [38.5%]), and vomiting (n=4 [30.8%]). Four serious adverse events were reported in 2 patients (Clostridium difficile colitis in 1 patient; malaise, septic shock, and influenza in 1 patient) and were determined not related or unlikely related to study drug. Conclusions: In a heterogeneous population of patients with HO secondary to treatment of hypothalamic tumors, 6 months of setmelanotide treatment was associated with sustained meaningful improvements in weight and BMI with no new safety signals. Presentation: Saturday, June 17, 2023

FRI065 Trial Design Of A Double-blind, Randomized, Placebo-controlled, Phase 3 Study Of Setmelanotide In Patients With Hypothalamic Obesity

Abstract Disclosure: C.L. Roth: Other; Self; Study funding from Rhythm Pharmaceuticals, Inc. A.H. Shoemaker: Advisory Board Member; Self; Rhythm Pharmaceuticals, Inc., Saniona. Other; Self; Study funding from Rhythm Pharmaceuticals, Inc. M. Gottschalk: Advisory Board Member; Self; Rhythm Pharmaceuticals, Inc. Consulting Fee; Self; Rhythm Pharmaceuticals, Inc. Other; Self; Study funding from Rhythm Pharmaceuticals, Inc. J.L. Miller: Other; Self; Study funding from Rhythm Pharmaceuticals, Inc. G. Yuan: Employee; Self; Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Rhythm Pharmaceuticals, Inc. S. Malhotra: Employee; Self; Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Rhythm Pharmaceuticals, Inc. C. Scimia: Employee; Self; Rhythm Pharmaceuticals, Inc. Stock Owner; Self; Rhythm Pharmaceuticals, Inc. S. McCormack: None. M.J. Abuzzahab: Consulting Fee; Self; Pfizer, Inc., Rhythm Pharmaceuticals, Inc., Consynance. Other; Self; Study funding from Rhythm Pharmaceuticals, Inc., Ascendis, NovoNordisk, Levo Pharmaceuticals, Lumos, Saniona, and Soleno. Background: Damage to the hypothalamus via physical trauma, tumors, surgical resection, and/or radiotherapy can impair the melanocortin signaling pathway and reduce energy expenditure, potentially leading to hypothalamic obesity, a severe form of obesity with no current specific therapeutic option. Recently, the selective melanocortin-4 receptor agonist setmelanotide demonstrated reduction in weight and hunger for patients with hypothalamic obesity who were treated in a Phase 2 trial. We describe the study design of a planned double-blind, randomized, placebo-controlled, Phase 3 trial of setmelanotide in patients with hypothalamic obesity. Methods: Approximately 120 patients will be enrolled across ≤35 sites globally. Eligibility criteria include age ≥4 years with documented evidence of acquired hypothalamic obesity with associated weight gain before and/or with tumor treatment and body mass index (BMI) ≥30 kg/m2 (≥18 years) or BMI ≥95th percentile (≥4 to <18 years). Key exclusion criteria include diagnosis of syndromic obesity, weight or BMI loss >2% based on age in the prior 3 months, bariatric surgery within the past 2 years, glycated hemoglobin >11.0%, and glomerular filtration rate <30 mL/min/1.73 m2. Patients will be randomized 2:1 and stratified by age to receive setmelanotide or placebo. Setmelanotide will be titrated over 2-8 weeks to a maximum daily subcutaneous dose of 1.5-3.0 mg based on age and weight. Double-blind treatment will continue for up to 60 weeks, with visits (in-clinic, at-home, or telehealth) at Weeks 20, 28, 36, 44, and 52 and at an end-of-treatment clinic visit at Week 60. The primary endpoint is the mean percent change in BMI after 52 weeks of treatment for setmelanotide versus placebo. Key secondary endpoints after 52 weeks are the proportion of patients aged ≥18 years with ≥5% reduction in BMI, proportion of patients aged <18 years with ≥0.2-point reduction in BMI Z score, and mean change in weekly average of the daily “most hunger” score in patients aged ≥12 years. Additional secondary endpoints include proportion of patients achieving ≥2-point reduction in weekly average of the daily maximal hunger score, Symptoms of Hyperphagia total score, additional weight-related parameters, and quality of life outcomes. Exploratory endpoints include change after 52 weeks in physical activity (measured by acDetigraphy), fatigue (via Functional Assessment of Chronic Illness Therapy [FACIT]-Fatigue©/Peds FACIT-F© scale), Impacts of Hyperphagia score, caregiver health status (via EuroQol-5-Dimension scale), cardiometabolic parameters, and waist circumference. Safety will be assessed by the frequency and severity of adverse events. Conclusions: This Phase 3 trial designed to assess the efficacy and safety of setmelanotide for weight loss and hunger reduction in patients ≥4 years of age with acquired hypothalamic obesity is planned to initiate in early 2023. Presentation: Friday, June 16, 2023

OR0303 Syndecans Enhance Ghrelin-Induced GHSR1a Signaling

Abstract Disclosure: K. Prins: None. M. Huisman: None. R. Mies: None. P.J. Delhanty: None. J.A. Visser: None. Ghrelin is a gut hormone that enhances food intake and growth hormone secretion through its receptor, the G-protein coupled receptor (GPCR) GHSR1a. Recently, we showed that ghrelin interacts with syndecans (SDCs), a family of four membrane proteins that have been linked to obesity in genome-wide association studies. Here, we investigated whether SDCs impact ghrelin signaling at GHSR1a. We used HEK293 cells that expressed GHSR1a and SDC1, SDC2, SDC3, or SDC4 through transient transfection. GHSR1a mainly signals through the Gαq pathway, so we first explored the effects of SDCs on ghrelin-induced intracellular Ca2+ influx. Using a mitochondrial-targeted aequorin, this Ca2+ response can be measured seconds after ghrelin stimulation. We found that all SDCs increased the maximum Ca2+ response at least 4.2-fold (P<0.001), without affecting the EC50. This enhanced Ca2+ response was not caused by increased receptor availability: SDCs reduced cell membrane receptor expression by 51-85% (P<0.001). This low GHSR1a membrane availability contrasted with the increased Ca2+ response, and raised the question whether SDCs could be an alternate receptor for ghrelin. To test this, we transfected cells with SDC1 alone, but this did not permit a ghrelin-stimulated Ca2+ response, indicating that the ghrelin-induced Ca2+ response was GHSR1a-dependent. Moreover, the SDC-potentiated response was also Gαq-dependent, since GNAQ knockout cells transfected with GHSR1a and SDC1 expression plasmids failed to generate a Ca2+ response. However, using a Gq biosensor, we found that the level of activation of Gαq by ghrelin was not affected by SDC1 transfection, suggesting an effect of SDC1 either downstream of Gαq or on the quenching of GHSR. Thus, we investigated the interaction between GHSR1a and β-arrestin 2. Β-arrestin 2 downregulates GHSR function following activation by sterically hindering G-protein coupling and mediating receptor internalization. Four minutes after ghrelin stimulation, SDCs reduced β-arrestin 2 recruitment by 69-87% (P<0.001). SDC-transfected cells also reached a maximum β-arrestin 2 response 8-14 minutes later than GHSR1a only-transfected cells. Notably, these differences in β-arrestin 2 recruitment occurred minutes after the Ca2+ response. These findings suggest that the reduction in β-arrestin 2 recruitment does not cause the increased Ca2+ response in the presence of SDCs, and that the effect of SDCs is probably downstream of Gαq. Interestingly, the effects of SDCs mimic those of melanocortin receptor accessory protein 2 (MRAP2). MRAP2 also enhances the ghrelin-stimulated activation of the Gαq signaling pathway, independently of β-arrestin recruitment. Altogether, we showed that SDCs enhanced Ca2+ influx and reduced β-arrestin 2 recruitment to GHSR1a in response to ghrelin. This could be a novel mechanism through which SDCs affect metabolism and obesity. Presentation: Thursday, June 15, 2023

Efficacy and Safety of Setmelanotide, a Melanocortin-4 Receptor Agonist, for Obese Patients: A Systematic Review and Meta-Analysis.

A malfunction in the melanocortin-4 receptor (MC4R) is associated with obesity in rare genetic syndromes; setmelanotide is a new drug that activates this receptor and is being used to treat severe obesity. This meta-analysis evaluated the efficacy and safety of setmelanotide for weight loss in severe obesity linked to human MC4R deficiency. We searched PubMed, Embase, and Cochrane for randomized and nonrandomized clinical trials using setmelanotide. We considered a p-value ≤ 0.05 statistically significant. We included 376 patients, of whom 328 (87.2%) received setmelanotide for a mean follow-up of 52 weeks. The mean age was 32.8 (14.67) years. Weight loss was significant (MD -3.52; 95% CI -3.98, -3.05; p = 0.01; I2 = 92%), with an average proportion of -6.91% weight loss during treatment. Changes in BMI showed an MD of -10.55 kg/m2 in patients > 18 years and -0.61 kg/m2 in patients < 18 years (BMI score). However, the drug was associated with a higher risk of skin hyperpigmentation (OR 0.69; 95% CI 0.55, 0.80; p = 0.08). Our results support the use of setmelanotide in treating severe obesity.

Association of the AspP298Asn polymorphism in the MC4R gene with fattening productivity of immunologically castrated and uncastrated gilts

The study was aimed at establishing the presence of MC4R (c.1426 A&gt;G) gene polymorphism among the population of immunologically castrated and native gilts (Large White × Landrace) × Maxgro. Asp298Asn polymorphism affects the economic rate of growth and fattening productivity of commercial lines of pigs. Since the polymorphism of the MC4R gene correlates with the concentration of androstenone, skatole, and indole, the possible use of polymorphism as a molecular DNA marker MC4R for genetic selection in order to reduce the level of boar odor in gilts meat and fat. This will establish which alleles and genotypes in general will be determined as desirable in the marker dilution of hybrid pigs. With this in mind, the purpose of the study was to determine the effect of the MC4R genotype on fattening performance indicators and correlation with boar odor in hybrid gilts based on population-genetic variability. Genomic DNA was isolated from the ear hair follicle of native gilts (n=42) and epithelial tissue from the ear of immunologically castrated ones (n=52). Genotyping was performed using polymorphism of the lengths of restrictive fragments (RFLP). Genetic-correlation parameters for native and immunologically castrated gilts were evaluated by ADG/AGE30 and ADG/AGE100. However, the polymorphism of the c.1426 A&gt;G gene did not significantly affect the ADG/AGE100 performance of hybrid gilts. The predominance of the frequency of allele A (0.55) is established above the frequency of the allele G (0.45). Analysis of the frequency distribution of genotypes showed the saturation of micropopulation with heterozygotes AG (0.51) with a small proportion of GG carriers (0.19). Immunologically castrated and uncastrated gilts with an MC4RA allele are characterized by a sufficiently high ADG/AGE100 and are probably characterized by a high level of boar-specific odor compared to gilts with lower ADG/AGE100 with an MC4RG allele. According to the data obtained, the desired genotype for an equilibrium of the concentration of boar odor without compromising physiological development, growth rate, and productivity formation is heterozygous offspring with the MC4RAG genotype.

Melanocortin-3 receptor expression in AgRP neurons is required for normal activation of the neurons in response to energy deficiency

The melanocortin-3 receptor (MC3R) is a negative regulator of the central melanocortin circuitry via presynaptic expression on agouti-related protein (AgRP) nerve terminals, from where it regulates GABA release onto secondary MC4R-expressing neurons. However, MC3R knockout (KO) mice also exhibit defective behavioral and neuroendocrine responses to fasting. Here, we demonstrate that MC3R KO mice exhibit defective activation of AgRP neurons in response to fasting, cold exposure, or ghrelin while exhibiting normal inhibition of AgRP neurons by sensory detection of food in the ad libitum-fed state. Using a conditional MC3R KO model, we show that the control of AgRP neuron activation by fasting and ghrelin requires the specific presence of MC3R within AgRP neurons. Thus, MC3R is a crucial player in the responsiveness of the AgRP soma to both hormonal and neuronal signals of energy need.

Acthar® Gel Treatment for Patients with Autoimmune and Inflammatory Diseases: An Historical Perspective and Characterization of Clinical Evidence.

Acthar® Gel (repository corticotropin injection) is a naturally sourced complex mixture of adrenocorticotropic hormone analogs and other pituitary peptides that is believed to have both steroidogenic and nonsteroidogenic immunomodulatory effects via activation of melanocortin receptors in various cells throughout the body. Since 1952, Acthar has been approved by the US Food and Drug Administration to treat a variety of autoimmune and inflammatory diseases. Since 2014, Mallinckrodt Pharmaceuticals has conducted a large number of preclinical, clinical, and real-world-evidence studies of Acthar for the treatment of rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis and polymyositis, multiple sclerosis relapse, ophthalmic disorders, sarcoidosis, and nephrotic syndrome. To date, Acthar has been the subject of more than 500 publications, many of which demonstrate the safety and efficacy of Acthar in patients with inflammatory diseases for whom standard treatments were ineffective or intolerable. Here, we review the history of Acthar and the findings of studies that have investigated the mechanism of action, safety, efficacy, and real-world effectiveness of Acthar for the treatment of inflammatory diseases.

S1014 Cellular and Molecular Impact of the Melanocortin Receptor Agonist PL8177 in Dextran Sulfate Sodium-Induced Colitis in Rats

S1014 Cellular and Molecular Impact of the Melanocortin Receptor Agonist PL8177 in Dextran Sulfate Sodium-Induced Colitis in Rats

Development of Melanocortin 4 Receptor Agonists by Exploiting Animal-Derived Macrocyclic, Disulfide-Rich Peptide Scaffolds.

G protein-coupled receptors are among the most widely studied classes of drug targets. A major challenge in this field is to develop ligands that will selectively modulate a single receptor subtype to overcome the disadvantages of undesired "off target" effects caused by lack of target and thus signaling specificity. In the current study, we explored ligand design for the melanocortin 4 receptor (MC4R) since it is an attractive target for developing antiobesity drugs. Endogenously, the receptor is activated by peptide ligands, i.e., three melanocyte-stimulating hormones (α-MSH, β-MSH, and γ-MSH) and by adrenocorticotropic hormone. Therefore, we utilized a peptide drug design approach, utilizing "molecular grafting" of pharmacophore peptide sequence motifs onto a stable nature-derived peptide scaffold. Specifically, protegrin-4-like-peptide-1 (Pr4LP1) and arenicin-1-like-peptide-1 (Ar3LP1) fully activated MC4R in a functional cAMP assay with potencies of 3.7 and 1.0 nM, respectively. In a nanoluciferase complementation assay with less signal amplification, the designed peptides fully recruited mini-Gs with subnanomolar and nanomolar potencies. Interestingly, these novel peptide MC4R ligands recruited β-arrestin-2 with ∼2-fold greater efficacies and ∼20-fold increased potencies as compared to the endogenous α-MSH. The peptides were inactive at related MC1R and MC3R in a cAMP accumulation assay. These findings highlight the applicability of animal-derived disulfide-rich scaffolds to design pathway and subtype selective MC4R pharmacological probes. In the future, this approach could be exploited to develop functionally selective ligands that could offer safer and more effective obesity drugs.