Dev Trivedi

Metabolic effects and cyclic AMP levels produced by glucagon, (1-Nα-trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes

[1-N alpha-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested.

Design and Microwave-Assisted Synthesis of Novel Macrocyclic Peptides Active at Melanocortin Receptors: Discovery of Potent and Selective hMC5R Receptor Antagonists

Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin receptors will require development of high affinity and selective antagonists. To date, a few synthetic antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized, and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues, compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9(PG-20N) is a selective and competitive hMC5R antagonist, with IC 50 of 130 +/- 11 nM, and a pA 2 value of 8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and 7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC 50 values of 38 +/- 3 nM and 58 +/- 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand the physiological roles played by the MC5 receptor in humans and other animals.

Design and synthesis of novel χ2-constrained phenylalanine, naphthylalanine, and tryptophan analogues and their use in biologically active melanotropin peptides

A series of novel hydrophobic, bulky χ2-constrained phenylalanine, naphthylalanine, and tryptophan derivatives was designed and synthesized. The key steps involved asymmetric hydrogenations of α-enamides using Burks DuPHOS-based Rh(I) catalysts to give high enantiomerically pure α-amino acid derivatives. The subsequent Suzuki cross couplings of the amino acid analogues with boronic acid derivatives afforded these aromatic substituted amino acids in high yields and with high enantioselectivity. The incorporation of these novel χ2-constrained amino acids into peptides and peptidomimetics provides fruitful information in the development of peptide and peptidomimetic ligands of melanotropins and an understanding of the interactions between ligands and receptors/acceptors.

Design of novel melanotropin agonists and antagonists with high potency and selectivity for human melanocortin receptors

alpha-MSH and gamma-MSH are the natural endogenous hormones for the human melanocortin-1, 3, 4 and 5 receptors (hMC1R, hMC3R, hMC4R and hMC5R). These and more potent, stable and prolonged acting analogues such as NDP-alpha-MSH, MT-II and SHU-9119 are not very receptor selective. To develop potent and selective agonist and antagonist ligands for the melanocortin receptors we have used state-of-the-art biophysical studies, computational chemistry, and design of conformational and topographical constraints with novel templates.

Pure Glucagon Antagonists: Biological Activities and cAMP Accumulation Using Phosphodiesterase Inhibitors

Five new glucagon analogues have been designed, synthesized, characterized and their biological activities tested. The investigation was centered on modifications in the N-terminal region in particular, residues at Thr5, Phe6 and Tyr10 positions, with the goal of obtaining pure glucagon antagonists in our newly developed high sensitivity cAMP accumulation assay. The structures of the designed compounds are: [des-His1, des-Phe6, Glu9] glucagon-NH2 (1); [des-His1, des-Phe6, Glu9, Phe10]glucagon-NH2 (2); [des-His1, Tyr5, des-Phe6, Glu9]glucagon-NH2 (3); [des-His1, Phe5, des-Phe6, Glu9]glucagon-NH2 (4) and [des-His1, des-Phe6, Glu9, D-Arg18]glucagon-NH2 (5). The binding potencies IC50 values in (nM) were 48.0, 27.4, 26.0, 20.0 and 416.0, respectively. All of these analogues when tested in the classical adenylate cyclase assay demonstrate antagonist properties, and in competition experiments, all caused a rightward-shift of the glucagon stimulated adenylate cyclase dose-response curve. The pA2 values for these analogues were 8.20 (1); 6.25 (2); 6.10 (3); 6.25 (4); and 6.08 (5), respectively. A newly revised assay has been developed to determine the intracellular cAMP accumulation levels in hepatocytes at the highest possible sensitivity. Four of the five glucagon analogues in this report (analogues 1, 2, 4 and 5), did not activate the adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 microM, and thus are pure antagonists.

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G‐protein‐coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine‐labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two‐photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists‐bound melanocortin receptors, on the other hand, did not recruit β‐arrestins, and remained in the cell membrane even after long‐term (30 min) treatment. Agonist‐mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin‐dependent endocytosis, but not to caveolae inhibitors. In summary, agonist‐mediated internalization of all subtypes of melanocortin receptors are dependent upon β‐arrestin‐mediated clathrin‐coated pits, whereas, β‐arrestin‐2 conjugated green fluorescence protein (β‐arrestin‐2‐GFP) recruitment is not dependent on protein kinase A activation. Real time two‐photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

Exploring the Stereostructural Requirements of Peptide Ligands for the Melanocortin Receptors

Abstract: The melanotropin peptides α‐MSH, γ‐MSH, and β‐MSH are believed to be the natural ligands for the four melanocortin receptors, MC1R, MC3R, MC4R, and MC5R. However, these peptides generally have low selectivity for these receptors. We report on some approaches to the development of selective agonists and antagonists peptide ligands for these receptors.

Effects of macrocycle size and rigidity on melanocortin receptor-1 and -5 selectivity in cyclic lactam α-melanocyte-stimulating hormone analogs

The effects of the linker arm rigidity and size on melanocortin receptor selectivity were explored in a series of compounds using cyclic lactam α‐melanocyte‐stimulating hormone template. A variety of dicarboxylic acid linkers introduced between the α‐amino group of His6 and the ɛ‐amino group of Lys10 lead to high‐affinity, selective human melanocortin receptor‐1 and ‐5 (hMC1R and hMC5R) antagonists. The incorporation of hydrophilic functions into the linker arm was found to be unfavorable for both binding potency and receptor selectivity. Analogs 8 and 9 containing highly conformationally constrained hydrophobic linkers (m‐ and p‐phthalic acids) were found to be selective nanomolar range hMC1R antagonists (IC50 = 7 and 4 nm, respectively), whereas the employment of a small conformationally constrained linker (maleic acid) resulted in a high‐affinity (IC50 = 19 nm) and selective hMC5R antagonist (analog 12). These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin‐1 and ‐5 receptors.

[des His1, des Phe6, Glu9]glucagon amide: A newly designed "pure" glucagon antagonist

Abstract We report the synthesis and biological activity of a new glucagon analog that was designed as a glucagon receptor antagonist by appropriate modifications in the N-terminal region of glucagon. The structure of the new analog is [des His 1 , des Phe 6 , Glu 9 ]glucagon amide , and its binding potency IC 50 value of 48 nM. The compound was found to be a pure antagonist in a new much more sensitive assay for glucagon stimulated cAMP accumulation activity and showed a pA 2 value of 8.20 in this assay. We report the sythesis and biological activity of a new glucagon analog that was designed as a glucagon receptor antagonist. The new analog, [ des His 1 , des Phe 6 , Glu 9 ]glucagon, amide , was found to be a pure antagonist in a new more sensitive assay for partial agonist activity, with a binding potency IC 50 of 48 nM and a pA 2 valueof 8.20.

Design of novel melanocortin receptor ligands: multiple receptors, complex pharmacology, the challenge.

The major pharmacophore for the melanocortin 1, 3, 4 and 5 receptors is the sequence -His-Phe-Arg-Trp-. There is a need for potent, biologically stable, receptor selective ligands, both agonists and antagonists, for these receptors. In this report we briefly examine the structural and biophysical approaches we have taken to develop selective agonist and antagonist ligands that can cross (or not) the blood brain barrier. Remaining questions and unmet needs are also discussed.