Structural Requirements for CNS Active Opioid Glycopeptides.

Glycopeptides related to β-endorphin penetrate the blood-brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful.

NMR studies of the dynamics of high-spin nitrophorins: comparative studies of NP4 and NP2 at close to physiological pH.

The β-barrel nitrophorin (NP) heme proteins are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands. NO is bound to iron of the NPs and is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4, which have sequence similarities in two pairs, NP1 and NP4 (90% identical) and NP2 and NP3 (80% identical).

Discovery of amphipathic dynorphin A analogues to inhibit the neuroexcitatory effects of dynorphin A through bradykinin receptors in the spinal cord.

We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well-known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg(10), Leu(9)]-kallidin (DALKD), and HOE140 showed different binding profiles at rat brain BRs than that previously reported.

Can amphipathic helices influence the CNS antinociceptive activity of glycopeptides related to β-endorphin?

Glycosylated β-endorphin analogues of various amphipathicity were studied in vitro and in vivo in mice. Opioid binding affinities of the O-linked glycopeptides (mono- or disaccharides) and unglycosylated peptide controls were measured in human receptors expressed in CHO cells. All were pan-agonists, binding to μ-, δ-, or κ-opioid receptors in the low nanomolar range (2.

NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A).

Nitrophorin 2 (NP2), one of the four NO-storing and NO-releasing proteins found in the saliva of the blood-sucking bug Rhodnius prolixus, has a more ruffled heme and a high preference for a particular heme orientation (B) compared with nitrophorin 1 and nitrophorin 4, which show not a preference (A to B ratio of approximately 1:1), suggesting that it fits more tightly in the β-barrel protein. In this work we have prepared a series of "belt" mutants of NP2(D1A) and (ΔM0)NP2 aimed at reducing the size of aromatic or other residues that surround the heme, and investigated them as the high-spin aqua and low-spin N-methylimidazole complexes. The belt mutants included Y38A, Y38F, F42A, F66A, Y85A, Y85F, Y104A, I120T, and a triple mutant of NP2(D1A), the F42L, L106F, I120T mutant.

NMR studies of the dynamics of nitrophorin 2 bound to nitric oxide.

The Rhodnius nitrophorins are β-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal.

An unusual conformation of γ-melanocyte-stimulating hormone analogues leads to a selective human melanocortin 1 receptor antagonist for targeting melanoma cells.

γ-MSH (γ-melanocyte-stimulating hormone, H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH), with its exquisite specificity and potency, has recently created much excitement as a drug lead. However, this peptide is like most peptides susceptible to proteolysis in vivo, which potentially decreases its beneficial activities. In our continued effort to design a proteolytically stable ligand with specific receptor binding, we have engineered peptides by cyclizing γ-MSH using a thioether bridge.

Native N-terminus nitrophorin 2 from the kissing bug: similarities to and differences from NP2(D1A).

The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli, the wild-type gene of the mature protein retains the methionine-0, which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E.

Approaches to the rational design of selective melanocortin receptor antagonists.

Introduction: When establishing the physiological roles of specific receptors in normal and disease states, it is critical to have selective antagonist ligands for each receptor in a receptor system with several subtypes. The melanocortin receptors have five subtypes referred to as the melanocortin 1 receptor, melanocortin 2 receptor, melanocortin 3 receptor, melanocortin 4 receptor and melanocortin 5 receptor, and they are of critical importance for many aspects of human health and disease.

Areas Covered: This article reviews the current efforts to design selective antagonistic ligands for the five human melanocortin receptors summarizing the currently published orthosteric and allosteric antagonists for each of these receptors.

Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins.

Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood-brain barrier.

Phosphorylation of enkephalins: NMR and CD studies in aqueous and membrane-mimicking environments.

Phosphorylation of l-serine-containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood-brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin-based peptides were modified for these studies, a set related to DTLES, a mixed μ/δ-agonist, and one related to DAMGO, a highly selective μ-agonist. Each unglycosylated peptide was compared to its phosphate, its mono-benzylphosphate ester, and its β-d-glucoside.

Effects of bulkiness and hydrophobicity of an aliphatic amino acid in the recognition helix of the GAGA zinc finger on the stability of the hydrophobic core and DNA binding affinity.

The GAGA factor of Drosophila melanogaster uses a single Cys 2His 2-type zinc finger for specific DNA binding. The conformation and DNA binding mode of the GAGA zinc finger are similar to those of other structurally characterized zinc fingers. In almost all Cys 2His 2-type zinc fingers, the fourth position of the DNA-recognizing helix is occupied by the Leu residue involved in the formation of the minimal hydrophobic core.

DNA-Binding ability of GAGA zinc finger depends on the nature of amino acids present in the beta-hairpin.

The GAGA factor of Drosophila melanogaster uses a single Cys2-His2-type zinc finger for specific DNA binding. Comparative sequence alignment of the GAGA zinc finger core with other structurally characterized zinc fingers reveals that the beta-hairpin of the GAGA zinc finger prefers amino acids with an aliphatic side-chain different from those of other zinc fingers. To probe the substitution effect of aromatic amino acids in the beta-hairpin on the DNA binding, three mutant peptides were designed by substituting consensus phenylalanine, an aromatic amino acid, at key positions in the beta-hairpin region.

Biomolecular mirror-image recognition: reciprocal chiral-specific DNA binding of synthetic enantiomers of zinc finger domain from GAGA factor.

To experimentally demonstrate the mirror-image recognition in protein and DNA interaction, we have designed a small DNA-binding peptide based on the zinc-finger domain of GAGA transcription factor. Circular dichroism data suggest that the conformations of peptide enantiomers as well as the DNA enantiomers have a mirror-image relationship. The gel mobility shift assay showed that the synthetic enantiomers of the peptide showed reciprocal chiral-specific interactions with the DNA; the natural L-peptide binds specifically with the natural D-DNA substrate, and the unnatural D-peptide binds specifically with the unnatural L-DNA substrate.

Designer zinc finger proteins: tools for creating artificial DNA-binding functional proteins.

The design of artificial functional DNA-binding proteins has long been a goal for several research laboratories. The zinc finger proteins, which typically contain many fingers linked in tandem fashion, are some of the most studied DNA-binding proteins. The zinc finger protein's tandem arrangement and its the ability to recognize a wide variety of DNA sequences make it an attractive framework to design novel DNA-binding peptides/proteins.

New prospects for glycopeptide based analgesia: glycoside-induced penetration of the blood-brain barrier.

Antinociceptive effects and BBB transport properties of glycosylated enkephalin derivatives are reviewed. Previously, the application of enkephalins as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood brain barrier. This shortcoming has been overcome by glycosylation, paradoxically leading to enhanced BBB transport via transcytosis.

Glycosylated neuropeptides: a new vista for neuropsychopharmacology?

The application of endogenous neuropeptides (e.g., enkephalins) as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood-brain barrier (BBB).

Glycopeptides related to beta-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers.

A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics.

Expected and unexpected results from combined beta-hairpin design elements.

A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains.

Antinociceptive structure-activity studies with enkephalin-based opioid glycopeptides.

Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase 9-fluorenylmethyloxy carbamate methods.

Small molecule inhibition of hepatitis C virus E2 binding to CD81.

The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear.

Solution and solid-state models of peptide CH...O hydrogen bonds.

Fumaramide derivatives were analyzed in solution by (1)H NMR spectroscopy and in the solid state by X-ray crystallography in order to characterize the formation of CH...