Shubh D. Sharma

Selectivity of Cyclic [d-Nal7] and [d-Phe7] Substituted MSH Analogues for the Melanocortin Receptor Subtypes

The binding of the 2 cyclic lactam MSH (4-10) analogues (MTII, SHU9119), and 5 cyclic [Cys4, Cys10] alpha-MSH analogues were tested on cells transiently expressing the human MC1, MC3, MC4 and MC5 receptors. The results indicate a differential importance of the C-terminal (Lys-Pro-Val) and N-terminal (Ser-Tyr-Ser) of cyclic [Cys4, Cys10] alpha-MSH analogues in binding to the MC receptor subtypes. Substitution of D-Phe7 by D-Nal(2)7 in both the cyclic lactam MSH (4-10) and the cyclic disulphide MSH (4-10) analogues resulted in a shift in favour of selectivity for the MC4 receptor; the disulphide analogue, [Cys4, D-Nal(2)7 Cys10] alpha-MSH (4-10) (HS9510), showing the highest selectivity for the MC4 receptor among all the substances tested. However, the cyclic lactams displayed an over all higher affinity for the MC receptors, than any of the cyclic disulphide MSH (4-10) analogues.

Design, synthesis, and conformation of superpotent and prolonged acting melanotropins.

a-Melanotropin (a-MSH, Ac-Ser-Tyr-Ser-Met-Glus-His-Phe-Arg-T~-GlyloLys-Pro-Val-NH,) has a long and storied history, being one of the first peptide hormones to be studied as a crude extract from the pituitary and to be isolated in pure form and have its structure determined. It was early recognized to have essentially the same N-terminal tridecapeptide sequence as adrenocorticotropic hormone (ACTH, corticotropin) except that the N-terminal was acetylated in the case of a-MSH but not in the case of ACTH, indicating that their biosyntheses were different. Subsequently it was discovered that a-MSH and ACTH were derived from the same gene, currently referred to as proopiomelanocortin (POMC), of which much more is discussed elsewhere in the proceedings of this meeting. ACTH does have weak melanotropinlike activities in certain a-MSH-specific assays such as the frog skin bioassay, but the acetyl group is important to maximize the biological potency of the native a-MSH (a-MSH is about 100 times more potent than ACTH in the classical pigmentary assays). It is now understood that though a-MSH was originally discovered as a peptide hormone, affecting pigmentation in peripheral tissues especially the skin, there is much anatomical and pharmacological evidence that a-MSH is found in the brain where it and/or ACTH and its fragments have a growing list of proposed neurotransmitter, neuromodulatory, neuroimmunological, behavioral, and other biological activities. Much more is said elsewhere in the proceedings of this meeting regarding these sites of action of this remarkable hormone. This paper, which is devoted to the chemistry and structure-biological activity relationships, will focus entirely on a-MSHs activities at peripheral receptors responsible for pigmentation, since this is the primary receptor for which extensive structure-biological activity studies have been done. Structure-activity studies on a-MSH began immediately with its isolation and

Melanotropic Peptides for Therapeutic and Cosmetic Tanning of the Skin

Many animals have the ability to rapidly or more slowly change color. Skin coloration is due to pigment cells (chromatophores) present within the integument. Physiological color changes result from the rapid movement (either centrifugal or centripetal) of pigment organelles within integumental chromatophores. Slower morphological color changes result from the increased production or destruction of pigment contained within the chromatophores. The rapid chameleonlike changes of the lizard are an example of physiological color change. The annual pelage molt of the varying hare and of the weasel from a summer brown color to a winter white coloration is an example of morphological color change. Both the rapid and the slower changes in color are regulated by hormones. Humans also possess pigment cells within their skin. The variable production and packaging of melanin pigment within these cells, melanocytes, is responsible for ethnic differences in skin coloration in humans.2 The ability to obtain a tan either by sun exposure or by ultraviolet Light (tanning booth) involves an increase in melanin production within epidermal melanocytes. A question that still remains unresolved is whether, like in the rabbit or in the weasel, enhanced melanin production of the skin is under hormonal control. The evidence for or against such an argument will be reviewed in this communication. What will be demonstrated, however, is the ability of human skin to tan in response to exogenously administered melanotropic peptides.3

Prevention of reflex natriuresis after acute unilateral nephrectomy by melanocortin receptor antagonists

gamma-Melanocyte-stimulating hormone (gamma-MSH), atrial natriuretic peptide (ANP), and oxytocin have been identified as candidate hormonal mediators of the reflex natriuresis that follows acute unilateral nephrectomy (AUN). Pharmacological characterization of the third melanocortin receptor (MC3-R) indicates that it uniquely responds to physiological concentrations of gamma-MSH. We tested the roles of gamma-MSH, ANP, and oxytocin in the postnephrectomy natriuresis by carrying out AUN during continuous intrarenal infusion of specific antagonists for their cognate receptors. In anesthetized Sprague-Dawley rats, urinary sodium excretion (UNaV) increased from 0.34 +/- 0.04 to 1.12 +/- 0.11 mu eq/min 90 min after AUN (P < 0.001). No change in UNaV occurred in rats undergoing a sham AUN procedure. Plasma immunoreactive gamma-MSH concentration was 53 +/- 8 fmol/ml after sham AUN but 112 +/- 17 fmol/ml after AUN (P < 0.01). SHU-9119 and SHU-9005 are substituted derivatives of alpha-MSH with potent antagonism at the MC3-R in vitro. Infusion of these compounds at 5 pmol/min completely blocked the natriuretic response to AUN despite a similar elevation in plasma gamma-MSH (111 +/- 12 vs. 49 +/- 8 fmol/ml in sham rats, P < 0.01). Intrarenal infusion of the ANP receptor antagonist A-71915 (5 pmol/min) or the oxytocin receptor antagonist [d(CH2)(5)1, Tyr(Me)2,Orn8] vasotocin (10 pmol/min) effectively inhibited the natriuresis induced by intravenous infusion of ANP or oxytocin (each at 1 pmol/min), respectively, but did not block the natriuresis after AUN. Plasma immunoreactivity of these peptides was not increased after AUN. These results indicate that reflex natriuresis after AUN is accompanied by an increase in plasma gamma-MSH but not ANP or oxytocin concentration and is prevented by intrarenal infusion of receptor antagonists with selectivity for MC3-R. The data indicate that gamma-MSH or a closely related peptide mediates postnephrectomy natriuresis and provide further support for the possibility that gamma-MSH may play a wider role in sodium homeostasis.γ-Melanocyte-stimulating hormone (γ-MSH), atrial natriuretic peptide (ANP), and oxytocin have been identified as candidate hormonal mediators of the reflex natriuresis that follows acute unilateral nephrectomy (AUN). Pharmacological characterization of the third melanocortin receptor (MC3-R) indicates that it uniquely responds to physiological concentrations of γ-MSH. We tested the roles of γ-MSH, ANP, and oxytocin in the postnephrectomy natriuresis by carrying out AUN during continuous intrarenal infusion of specific antagonists for their cognate receptors. In anesthetized Sprague-Dawley rats, urinary sodium excretion (UNaV) increased from 0.34 ± 0.04 to 1.12 ± 0.11 μeq/min 90 min after AUN ( P < 0.001). No change in UNaV occurred in rats undergoing a sham AUN procedure. Plasma immunoreactive γ-MSH concentration was 53 ± 8 fmol/ml after sham AUN but 112 ± 17 fmol/ml after AUN ( P < 0.01). SHU-9119 and SHU-9005 are substituted derivatives of α-MSH with potent antagonism at the MC3-R in vitro. Infusion of these compounds at 5 pmol/min completely blocked the natriuretic response to AUN despite a similar elevation in plasma γ-MSH (111 ± 12 vs. 49 ± 8 fmol/ml in sham rats, P < 0.01). Intrarenal infusion of the ANP receptor antagonist A-71915 (5 pmol/min) or the oxytocin receptor antagonist [d(CH2)51, Tyr(Me)2,Orn8] vasotocin (10 pmol/min) effectively inhibited the natriuresis induced by intravenous infusion of ANP or oxytocin (each at 1 pmol/min), respectively, but did not block the natriuresis after AUN. Plasma immunoreactivity of these peptides was not increased after AUN. These results indicate that reflex natriuresis after AUN is accompanied by an increase in plasma γ-MSH but not ANP or oxytocin concentration and is prevented by intrarenal infusion of receptor antagonists with selectivity for MC3-R. The data indicate that γ-MSH or a closely related peptide mediates postnephrectomy natriuresis and provide further support for the possibility that γ-MSH may play a wider role in sodium homeostasis.

Melanotropic peptide receptors: membrane markers of human melanoma cells

Abstract The objectives of this research were to determine whether melanotropin receptors are characteristic (constant) membrane markers of human melanoma cells. Methodologies were developed to visualize these receptors by fluorescence microscopy. Multiple copies (10–20) of both [Nle4.D‐Phe7]α‐MSH, a superpotent analog of α‐melanocyte stimulating hormone (α‐MSH). and a fluorophore, were conjugated to polyvinyl alcohol (PVA). Incubation in the presence of the multivalent macromolecular conjugate (FITC‐PVA‐MSH) resulted in binding of human epidermal melanocytes and keratinocytes and human melanoma cells (both melanotic and amelanotic) to the fluorescent conjugate. Binding of the conjugate to the cells exhibited a unique cluster pattern (capping) suggesting a receptor internalization related phenomenon. Most importantly, every cell of every melanoma cell line, melanotic or amelanotic. possessed receptors as visualized by fluorescence microscopy. Since the cells were not synchronized, some binding apparently took place during all phases of the cell cycle. Therefore, receptor expression appears not to be cell‐cycle dependent. Specificity of binding of FITC‐PVA‐MSH was demonstrated by several studies, (i) Binding of the conjugate to melanoma cells could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog: [Nle4,D‐Phe7]α‐MSH. (ii) The macromolecular conjugate lacking bound ligand (FITC‐PVA) did not bind to the melanoma cells, (iii) Another peptide, a substance‐P analog, attached to the substrate (FITC‐PVA‐SP) failed to bind to the cells, (iv) With the exception of keratinocytes, other cells of nonmelanocyte origin (e.g., fibroblasts, spleen, liver, kidney cells, and mammary cancer cells, lung cancer cells) did not bind to the conjugate. Thus, cell‐specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes, keratinocytes, and melanoma cells. In several human melanoma cell lines these receptors appeared to be functional since [Nle4,α‐Phe7]α‐MSH stimulated tyrosinase activity. Fluorescent melanotropin conjugates might prove useful in determining whether all human melanoma (primary and metastatic) tumors possess such receptors. These receptors might then provide targets for melanotropic peptides for the identification, localization, and chemotherapy of melanoma.

Preclinical gastrointestinal prokinetic efficacy and endocrine effects of the ghrelin mimetic RM-131

AIMSnThe 28 amino acid hormone ghrelin, the natural ligand for the growth hormone secretagogue, or ghrelin receptor (GHR), has diverse physiological functions, including a possible role as a gastrointestinal prokinetic. The synthetic ghrelin mimetic RM-131 is in Phase II clinical trials for treatment of diabetic gastroparesis and other gastrointestinal (GI) disorders. We aimed to determine the relative potency of RM-131, when compared to other GI ghrelin mimetics, to predict efficacy and determine the role of RM-131 in models of inflammatory bowel disease.nnnMAIN METHODSnWe evaluated and compared ghrelin, RM-131 and other synthetic ghrelin mimetics for their prokinetic potency in models of gastrointestinal disorders in the rat and we evaluated the endocrine (rats and dogs) and anti-inflammatory effects (mice) of the ghrelin mimetic RM-131.nnnKEY FINDINGSnThe pentapeptide RM-131 increased gastric emptying in rodent models of ileus. RM-131 is about 100-fold more potent than human ghrelin and is 600 to 1800-fold more potent, when compared to several investigational ghrelin mimetics tested in clinical trials. RM-131 has anti-inflammatory effects and significantly increases survival and reduces macroscopic markers of tissue damage in a TNBS model of inflammatory bowel disease. RM-131 treatment shows a transient increase in growth hormone levels in Beagle dogs and rats, returning to baseline upon chronic treatment. Significant effects on glucose and insulin are not observed in chronic studies.nnnSIGNIFICANCEnRM-131s potency, efficacy and endocrine profile, are promising attributes for the treatment of diverse functional gastrointestinal disorders in humans.

Characterisation of D117A and H260A mutations in the melanocortin 1 receptor

Recent site directed mutagenesis studies on the melanocortin 1 (MC1) receptor have indicated the importance of D117 and H260 amino acid residues for the binding of alpha-MSH (melanocyte stimulating hormone). Here, we report the testing of 12 cyclic and linear MSH peptides on the D117A and H260A mutant receptors. Moreover, we constructed a double mutant which displayed a major loss in affinity for [Nle4, D-Phe7]alpha-MSH. Our new data of His6 and Phe7 substituted MSH peptides are compared with previous results and the hypothesis of putative interactions of D117 and H260 with single amino acids in the MSH peptide. Our conclusions are that the D117A and the H260A mutations may cause conformational changes in the receptor which can not be linked to any specific amino acid in the MSH-peptides.

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for alpha-MSH (alpha-melanocyte stimulating hormone, Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9- Gly10-Lys11-Pro12-Val13-NH2) and [D-Phe7] alpha-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly10 residue, as substitutions for L-Ala, D-Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of alpha-MSH(4-11) and alpha-MSH(5-11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly10 residue with L-Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp message sequence within the sequences of alpha-MSH and [D-Phe7]alpha-MSH. In such conformations the aromatic moieties of the side chains of the His6, L/D-Phe7, and Trp9 residues form a continuous hydrophobic surface, presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for alpha-MSH and [D-Phe7]alpha-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations.

Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency.

Objective Pro-opiomelanocortin (POMC)-derived peptides act on neurons expressing the Melanocortin 4 receptor (MC4R) to reduce body weight. Setmelanotide is a highly potent MC4R agonist that leads to weight loss in diet-induced obese animals and in obese individuals with complete POMC deficiency. While POMC deficiency is very rare, 1–5% of severely obese individuals harbor heterozygous mutations in MC4R. We sought to assess the efficacy of Setmelanotide in human MC4R deficiency. Methods We studied the effects of Setmelanotide on mutant MC4Rs in cells and the weight loss response to Setmelanotide administration in rodent studies and a human clinical trial. We annotated the functional status of 369 published MC4R variants. Results In cells, we showed that Setmelanotide is significantly more potent at MC4R than the endogenous ligand alpha-melanocyte stimulating hormone and can disproportionally rescue signaling by a subset of severely impaired MC4R mutants. Wild-type rodents appear more sensitive to Setmelanotide when compared to MC4R heterozygous deficient mice, while MC4R knockout mice fail to respond. In a 28-day Phase 1b clinical trial, Setmelanotide led to weight loss in obese MC4R variant carriers. Patients with POMC defects upstream of MC4R show significantly more weight loss with Setmelanotide than MC4R deficient patients or obese controls. Conclusions Setmelanotide led to weight loss in obese people with MC4R deficiency; however, further studies are justified to establish whether Setmelanotide can elicit clinically meaningful weight loss in a subset of the MC4R deficient obese population.

Human epidermal melanocyte and keratinocyte melanotropin receptors: visualization by melanotropic peptide conjugated macrospheres (polyamide beads)

Abstract The objectives of this research were to determine whether melanotropin receptors are characteristic membrane markers of human epidermal melanocytes. Methodologies were developed to visualize these receptors by light microscopy. Multiple copies (up to a thousand) of [Nle4,D‐Phe7]α‐MSH, a superpotent analog of α‐melanocyte stimulating hormone (α‐MSH), were conjugated to a macromolecular carrier, large polyamide beads (macrospheres). Incubation in (the presence of the I conjugated macrospheres resulted in binding of human epidermal melanocytes to the macrospheres. Specificity of the binding of melanocytes of the melanotro‐pin‐conjugated macrospheres was demonstrated by several studies: (i) Binding of melanocytes to the conjugate was specific since it could be blocked by prior incubation of the cells in the presence of (the unconjugated hormone analog: (ii) The macrospheres after removal of the bound ligand did not bind to the melanocytes: (iii) Another peptide hormone ligand (e.g., a substance‐P analog) attached to the macrospheres failed to bind to the melanocytes: (iv) BI6/F10 mouse melanoma cells known lo express melanotropin receptors bound to the macrospheres; (v) Cells of nomnelanocyte origin (e.g., mammary cancer cells, lung cancer cells, fibroblasts) did not bind lo the macrospheres. One exception was that human epidermal keratinocytes also expressed melanotropin receptors as determined by all the criteria established for epidermal melanocyles. Thus, cell specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.