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The melanocortin pathway is an important participant in the regulation of skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. Melanocortin agonists contain the putative sequence ‘His-Phe-Arg-Trp’, which has been designated as the ‘message’ sequence for melanocortin peptides, and this sequence has been hypothesized to adopt a bioactive reverse turn conformation. Exploring the relationship between its structure and biological activity, we report the synthesis and evaluation of seven aza-analogs of the potent melanocortin receptor agonist Ac-His-d-Phe-Arg-Trp-NH2. Aza-amino acids, in which the α-carbon was replaced by nitrogen, were inserted along the peptide sequence to probe the importance of local configuration and turn conformation on the biology of this tetrapeptide. Although systematic substitution of aza-amino acids for thed-Phe and Arg residues led to a significant loss of activity relative to the parent peptide for all melanocortin receptor subtypes examined, substitution of aza-amino acids at the C-terminal Trp residue gave analogs equipotent to the parent peptide. In summary, the aza-scan has demonstrated that recognition of this tetrapeptide by the melanocortin receptors is particularly sensitive to modifications of configuration and conformation at thed-Phe and Arg residues versus the Trp amino acid. In light of aza-residues imparting resistance from enzymatic degradation, C-terminal aza-amino acid analogs may be used to design new peptide mimics with enhanced metabolic stability. [ABSTRACT FROM AUTHOR]

The melanocortin pathway is an important participant in the regulation of skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. Melanocortin agonists contain the putative sequence ‘His-Phe-Arg-Trp’, which has been designated as the ‘message’ sequence for melanocortin peptides, and this sequence has been hypothesized to adopt a bioactive reverse turn conformation. Exploring the relationship between its structure and biological activity, we report the synthesis and evaluation of seven aza-analogs of the potent melanocortin receptor agonist Ac-His-d-Phe-Arg-Trp-NH2. Aza-amino acids, in which the α-carbon was replaced by nitrogen, were inserted along the peptide sequence to probe the importance of local configuration and turn conformation on the biology of this tetrapeptide. Although systematic substitution of aza-amino acids for thed-Phe and Arg residues led to a significant loss of activity relative to the parent peptide for all melanocortin receptor subtypes examined, substitution of aza-amino acids at the C-terminal Trp residue gave analogs equipotent to the parent peptide. In summary, the aza-scan has demonstrated that recognition of this tetrapeptide by the melanocortin receptors is particularly sensitive to modifications of configuration and conformation at thed-Phe and Arg residues versus the Trp amino acid. In light of aza-residues imparting resistance from enzymatic degradation, C-terminal aza-amino acid analogs may be used to design new peptide mimics with enhanced metabolic stability. [ABSTRACT FROM AUTHOR]

We previously reported Chalcone-4 (1) that binds the chemokine CXCL12, not its cognate receptors CXCR4 or CXCR7, and neutralizes its biological activity. However, this neutraligand suffers from limitations such as poor chemical stability, solubility, and oral activity. Herein, we report on the discovery of pyrimidinone 57 (LIT-927), a novel neutraligand of CXCL12 which displays a higher solubility than 1 and is no longer a Michael acceptor. While both 1 and 57 reduce eosinophil recruitment in a murine model of allergic airway hypereosinophilia, 57 is the only one to display inhibitory activity following oral administration. Thereby, we here describe 57 as the first orally active CXCL12 neutraligand with anti-inflammatory properties. Combined with a high binding selectivity for CXCL12 over other chemokines, 57 represents a powerful pharmacological tool to investigate CXCL12 physiology in vivo and to explore the activity of chemokine neutralization in inflammatory and related diseases.

Ghrelin is a 28-residue peptide acylated with an n-octanoyl group on the Ser 3 residue, predominantly produced by the stomach. Ghrelin displays strong growth hormone (GH) releasing activity, which is mediated by the activation of the so-called GH secretagogue receptor type 1a (GHS-R1a). Given the wide spectrum of biological activities of Ghrelin in neuroendocrine and metabolic pathways, many research groups, including our group, developed synthetic peptide, and nonpeptide GHS-R1a ligands, acting as agonists, partial agonists, antagonists, or inverse agonists. In this highlight article, we will focus on the discovery of a GHS-R1a antagonist compound, JMV 2959, which has been extensively studied in different in vitro and in vivo models. We will first describe the peptidomimetic approach that led us to discover this compound. Then we will review the results obtained with this compound in different studies in the fields of food intake and obesity, addictive behaviors, hyperactivity and retinopathy.

The synthetic hexapeptide growth hormone releasing peptide-6 (GHRP-6) exhibits dual affinity for the growth hormone secretagogue receptor 1a (GHS-R1a) and the cluster of differentiation 36 (CD36) receptor. Azapeptide GHRP-6 analogues have been synthesized, exhibiting micromolar affinity to the CD36 receptor with reduced affinity toward the GHS-R1a. A combinatorial split-and-mix approach furnished aza-GHRP-6 leads, which were further examined by alanine scanning. Incorporation of an aza-amino acid residue respectively at the D-Trp(2), Ala(3), or Trp(4) position gave aza-GHRP-6 analogues with reduced affinity toward the GHS-R1a by at least a factor of 100 and in certain cases retained affinity for the CD36 receptor. In the latter cases, the D-Trp(2) residue proved important for CD36 receptor affinity; however, His(1) could be replaced by Ala(1) without considerable loss of binding. In a microvascular sprouting assay using a choroid explant, [azaTyr(4)]-GHRP-6 (15), [Ala(1), azaPhe(2)]-GHRP-6 (16), and [azaLeu(3), Ala(6)]-GHRP-6 (33) all exhibited antiangiogenic activity.

A peptide self-assembled monolayer (SAM) was designed to bind His-tagged biomolecules for surface plasmon resonance (SPR) bioanalysis, which was applied for the determination of K(d) for small ligand screening against CD36. Nonspecific adsorption could be minimized using penta- and hexa-peptide monolayers. In particular, monolayers consisting of 3-mercaptopropionyl-leucinyl-histidinyl-aspartyl-leucinyl-histidinyl-aspartic acid (3-Mpa-LHDLHD) exhibited little (12 ng cm(-2)) nonspecific adsorption in crude serum. Modification of this peptide monolayer with Nα,Nα-bis(carboxymethyl)-L-lysine gave a surface competent for binding His-tagged proteins, as demonstrated using enzyme (human dihydrofolate reductase), protein/antibody and receptor (CD36) examples. Immobilization featured chelation of copper and the His-tagged protein by the peptide monolayer, which could be recycled by removing the copper using imidazole washes prior to reuse.

Purpose: We performed a phase I clinical trial of adenovirus/prostate-specific antigen (PSA) vaccine in men with measurable metastatic hormone-refractory disease., Experimental Design: Men with measurable metastatic disease received one vaccine injection. Toxicity, immune responses, changes in PSA doubling times, and patient survival were assessed. Thirty-two patients with hormone-refractory metastatic prostate cancer were treated with a single s.c. vaccine injection at one of three dose levels, either as an aqueous solution or suspended in a Gelfoam matrix. All patients returned for physical and clinical chemistry examinations at regular intervals up to 12 months after injections., Results: The vaccine was deemed safe at all doses in both administration forms. There were no serious vaccine-related adverse events; the most prevalent were localized erythema/ecchymoses and cold/flu-like symptoms. Anti-PSA antibodies were produced by 34% of patients and anti-PSA T-cell responses were produced by 68%. PSA doubling time was increased in 48%, whereas 55% survived longer than predicted by the Halabi nomogram., Conclusions: The adenovirus/PSA vaccine was proven safe with no serious vaccine-related adverse events. The majority of vaccinated patients produced anti-PSA T-cell responses and over half survived longer than predicted by nomogram. Although the latter data are only derived from a small number of patients in this phase I trial, they are encouraging enough to pursue further studies.

Submonomer synthesis of aza-peptides featuring regioselective alkylation of peptide-bound aza-Gly residues provided ten aza-analogues of the Growth Hormone Releasing Peptide-6 (GHRP-6) in 15-42% yield and purity generally >or=90%. Circular dichroism demonstrated that azaPhe-peptide 7a induced a beta-turn conformation which may be responsible for its 1000-fold improvement in GHRP-6 selectivity for the CD36 receptor. This versatile method for making aza-peptides avoids solution-phase hydrazine synthesis and is well suited for studying side-chain-activity relationships of biologically active peptides.

A convenient and reliable solid-phase strategy for the synthesis of di- and trisubstituted benzazepine derivatives was developed. 5-Amino-1-tert-butoxycarbonyl-2,3,4,5-tetrahydro-1H-benzo[b]azepine and 5-amino-1-tert-butoxycarbonyl-7-bromo-2,3,4,5-tetrahydro-1H-benzo[b]azepine G-protein coupled receptor-targeted (GPCR-targeted) scaffolds were efficiently synthesized in a six-step solution-phase process, immobilized on the acid-labile FMPB-AM resin, and further functionalized through acylation, sulfonation, reductive amination, alkylation, and Suzuki or Buchwald-Hartwig cross-coupling reactions. The efficacy of this strategy was exemplified by the preparation of an original pilot library of di- and trisubstituted benzazepines obtained in high purity as assessed by both 1H NMR and liquid chromatography/mass spectrometry (LC/MS) analysis.

A new series of growth hormone secretagogue (GHS) analogues based on the 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and their ability to stimulate intracellular calcium release to the cloned hGHS-1a ghrelin receptor expressed in LLC PK-1 cells. We have synthesized potent ligands of this receptor, some of them behaving as agonists, partial agonists, or antagonists. Some compounds among the most potent, i.e., agonist 29c (JMV2873), partial agonists including 21b (JMV2810), antagonists 19b (JMV2866) and 19c (JMV2844), were evaluated for their in vivo activity on food intake, after sc injection in rodents. Some compounds were found to stimulate food intake like hexarelin; some others were identified as potent hexarelin antagonists in this assay. Among the tested compounds, 21b was identified as an in vitro ghrelin receptor partial agonist, as well as a potent in vivo antagonist of hexarelin-stimulated food intake in rodents. Compound 21b was without effect on GH release from rat. However, in this series of compounds, it was not possible to find a clear correlation between in vitro and in vivo results.

Calcitonin gene-related peptide antagonists have potential for the treatment and prevention of disease states such as non-insulin-dependent diabetes mellitus, migraine headache, pain, and inflammation. To gain insight into the spatial requirements for CGRP antagonism, three strategies were employed to restrict the conformation of the potent undecapeptide antagonist, [D31,P34,F35]CGRP27-37. First, aza-amino acid scanning was performed, and ten aza-peptide analogues were synthesized and examined for biological activity. Second, (3S,6S,9S)-2-oxo-3-amino-indolizidin-2-one amino acid (I2aa) and (2S,6S,8S)-9-oxo-8-amino-indolizidin-9-one amino acid (I9aa) both were introduced at positions 31-32, 32-33, 33-34, and 34-35, regions of the backbone expected to adopt turns. Finally, the conformation of the backbone and side-chain of the C-terminal residue, Phe35-Ala36-Phe37-NH2, was explored employing (2S,4R,6R,8S)-9-oxo-8-amino-4-phenyl-indolizidin-9-one amino acid (4-Ph-I9aa) as a constrained phenylalanine mimic. The structure-activity relationships exhibited by our 26 analogues illustrate conformational requirements important for designing CGRP antagonists and highlight the importance of beta-turns centered at Gly33-Pro34 for potency.

In order to develop a fluorescence polarization (FP) assay for calcium binding proteins, a fluorescent peptides based library of 1328 compounds has been synthesized. The use of this library has been validated by setting up a FP-high-throughput screening (FP-HTS) assay for calmodulin using the synthetic gene product (synCaM). With this assay, a set of 880 FDA approved compounds was screened. Besides the promazine class, we discovered two new classes of compounds that interact with calmodulin in a calcium dependent manner. One class has compounds with anti-histaminic/spasmolytic activities, and the other one are detergents with antibacterial activities.

To examine the structural determinants necessary for TC10 trafficking, localization, and function in adipocytes, we generated a series of point mutations in the carboxyl-terminal targeting domain of TC10. Wild-type TC10 (TC10/WT) localized to secretory membrane compartments and caveolin-positive lipid raft microdomains at the plasma membrane. Expression of a TC10/C206S point mutant resulted in a trafficking and localization pattern that was indistinguishable from that of TC10/WT. In contrast, although TC10/C209S or the double TC10/C206,209S mutant was plasma membrane localized, it was excluded from both the secretory membrane system and the lipid raft compartments. Surprisingly, inhibition of Golgi membrane transport with brefeldin A did not prevent plasma membrane localization of TC10 or H-Ras. Moreover, inhibition of trans-Golgi network exit with a 19 degrees C temperature block did not prevent the trafficking of TC10 or H-Ras to the plasma membrane. These data demonstrate that TC10 and H-Ras can both traffic to the plasma membrane by at least two distinct transport mechanisms in adipocytes, one dependent upon intracellular membrane transport and another independent of the classical secretory membrane system. Moreover, the transport through the secretory pathway is necessary for the localization of TC10 to lipid raft microdomains at the plasma membrane.

Growth hormone-releasing peptides (GHRPs) or growth hormone secretagogues (GHSs) of peptide or non-peptide structure are able to stimulate growth hormone secretion. They act through a growth hormone-releasing hormone (GHRH)/somatotropin release inhibiting factor (SRIF) independent pathway. A receptor able to bind to these compounds was cloned in 1996, and a natural ligand of this receptor, named ghrelin, was characterized in 1999. The potential therapeutic value of such compounds is considerable as they offer an alternative therapy for treatment of deficiency in growth hormone secretion. As growth hormone (GH) also participates in many anabolic effects, interest in these compounds is increasing and many clinical applications are expected in the future.

Previously we have demonstrated that striated muscle GLUT4 gene expression decreased following streptozotocin-induced diabetes due to a loss of MEF2A transcription factor expression without any significant effect on the MEF2D isoform (Mora, S. and J. E. Pessin (2000) J Biol Chem, 275:16323-16328). In contrast to both cardiac and skeletal muscle, adipose tissue displays a selective decrease in MEF2D expression in diabetes without any significant alteration in MEF2A protein content. Adipose tissue also expresses very low levels of the MEF2 transcription factors and nuclear extracts from white adipose tissue exhibit poor in vitro binding to the MEF2 element. However, addition of in vitro synthesized MEF2A to adipose nuclear extracts results in the formation of the expected MEF2/DNA complex. More importantly, binding to the MEF2 element was also compromised in the diabetic condition. Furthermore, in vivo overexpression of MEF2A selectively in adipose tissue did not affect GLUT4 or MEF2D expression and was not sufficient to prevent GLUT4 down-regulation that occurred in insulin-deficient states.