Absorption, metabolism, and excretion of [14 C]dersimelagon, an investigational oral selective melanocortin 1 receptor agonist, in preclinical species and healthy volunteers.

Dersimelagon (formerly MT-7117) is a novel, orally administered nonpeptide small molecule selective agonist for melanocortin 1 receptor currently being investigated for the treatment of erythropoietic protoporphyria, X-linked protoporphyria, and diffuse cutaneous systemic sclerosis (dcSSc). Findings of studies evaluating the absorption, distribution, metabolism, and excretion (ADME) of dersimelagon following a single dose of [14 C]dersimelagon in healthy adult volunteers (N = 6) who participated in phase 1, single-center, open-label, mass balance study (NCT03503266), and in preclinical animal models are presented. Rapid absorption and elimination were observed following oral administration of [14 C]dersimelagon in clinical and nonclinical studies, with a mean Tmax of 30 min in rats and 1.5 h in monkeys, and a median Tmax of 2 h in humans. In rats, there was a widespread distribution of [14 C]dersimelagon-related material, but little or no radioactivity was detected in the brain or fetal tissues. In humans, elimination of radioactivity in urine was negligible (excretion of radioactivity into the urine: 0.31% of dose), and the primary route of excretion was feces, with more than 90% of the radioactivity recovered through 5 days postdose. Based on these findings, dersimelagon is not retained in the human body. Findings from humans and animals suggest dersimelagon is extensively metabolized to the glucuronide in the liver, which is eliminated in bile, and hydrolyzed to unchanged dersimelagon in the gut. The results to date for this orally administered agent elucidate the ADME of dersimelagon in human and animal species and support its continued development for the treatment of photosensitive porphyrias and dcSSc.

Dersimelagon in Erythropoietic Protoporphyrias.

BACKGROUND: Erythropoietic protoporphyria and X-linked protoporphyria are inborn errors of heme biosynthesis that cause elevated circulating levels of metal-free protoporphyrin and phototoxicity. Both disorders are characterized by excruciating phototoxic attacks after exposure to visible light. Dersimelagon is a new, orally administered, selective melanocortin 1 receptor agonist that increases levels of skin eumelanin. METHODS: We conducted a randomized, placebo-controlled, phase 2 trial to investigate the efficacy and safety of dersimelagon with respect to the time to onset and the severity of symptoms associated with sunlight exposure in patients with erythropoietic protoporphyria or X-linked protoporphyria. Patients 18 to 75 years of age were randomly assigned in a 1:1:1 ratio to receive placebo or dersimelagon at a dose of 100 or 300 mg once daily for 16 weeks. The primary end point was the change from baseline to week 16 in the time to the first prodromal symptom associated with sunlight exposure. Patients recorded daily sunlight exposure and symptom data in an electronic diary. Quality of life and safety were also assessed. RESULTS: Of the 102 patients (93 with erythropoietic protoporphyria and 9 with X-linked protoporphyria) who underwent randomization, 90% completed the treatment period. The mean daily time to the first prodromal symptom associated with sunlight exposure increased significantly with dersimelagon: the least-squares mean difference from placebo in the change from baseline to week 16 was 53.8 minutes in the 100-mg dersimelagon group (P = 0.008) and 62.5 minutes in the 300-mg dersimelagon group (P = 0.003). The results also suggest that quality of life improved in patients receiving dersimelagon as compared with placebo. The most common adverse events that occurred or worsened during treatment were nausea, freckles, headache, and skin hyperpigmentation. CONCLUSIONS: At both doses evaluated, dersimelagon significantly increased the duration of symptom-free sunlight exposure in patients with erythropoietic protoporphyria or X-linked protoporphyria. (Funded by Mitsubishi Tanabe Pharma; Endeavor ClinicalTrials.gov number, NCT03520036.).

Negative Modulation of B Cell Activation by Melanocortin 1 Receptor Signaling Protects against Membranous Nephropathy.

SIGNIFICANCE STATEMENT: Emerging evidence suggests that melanocortin neuropeptides-specifically adrenocorticotropic hormone-offer a novel, steroidogenic-independent therapeutic modality for membranous nephropathy (MN). The molecular mechanism underlying this beneficial effect, however, remains largely elusive. To investigate whether melanocortins modulate humoral immunity, the authors induced passive Heymann nephritis, a model of human MN, in wild-type and melanocortin 1 receptor (MC1R) knockout rats and treated them with melanocortin agents. Additional rats received adoptive transfer of bone marrow-derived cells beforehand from wild-type or MC1R knockout rats. The findings indicate that MC1R signaling plays a key role in negative modulation of B-cell activation and thereby suppresses humoral immune responses in passive Heymann nephritis, and suggest that MC1R signaling might offer a novel B cell-targeted therapeutic strategy for MN. BACKGROUND: Emerging evidence suggests that the pituitary neuropeptide melanocortins-specifically, adrenocorticotropic hormone-offer a novel nonsteroidogenic therapeutic modality for membranous nephropathy (MN). However, the mechanism(s) of action remains elusive. METHODS: To investigate whether melanocortins modulate humoral immunity, we induced passive Heymann nephritis (PHN), a model of MN, in wild-type (WT) and melanocortin 1 receptor (MC1R) knockout (KO) rats. We treated the animals with melanocortin agents-repository corticotropin injection, the nonsteroidogenic pan-melanocortin receptor agonist [Nle 4 , DPhe 7 ]-α-melanocyte stimulating hormone, the selective MC1R agonist MS05, vehicle gel, or phosphate-buffered saline-and evaluated kidney function, histology, and molecular changes. Additional rats received adoptive transfer of syngeneic bone marrow-derived cells beforehand from WT or MC1R KO rats. RESULTS: KO of MC1R worsened PHN and this was associated with increased deposition of autologous immunoglobulin G (IgG) and complement C5b-9 in glomeruli and higher circulating levels of autologous IgG-evidence of a sensitized humoral immune response. Melanocortin therapy ameliorated PHN in WT rats, coinciding with reduced glomerular deposition of autologous IgG and C5b -9. The beneficial efficacy of melanocortins was blunted in KO rats but restored by adoptive transfer of syngeneic bone marrow-derived cells derived from WT rats. Mechanistically, MC1R was expressed in B lymphocytes and was negatively associated with B cell activation. MC1R agonism triggered the expression of microphthalmia-associated transcription factor in activated B cells in a cAMP-dependent mode and also repressed the expression of interferon regulatory factor 4 (a lymphoid transcription factor essential for B-cell development and maturation), resulting in suppressed plasma cell differentiation and IgG production. CONCLUSIONS: MC1R signaling negatively modulates B cell activation and suppresses humoral immune responses in PHN, suggesting that MC1R signaling might offer a novel therapeutic target for MN.

Late-Stage Functionalization with Cysteine Staples Generates Potent and Selective Melanocortin Receptor-1 Agonists.

In this work, cysteine staples were used as a late-stage functionalization strategy to diversify peptides and build conjugates targeting the melanocortin G-protein-coupled receptors [melanocortin receptor-1 (MC1R) and MC3R-MC5R]. Monocyclic and bicyclic agonists based on sunflower trypsin inhibitor-1 were used to generate a selection of stapled peptides that were evaluated for binding (pKi) and functional activation (pEC50) of the melanocortin receptor subtypes. Stapled peptides generally had improved activity, with aromatic stapled peptides yielding selective MC1R agonists, including a xylene-stapled peptide (2) with an EC50 of 1.9 nM for MC1R and >150-fold selectivity for MC3R and MC4R. Selected stapled peptides were further functionalized with linkers and payloads, generating a series of conjugated peptides with potent MC1R activity, including one pyridazine-functionalized peptide (21) with picomolar activity at MC1R (Ki 58 pM; EC50 < 9 pM). This work demonstrates that staples can be used as modular synthetic tools to tune potency and selectivity in peptide-based drug design.

BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model.

Neuronal apoptosis induced by oxidative stress plays an important role in the pathogenesis and progression of hypoxic-ischemic encephalopathy (HIE). Previous studies reported that activation of melanocortin-1 receptor (MC1R) exerts antioxidative stress, antiapoptotic, and neuroprotective effects in various neurological diseases. However, whether MC1R activation can attenuate oxidative stress and neuronal apoptosis after hypoxic-ischemic- (HI-) induced brain injury remains unknown. Herein, we have investigated the role of MC1R activation with BMS-470539 in attenuating oxidative stress and neuronal apoptosis induced by HI and the underlying mechanisms. 159 ten-day-old unsexed Sprague-Dawley rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. The novel-selective MC1R agonist BMS-470539 was administered intranasally at 1 h after HI induction. MC1R CRISPR KO plasmid and Nurr1 CRISPR KO plasmid were administered intracerebroventricularly at 48 h before HI induction. Percent brain infarct area, short-term neurobehavioral tests, Western blot, immunofluorescence staining, Fluoro-Jade C staining, and MitoSox Staining were performed. We found that the expression of MC1R and Nurr1 increased, peaking at 48 h post-HI. MC1R and Nurr1 were expressed on neurons at 48 h post-HI. BMS-470539 administration significantly attenuated short-term neurological deficits and infarct area, accompanied by a reduction in cleaved caspase-3-positive neurons at 48 h post-HI. Moreover, BMS-470539 administration significantly upregulated the expression of MC1R, cAMP, p-PKA, Nurr1, HO-1, and Bcl-2. However, it downregulated the expression of 4-HNE and Bax, as well as reduced FJC-positive cells, MitoSox-positive cells, and 8-OHdG-positive cells at 48 h post-HI. MC1R CRISPR and Nurr1 CRISPR abolished the antioxidative stress, antiapoptotic, and neuroprotective effects of BMS-470539. In conclusion, our findings demonstrated that BMS-470539 administration attenuated oxidative stress and neuronal apoptosis and improved neurological deficits in a neonatal HI rat model, partially via the MC1R/cAMP/PKA/Nurr1 signaling pathway. Early administration of BMS-470539 may be a novel therapeutic strategy for infants with HIE.

MC1R Functions, Expression, and Implications for Targeted Therapy.

The G protein-coupled MC1R is expressed in melanocytes and has a pivotal role in human skin pigmentation, with reduced function in human genetic variants exhibiting a red hair phenotype and increased melanoma predisposition. Beyond its role in pigmentation, MC1R is increasingly recognized as promoting UV-induced DNA damage repair. Consequently, there is mounting interest in targeting MC1R for therapeutic benefit. However, whether MC1R expression is restricted to melanocytes or is more widely expressed remains a matter of debate. In this paper, we review MC1R function and highlight that unbiased analysis suggests that its expression is restricted to melanocytes, granulocytes, and the brain.

Melanocortin 1 Receptor Agonists Based on a Bivalent, Bicyclic Peptide Framework.

We have designed a new class of highly potent bivalent melanocortin receptor ligands based on the nature-derived bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1). Incorporation of melanotropin pharmacophores in each of the two turn regions of SFTI-1 resulted in substantial gains in agonist activity particularly at human melanocortin receptors 1 and 3 (hMC1R/hMC3R) compared to monovalent analogues. In in vitro binding and functional assays, the most potent molecule, compound 6, displayed low picomolar agonist activity at hMC1R (pEC50 > 10.3; EC50 < 50 pM; pKi: 10.16 ± 0.04; Ki: 69 ± 5 pM) and is at least 30-fold more selective for this receptor than for hMC3R, hMC4R, or hMC5R. The results are discussed in the context of structural homology models of hMCRs in complex with the developed bivalent ligands.

Development of hMC1R Selective Small Agonists for Sunless Tanning and Prevention of Genotoxicity of UV in Melanocytes.

Activation of the human melanocortin 1 receptor (hMC1R) expressed on melanocytes by α-melanocortin plays a central role in regulating human pigmentation and reducing the genotoxicity of UV by activating DNA repair and antioxidant defenses. For the development of a hMC1R-targeted photoprotection strategy, we designed tetra- and tripeptide agonists with modifications that provide the necessary lipophilicity and hMC1R selectivity to be effective drugs. These peptides proved to be superior to most of the existing analogs of the physiological tridecapeptide α-melanocortin because of their small size and high hMC1R selectivity. Testing on primary cultures of human melanocytes showed that these peptides are highly potent with prolonged stimulation of melanogenesis, enhanced repair of UV-induced DNA photoproducts, and reduced apoptosis. One of the tripeptides, designated as LK-514 (5), with a molecular weight of 660 Da, has unprecedented (>100,000) hMC1R selectivity when compared with the other melanocortin receptors hMC3R, hMC4R, and hMC5R, and increases pigmentation (sunless tanning) in a cultured, three-dimensional skin model. These new analogs should be efficacious in preventing skin cancer, including melanoma, and treatment of skin disorders, such as vitiligo and polymorphic light eruptions.

Activation of the Melanocortin-1 Receptor by NDP-MSH Attenuates Oxidative Stress and Neuronal Apoptosis through PI3K/Akt/Nrf2 Pathway after Intracerebral Hemorrhage in Mice.

Oxidative stress and neuronal apoptosis play crucial roles in secondary brain injury (SBI) after intracerebral hemorrhage (ICH). Recently, Nle4-D-Phe7-α-melanocyte-stimulating hormone (NDP-MSH), a synthetic agonist of the melanocortin-1 receptor (Mc1r), has been proved to inhibit neuroinflammatory in several diseases. This study is aimed at exploring if NDP-MSH could reduce oxidative stress and neuronal apoptosis following ICH, as well as the potential mechanism. A mouse ICH model was induced by autologous blood injection. NDP-MSH was intraperitoneally injected at 1 h after ICH. Mc1r siRNA and PI3K inhibitor LY294002 were administrated to inhibit the expression of Mc1r and phosphorylation of PI3K, respectively. Neurological test, brain water content, enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), immunofluorescence, and Western blot analysis were utilized in this study. The results exhibited that Mc1r was mainly expressed in neurons, and its level in the ipsilateral hemisphere was significantly elevated after ICH. NDP-MSH treatment significantly attenuated the neurological deficits and brain water content 24 hours after ICH, which was accompanied by the inhibition of oxidative stress and neuronal apoptosis. The administration of NDP-MSH after ICH significantly promoted the expression of Mc1r, p-PI3K, p-Akt, and p-Nrf2, followed by an increase of Bcl-2 and reduction of cleaved caspase-3. Conversely, downregulating the expression of Mc1r and phosphorylation of PI3K aggravated the neurological deficits and brain edema at 24 hours after ICH, meanwhile, the effect of NDP-MSH on the expression of Mc1r, p-PI3K, p-Akt, p-Nrf2, Bcl-2, and cleaved caspase 3 was also abolished. In conclusion, our data suggest that the activation of Mc1r by NDP-MSH ameliorates oxidative stress and neuronal apoptosis through the PI3K/Akt/Nrf2 signaling pathway after ICH in mice.

Melanocortin therapy ameliorates podocytopathy and proteinuria in experimental focal segmental glomerulosclerosis involving a podocyte specific non-MC1R-mediated melanocortinergic signaling.

The clinical effectiveness of adrenocorticotropin in inducing remission of steroid-resistant nephrotic syndrome points to a steroidogenic-independent anti-proteinuric activity of melanocortins. However, which melanocortin receptors (MCR) convey this beneficial effect and if systemic or podocyte-specific mechanisms are involved remain uncertain. In vivo, wild-type (WT) mice developed heavy proteinuria and kidney dysfunction following Adriamycin insult, concomitant with focal segmental glomerulosclerosis (FSGS) and podocytopathy, marked by loss of podocin and synaptopodin, podocytopenia and extensive foot process effacement on electron microscopy. All these pathologic findings were prominently attenuated by NDP-MSH, a potent non-steroidogenic pan-MCR agonist. Surprisingly, MC1R deficiency in MC1R-null mice barely affected the severity of Adriamycin-elicited injury. Moreover, the beneficial effect of NDP-MSH was completely preserved in MC1R-null mice, suggesting that MC1R is likely non-essential for the protective action. A direct podocyte effect seems to contribute to the beneficial effect of NDP-MSH, because Adriamycin-inflicted cytopathic signs in primary podocytes prepared from WT mice were all mitigated by NDP-MSH, including apoptosis, loss of podocyte markers, de novo expression of the podocyte injury marker desmin, actin cytoskeleton derangement and podocyte hypermotility. Consistent with in vivo findings, the podoprotective activity of NDP-MSH was fully preserved in MC1R-null podocytes. Mechanistically, MC1R expression was predominantly distributed to glomerular endothelial cells in glomeruli but negligibly noted in podocytes in vivo and in vitro, suggesting that MC1R signaling is unlikely involved in direct podocyte protection. Ergo, melanocortin therapy protects against podocyte injury and ameliorates proteinuria and glomerulopathy in experimental FSGS, at least in part, via a podocyte-specific non-MC1R-mediated melanocortinergic signaling.

Novel anti-inflammatory and chondroprotective effects of the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and human melanocortin MC3 receptor agonist PG-990 on lipopolysaccharide activated chondrocytes.

Human melanocortin MC1 and MC3 receptors expressed on C-20/A4 chondrocytes exhibit chondroprotective and anti-inflammatory effects when activated by melanocortin peptides. Nearly 9 million people in the UK suffer from osteoarthritis, and bacterial infections play a role in its development. Here, we evaluate the effect of a panel of melanocortin peptides with different selectivity for human melanocortin MC1 (α-MSH, BMS-470539 dihydrochloride) and MC3 ([DTrp8]-γ-MSH, PG-990) receptors and C-terminal peptide α-MSH11-13(KPV), on inhibiting LPS-induced chondrocyte death, pro-inflammatory mediators and induction of anti-inflammatory proteins. C-20/A4 chondrocytes were treated with a panel of melanocortin peptides prophylactically and therapeutically in presence of LPS (0.1 µg/ml). The chondroprotective properties of these peptides determined by cell viability assay, RT-PCR, ELISA for detection of changes in inflammatory markers (IL-6, IL-8 and MMP-1, -3 and -13) and western blotting for expression of the anti-inflammatory protein heme-oxygenase-1. C-20/A4 expressed human melanocortin MC1 and MC3 receptors and melanocortin peptides elevated cAMP. LPS stimulation caused a reduction in C-20/A4 viability, attenuated by the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride, and MC3 receptor agonists PG-990 and [DTrp8]-γ-MSH. Prophylactic and therapeutic regimes of [DTrp8]-γ-MSH significantly inhibited LPS-induced modulation of cartilage-damaging IL-6, IL-8, MMPs -1,-3 and -13 mediators both prophylactically and therapeutically, whilst human melanocortin MC1 and MC3 receptor agonists promoted an increase in HO-1 production. In the presence of LPS, activation of human melanocortin MC1 and MC3 receptors provided potent chondroprotection, upregulation of anti-inflammatory proteins and downregulation of inflammatory and proteolytic mediators involved in cartilage degradation, suggesting a new avenue for osteoarthritis treatment.

Therapeutic senescence via GPCR activation in synovial fibroblasts facilitates resolution of arthritis.

Rheumatoid arthritis affects individuals commonly during the most productive years of adulthood. Poor response rates and high costs associated with treatment mandate the search for new therapies. Here we show that targeting a specific G-protein coupled receptor promotes senescence in synovial fibroblasts, enabling amelioration of joint inflammation. Following activation of the melanocortin type 1 receptor (MC1), synovial fibroblasts acquire a senescence phenotype characterized by arrested proliferation, metabolic re-programming and marked gene alteration resembling the remodeling phase of wound healing, with increased matrix metalloproteinase expression and reduced collagen production. This biological response is attained by selective agonism of MC1, not shared by non-selective ligands, and dependent on downstream ERK1/2 phosphorylation. In vivo, activation of MC1 leads to anti-arthritic effects associated with induction of senescence in the synovial tissue and cartilage protection. Altogether, selective activation of MC1 is a viable strategy to induce cellular senescence, affording a distinct way to control joint inflammation and arthritis.

Discovery of a Highly Selective MC1R Agonists Pentapeptide to Be Used as a Skin Pigmentation Enhancer and with Potential Anti-Aging Properties.

One of the first lines of cutaneous defense against photoaging is a) the synthesis of melanin and b) the initiation of an oxidative stress response to protect skin against the harmful effects of solar radiation. Safe and selective means to stimulate epidermal pigmentation associated with oxidative stress defense are; however, scarce. Activation of the melanocortin-1 receptor (MC1R) on epidermal melanocytes represents a key step in cutaneous pigmentation initiation and, additionally, it regulates cellular defense mechanisms like oxidative stress and DNA-repair. Thus, making the activation of MC1R an attractive strategy for modulating skin pigmentation and oxidative stress. In this context, we designed and synthesized pentapeptides that act as MC1R agonists. These peptides bound, with high potency, to MC1R and activated cAMP synthesis in CHO cells expressing human MC1R. Using one lead pentapeptide, we could show that this activation of MC1R was specific as testing the activation of other G-protein coupled receptors, including the MC-receptor family, was negative. In vitro efficacy on mouse melanoma cells showed similar potency as for the synthetic MC1R agonist alpha-melanocyte stimulating hormone (NDP-alpha-MSH). Moreover, we could reproduce this activity in human skin tissue culture. The lead pentapeptide was able to induce ex-vivo protein expression of key melanogenesis markers melanocyte inducing transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP-1). Concerning oxidative stress response, we found that the pentapeptide enhanced the activation of Nrf2 after UVA-irradiation. Our results make this pentapeptide an ideal candidate as a skin pigmentation enhancer that mimics alpha-MSH and may also have anti-photoaging effects on the skin.

Loss-of-function mutations in the melanocortin 1 receptor cause disruption of dorso-ventral countershading in teleost fish.

The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in vertebrate pigmentation. Mutations in this gene cause variations in coat coloration in amniotes. Additionally, in mammals MC1R is the main receptor for agouti-signaling protein (ASIP), making it the critical receptor for the establishment of dorsal-ventral countershading. In fish, Mc1r is also involved in pigmentation, but it has been almost exclusively studied in relation to melanosome dispersion activity and as a putative genetic factor involved in dark/light adaptation. However, its role as the crucial component for the Asip1-dependent control of dorsal-ventral pigmentation remains unexplored. Using CRISPR/Cas9, we created mc1r homozygous knockout zebrafish and found that loss-of-function of mc1r causes a reduction of countershading and a general paling of the animals. We find ectopic development of melanophores and xanthophores, accompanied by a decrease in iridophore numbers in the ventral region of mc1r mutants. We also reveal subtle differences in the role of mc1r in repressing pigment cell development between the skin and scale niches in ventral regions.

Current and innovative emerging therapies for porphyrias with hepatic involvement.

Porphyrias are rare inherited disorders caused by specific enzyme dysfunctions in the haem synthesis pathway, which result in abnormal accumulation of specific pathway intermediates. The symptoms depend upon the chemical characteristics of these substances. Porphyrins are photoreactive and cause photocutaneous lesions on sunlight-exposed areas, whereas accumulation of porphyrin precursors is related to acute neurovisceral attacks. Current therapies are suboptimal and mostly address symptoms rather than underlying disease mechanisms. Advances in the understanding of the molecular bases and pathogenesis of porphyrias have paved the way for the development of new therapeutic strategies. In this Clinical Trial Watch we summarise the basic principles of these emerging approaches and what is currently known about their application to porphyrias of hepatic origin or with hepatic involvement.

ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5.

ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR5) PG-901 (10-10M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR1 agonist BMS-470539 (10-5 M) or with the mixed MCR3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGF-containing exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10-10M) but not by the MCR3/4 agonist MTII (0.30 nmol) or the MCR1 agonist BMS-470539 (10-5 M). The MCR5-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR5 agonist in the ARPE-19 cells. Altogether, these data suggest that MCR5 is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo.

Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization.

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.

Arg-Phe-Phe d-Amino Acid Stereochemistry Scan in the Macrocyclic Agouti-Related Protein Antagonist Scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro] Results in Unanticipated Melanocortin-1 Receptor Agonist Profiles.

The melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), endogenous agonists derived from the proopiomelanocortin gene transcript, and naturally occurring antagonists agouti and agouti-related protein (AGRP) have been linked to biological pathways associated with energy homeostasis. The active tripeptide sequence of AGRP, Arg111-Phe112-Phe113, is located on a hypothesized ß-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was Asn or diaminopropionic acid (Dap). Macrocyclic peptides were synthesized with one, two, or three residues of the Arg-Phe-Phe sequence substituted with the corresponding d-isomer(s), generating a 14 compound library. While l-to-d inversions of the Arg-Phe-Phe sequence in a 20-residue AGRP-derived ligand previously resulted in agonist activity at the MC1R, MC3R, MC4R, and MC5R, only the MC1R was consistently stimulated by the macrocyclic ligands in the present study, with varying ligand potencies and efficacies observed at the MC1R. A general trend of increased MC4R antagonist potency was observed for Dap-containing compounds, while MC5R inverse agonist activity was observed for select ligands. It was observed that stereochemical modification of the Arg-Phe-Phe active tripeptide sequence was insufficient to convert melanocortin antagonist into agonists. Overall, these observations are important in the design of melanocortin ligands possessing potent and selective agonist and antagonist activities.

Replacement of Arg with Nle and modified D-Phe in the core sequence of MSHs, Ac-His-D-Phe-Arg-Trp-NH2, leads to hMC1R selectivity and pigmentation.

Melanoma skin cancer is the fastest growing cancer in the US [1]. A great need exists for improved formulations and mechanisms to prevent and protect human skin from cancers and other skin damage caused by sunlight exposure. Current efforts to prevent UV damage to human skin, which in many cases leads to melanoma and other skin cancers. The primordial melanocortin-1 receptor (MC1R) is involved in regulating skin pigmentation and hair color, which is a natural prevention from UV damage. The endogenous melanocortin agonists induce pigmentation and share a core pharmacophore sequence "His-Phe-Arg-Trp", and it was found that substitution of the Phe by D-Phe results in increasing melanocortin receptor potency. To improve the melanocortin 1 receptor (MC1R) selectivity a series of tetra-peptides with the moiety of Ac-Xaa-Yaa-Nle-Trp-NH2, and structural modifications to reduce electrostatic ligand-receptor interactions have been designed and synthesized. It is discovered that the tetrapeptide Ac-His-D-Phe(4-CF3)-Nle-Trp-NH2 resulted in a potent and selective hMC1R agonist at the hMC1R (EC50: 10 nM). Lizard anolis carolinensis pigmentation study shows very high potency in vivo. NMR studies revealed a reversed ß turn structure which led to the potency and selectivity towards the hMC1R.