Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

Blockade of MCH1 receptor signalling ameliorates obesity and related hepatic steatosis in ovariectomized mice.

BACKGROUND AND PURPOSE: Melanin-concentrating hormone (MCH) is a cyclic orexigenic neuropeptide predominantly expressed in the lateral hypothalamus. We investigated the roles of MCH1 receptor signalling in ovariectomy (OVX)-induced obesity in female C57BL/6J mice, an animal model of postmenopausal obesity. EXPERIMENTAL APPROACH: The effects of blocking signalling via the MCH1 receptor on OVX-induced obesity was investigated by using Mch1r deficient (KO) mice and chronic treatment with a selective MCH1 receptor antagonist. KEY RESULTS: OVX induced body weight gain and increases in the weight of visceral fat and of liver; these effects were attenuated following OVX in Mch1r KO mice. OVX-induced triglyceride (TG) accumulation and elevated expression of lipogenic genes were significantly ameliorated in the liver of Mch1r KO mice. In agreement with these results, chronic i.c.v. infusion of a selective MCH1 receptor antagonist significantly reduced body weight gain, visceral fat and liver weights in OVX mice, and hepatic TG contents and lipogenic gene expression levels were normalized. CONCLUSION AND IMPLICATIONS: Our results indicate that MCH1 receptor signalling is involved in the development of fatty liver, as well as obesity, in OVX mice, and suggest a therapeutic potential for MCH1 receptor antagonists in the treatment of obesity and fatty liver.

1H NMR structural analysis of human ghrelin and its six truncated analogs.

Human ghrelin, the first recognized natural ligand of growth hormone secretagogue growth hormone secretagogue receptors (GHS-Rs) (M. Kojima, H. Hosada, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa, Nature, 1999, Vol. 402, pp. 656-660), consists of 28 amino acids of which Ser3 is modified by n-octanoylation. This new peptide hormone has been implicated not only in regulation of the GH secretion but also in regulation of food intake. The discovery of ghrelin opens up more opportunities to study the relationship of ghrelin with metabolic diseases. Until now, only mass spectometry analysis has been reported on the structure of ghrelin. NMR analysis is a suitable way to study if any tertiary structure of unbound ghrelin is present in solution. NMR studies were carried out on human ghrelin and its five truncated analogs. The full-length ghrelin and its fragments exhibited random coil behavior in aqueous solution. Additional studies were carried out on the shortest active segment of human ghrelin, which consists of the first five amino acids of the ghrelin sequence (M. A. Bednarek, S. D. Feighner, S.-S. Pong, K. K. McKee, D. L. Hreniuk, M. V. Silva, V. A. Warrem, A. D. Howard, L. H. Y. Van der Ploeg, and J. V. Heck, Journal of Medical Chemistry, 2000, Vol. 43, pp. 4370-4376), to compare the spectral features with their counterparts in the full-length ghrelin. The NMR data showed behavior similar to ghrelin except for two additional nuclear Overhauser effects (NOEs) between the Phe4 NH and the protons of the beta-methylene of Ser3. CD on human ghrelin and its short active analog in water were indicative of random coil peptides. Molecular modeling based on NMR data was carried out to probe which structural features were similar to growth hormone-releasing peptide-6 (GHRP-6), a hexapeptide that binds to GHS-R releasing GH and stimulating food intake. Modeling suggested some similarities, but they were not of a nature to account for binding properties of these compounds.

Selective, high affinity peptide antagonists of alpha-melanotropin action at human melanocortin receptor 4: their synthesis and biological evaluation in vitro.

Peptide Ac-Nle(4)-cyclo(5beta-->10epsilon)(Asp(5)-His(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), compound 1, a cyclic derivative of alpha-melanotropin, is a nonselective high affinity antagonist at human melanocortin receptors 3 and 4, and an agonist at melanocortin receptors 1 and 5. To differentiate between the physiological functions of these receptors, antagonists with improved receptor selectivity are needed. In this study, analogues of compound 1 without Ac-Nle(4) or His(6) and/or the amino group of Asp(5) were prepared and tested in binding assays and in functional assays on CHO cells expressing hMC3-5R. Several of these peptides were to be selective, high affinity hMC-4R antagonists. The most interesting was compound 10, named MBP10, cyclo(6beta-->10epsilon)(succinyl(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), an antagonist (IC(50) = 0.5 nM) with 125-fold selectivity over hMC-3R (and of >300-fold selectivity over MC-1RB). This compound had no agonist activity at hMC-3R or hMC-4R and only weak agonist activity at hMC-5R. Examination of the sequences of these new peptides revealed that the D-(2')Nal(7)-Arg(8)-Trp(9) segment of peptide 1 forms the "essential core" required for high affinity and high selectivity of analogues of peptide 1 at hMC-4R, but the "extended core", His(6)-D-(2')Nal(7)-Arg(8)-Trp(9), is necessary for the maximum affinity for hMC-3R and hMC-5R.

Short segment of human melanin-concentrating hormone that is sufficient for full activation of human melanin-concentrating hormone receptors 1 and 2.

Human melanin-concentrating hormone (hMCH) is a potent but nonselective agonist at human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R, respectively). To determine the structural features of this neuropeptide which are necessary for efficient binding to and activation of the receptors, Ala-substituted, open-chain, and truncated analogues were synthesized and tested in the binding assays in CHO cells expressing hMCH-1R and hMCH-2R, and in functional assays measuring the level of intracellular calcium mobilization in human HEK-293 cells expressing these receptors. A compound consisting merely of the cyclic core of hMCH with the Arg attached to the N-terminus of the disulfide ring was found to activate both hMCH-1R and hMCH-2R about as effectively as full-length hMCH. Thus, the sequence Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys) appears to constitute the "active core" that is necessary for agonist potency at hMCH-1R and hMCH-2R. A potent and approximately 4-fold more selective agonist at hMCH-1R than at hMCH-2R is also reported.

Potent and selective peptide agonists of alpha-melanotropin action at human melanocortin receptor 4: their synthesis and biological evaluation in vitro.

alpha-Melanotropin (alphaMSH) and several of its derivatives are potent but not selective agonists at melanocortin receptors 3, 4, and 5 present in the brain (MC3-5R). To differentiate between the physiological role of hMC-4R (believed to be involved in regulation of energy balance) from those of melanocortin receptors 3 and 5, potent and receptor-specific agonists are needed. Therefore, the cyclic derivatives of alphaMSH of a general structure, cyclo(X-His-d-Phe-Arg-Trp-Y)-NH(2), where X is succinic acid or an omega-amino-carboxylic acid, and Y is an alpha,omega-di-amino-carboxylic acid or an omega-carboxy-alpha-amino acid, were prepared and tested in binding assays and in cAMP assays on CHO cells expressing hMC3-5R. Several of the 21-membered or larger lactams turned out to be potent and hMC-4R-selective agonists. For instance, cyclo(CO-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Dab)-NH(2) (Dab: 2,4-di-amino-butyric acid) was a potent agonist at hMC-4R (EC(50) = 4 nM) with 55-fold selectivity over hMC-3R and greater than 1000-fold selectivity over hMC-5R. Another potent and selective compound was cyclo(NH-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Glu)-NH(2): EC(50) about 1 nM at hMC-4R, with 90-fold selectivity over hMC-3R and greater than 2000-fold selectivity over hMC-5R.

Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a.

The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.

Analogs of lactam derivatives of alpha-melanotropin with basic and acidic residues.

A role of the aromatic and of the basic residues of the potent agonist (MTII) and antagonist (SHU9119) at the human melanocortin receptors 4 in the formation and stabilization of ligand-receptor complexes was examined. Analogs of MTII and SHU9119 with glutamic acid replacing one amino acid at a time were synthesized and tested for their ability to bind to and activate human melanocortin receptors 3, 4, and 5. Replacement of Phe (Nal) or Trp with Glu resulted in analogs of MTII and SHU9119 which were practically inactive at the receptors studied. The rather large (and unexpected) tolerance toward the presence of Glu in the position of His or Arg of MTII and SHU9119 clearly suggested that in the ligand receptor complexes these basic residues are not in contact with the receptors but probably face the extracellular environment. This identified the aromatic residues of MTII and SHU9119 as the primary structural features determining interactions of the agonist/antagonist with hMCR3-5.

Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.

Structure-function studies on the cyclic peptide MT-II, lactam derivative of alpha-melanotropin.

The alanine-substituted and the retro, enantio, and retro-enantio analogs of MT-II, a potent agonist at melanocortin (MC) receptors, were prepared by solid-phase synthesis and evaluated for their ability to bind and activate human MC3, MC4, and MC5 receptors. Replacement of His with Ala resulted in [Ala6]-MT-II with affinity and agonist potency at human MC3, MC4, and MC5 receptors similar to MT-II. Substitution of Arg with Ala gave compound 100-fold less potent than MT-II, but replacement of Phe or Trp with Ala led to inactive compounds (at the micromolar concentrations). The significant drop of potency of the retro, enantio, and retro-enantio analogs of MT-II, demonstrated a crucial role of side-chain topology, and to a lesser degree, of peptide backbone in interactions of MT-II with the melanocortin receptors. The nuclear magnetic resonance analysis of MT-II suggested involvement of Phe and Arg residues in H-bonds stabilizing the bent conformations of the peptide backbone.

Analogs of MTII, lactam derivatives of alpha-melanotropin, modified at the N-terminus, and their selectivity at human melanocortin receptors 3, 4, and 5.

In search for selective agonists at human melanocortin-4 receptor, proline-substituted analogs of MTII, a potent nonselective agonist at melanocortin receptors, were prepared by solid-phase syntheses and evaluated for their ability to bind and activate human MC-3, MC-4, and MC-5 receptors. Replacement of Nle(4) with Pro resulted in [Pro(4)]MTII with affinity to and agonist potency at hMC-4R similar to MTII, but with about 400-fold lower potency at hMC-5R and about 20-fold lower potency at hMC-3R. The substantial increase in selectivity of [Pro(4)]MTII with respect to hMC-5R prompted us to investigate additional analogs of MTII with modified N-termini. The Ac-Nle(4) segment, not encompassed in the lactam ring, was substituted with flexible, hydrophobic, or hydrophilic substituents, and also, with residues resembling proline. The similar agonist potency of these peptides to that of MTII at hMC-4R but significantly lower activity of these compounds at hMC-5R demonstrated that the N-terminal fragment of MTII has virtually no effect on the binding affinity and activation at hMC-4R, but it is essential for full potency at hMC-5R.

Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4.

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.

Amino acids with aryl-keto function in their side chains.

Amino acids with aryl-keto function in their side-chains were obtained in excellent yields in the reaction of omega-carboxyamino acids with liquid HF in the presence of aromatic compounds susceptible to electrophilic substitution, such as anisole, 2-methoxybiphenyl, butyl phenyl ether or 1,3-dimethoxybenzene. The new amino acids were converted smoothly into N-tert-butyloxycarbonyl or N-fluorenylmethoxycarbonyl derivatives and then incorporated into peptides by conventional coupling methods. During the coupling step, no formation of cyclic Schiff bases was observed for aryl-keto amino acids derived from DL-alpha-aminopimelic acid and from L-alpha-aminosuberic acid. In the crude products, truncated peptides terminated at the keto amino acids were not detected by liquid chromatography-mass spectrometry.

Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase.

A novel fungal metabolite, apicidin [cyclo(N-O-methyl-L-tryptophanyl-L -isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], that exhibits potent, broad spectrum antiprotozoal activity in vitro against Apicomplexan parasites has been identified. It is also orally and parenterally active in vivo against Plasmodium berghei malaria in mice. Many Apicomplexan parasites cause serious, life-threatening human and animal diseases, such as malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis, and new therapeutic agents are urgently needed. Apicidin's antiparasitic activity appears to be due to low nanomolar inhibition of Apicomplexan histone deacetylase (HDA), which induces hyperacetylation of histones in treated parasites. The acetylation-deacetylation of histones is a thought to play a central role in transcriptional control in eukaryotic cells. Other known HDA inhibitors were also evaluated and found to possess antiparasitic activity, suggesting that HDA is an attractive target for the development of novel antiparasitic agents.

Observation of noncovalent complexes between margatoxin and the Kv1.3 peptide ligands: A model investigation using ion-spray mass spectrometry.

Margatoxin (MgTX), a 39 amino acid peptide present in the venom of the new world scorpion Centruroides margaritatus, is a potent inhibitor of the voltage-gated potassium channel (Kv1.3) in human peripheral T lymphocytes. Peptide analogs corresponding to the amino acid segments that are located at the rat Kv1.3 putative binding site for the ion channel blockers were synthesized. Gas phase noncovalent complexes of the synthetic analogs of the rat Kv1.3 peptide ligands with MgTX were detected using ion-spray mass spectrometry.

Coupling in the absence of tertiary amines. V. Removal of the 2-nitrobenzenesulfenyl (NPS) group with 1-hydroxybenzotriazole in the presence of aniline or N-methylaniline.

In a search for conditions of acidolytic removal of amine protecting groups leading to salts of the deblocked amine that can be acylated without addition of a tertiary amine, cleavage of the 2-nitrobenzenesulfenyl (Nps) group with hydroxybenzotriazole (HOBt) in 2,2,2-trifluoroethanol was attempted. The Nps group was smoothly removed, but the resulting salt of the amine component could not be acylated unless deprotonated with a tertiary base. A rationale is now proposed for this unsatisfactory outcome of the cleavage reaction and for the concomitant surprising reduction of HOBt to benzotriazole. Based on the proposed mechanism, a new approach was designed for the removal of the Nps group. It was cleaved with HOBt in the presence of weakly basic nucleophiles such as aniline, N-methylaniline or 8-aminoquinoline. The protecting group was transferred smoothly to the amino group of the nucleophilic acceptor leaving the deblocked amine component in the form of its HOBt salt. This was then readily acylated without addition of a tertiary amine.

Chemical synthesis and structure-function studies of margatoxin, a potent inhibitor of voltage-dependent potassium channel in human T lymphocytes.

The 39 amino acid peptide, margatoxin (MgTX), a potent inhibitor of the voltage-activated potassium channel (Kv 1.3) in human T lymphocytes, was synthesized by a solid phase technique. Formation of the disulfide bridges was rapid at pH 8.2. The final product was purified to homogeneity and was physically and biologically indistinguishable from the toxin prepared biosynthetically. The disulfide bridge pairing was similar to that found previously for the related toxin-charybdotoxin (3): from Cys7 to Cys29, from tested for inhibition of 125I margatoxin binding to voltage-activated potassium channels. The results indicate that the three C-terminal residues of MgTX are important for the efficient toxin binding to Kv1.3.

Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains.

A method to predict the relative binding strengths of all possible nonapeptides to the MHC class I molecule HLA-A2 has been developed based on experimental peptide binding data. These data indicate that, for most peptides, each side-chain of the peptide contributes a certain amount to the stability of the HLA-A2 complex that is independent of the sequence of the peptide. To quantify these contributions, the binding data from a set of 154 peptides were combined together to generate a table containing 180 coefficients (20 amino acids x 9 positions), each of which represents the contribution of one particular amino acid residue at a specified position within the peptide to binding to HLA-A2. Eighty peptides formed stable HLA-A2 complexes, as assessed by measuring the rate of dissociation of beta 2m. The remaining 74 peptides formed complexes that had a half-life of beta 2m dissociation of less than 5 min at 37 degrees C, or did not bind to HLA-A2, and were included because they could be used to constrain the values of some of the coefficients. The "theoretical" binding stability (calculated by multiplying together the corresponding coefficients) matched the experimental binding stability to within a factor of 5. The coefficients were then used to calculate the theoretical binding stability for all the previously identified self or antigenic nonamer peptides known to bind to HLA-A2. The binding stability for all other nonamer peptides that could be generated from the proteins from which these peptides were derived was also predicted. In every case, the previously described HLA-A2 binding peptides were ranked in the top 2% of all possible nonamers for each source protein. Therefore, most biologically relevant nonamer peptides should be identifiable using the table of coefficients. We conclude that the side-chains of most nonamer peptides to the first approximation bind independently of one another to the HLA-A2 molecule.

Recognition by HLA-A2-restricted cytotoxic T lymphocytes of endogenously generated and exogenously provided synthetic peptide analogues of the influenza A virus matrix protein.

Experiments were carried out to determine whether complexes between MHC class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensitized with exogenously provided and endogenously generated peptide analogues of the optimal nonameric peptide 58-66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading was accomplished by expressing minigene DNA coding for alanine-substituted analogues of peptide 58-66 in HLA-A2-positive cells. Susceptibility to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocytes was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were observed. The endogenously presented analogues 58-66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenously presented analogues. This difference in recognition was most striking for peptide 58-66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Additional experiments with endogenously expressed analogues of 58-66 with substitutions other than alanine were carried out to define the interaction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these interactions contribute to peptide binding.

Reactions of C-terminal omega-amino acid residues in liquid hydrogen fluoride.

The benzyl-ester bond linking the C-terminal delta-aminovaleric acid residue of a peptide to a polymeric support was cleaved with liquid hydrogen fluoride in the presence of anisole, added as scavenger. Instead of the expected peptide with a free carboxyl group at the C-terminus, a peptide terminating in a ketone derivative was obtained. The unusual extent of this known side-reaction was attributed to the effect of the distance between the amino group and the carboxyl group in the C-terminal residue. The results of model experiments corroborated this view.