Circular dichroism study of the carbohydrate-modified opioid peptides

Horvat, Štefica; Ötvös, László; Ürge, László; Horvat, Jaroslav; Čudić, Mare; Varga-Defterdarović, Lidija

doi:10.1016/s1386-1425(99)00132-8

Cited by 7 publications

(6 citation statements)

References 28 publications

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“…Curves were smoothed by the algorithm provided by Jasco. The CD spectra obtained were evaluated by comparing them to the spectra of known peptide conformations (23), and ␣-helicity was calculated according to the algorithm of Greenfield and Fasman (24).…”

Section: Methodsmentioning

confidence: 99%

“…To do this, we first built a model of the three-dimensional structure of INSL3 based on the structure of relaxin, as the structure of INSL3 is yet to be determined by experimental methods. We then examined this model for potential sites to insert a disulfide constraint between residues of the B-chain ␤-strand (residues 1-9) and ␣-helix (residues [13][14][15][16][17][18][19][20][21][22][23][24][25][26] 9 and 16, respectively, Fig. 2, D and E).…”

Section: Design and Synthesis Of Cyclic Disulfide-constrained B-chainmentioning

confidence: 99%

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Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo

Borgo

Hughes

Bathgate

et al. 2006

Journal of Biological Chemistry

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Insulin-like peptide 3 (INSL3) is a member of the insulin superfamily that plays an important role in mediating testes descent during fetal development. More recently, it has also been demonstrated to initiate oocyte maturation and suppress male germ cell apoptosis. These actions are mediated via a specific G-protein-coupled receptor, LGR8. Little is known regarding the structure and function relationship of INSL3, although it is believed that the principal receptor binding site resides within its B-chain. We subsequently observed that the linear B-chain alone (INSL3B-(1-31)) bound to LGR8 and was able to antagonise INSL3 stimulated cAMP accumulation in HEK-293T cells expressing LGR8. Sequentially N-and C-terminally shortened linear analogs were prepared by solid phase synthesis and subsequent assay showed that the minimum length required for binding was residues 11-27. It was also observed that increased binding affinity correlated with a corresponding increase in ␣-helical content as measured by circular dichroism spectroscopy. Molecular modeling studies suggested that judicious placement of a conformational constraint within this peptide would increase its ␣-helix content and result in increased structural similarity to the B-chain within native INSL3. Consequently, intramolecularly disulfide-linked analogs of the B-chain showed a potentiation of INSL3 antagonistic activity, as well as exhibiting increased proteolytic stability, as assessed in rat serum in vitro. Administration of one of these peptides into the testes of rats resulted in a substantial decrease in testis weight probably due to the inhibition of germ cell survival, suggesting that INSL3 antagonists may have potential as novel contraceptive agents. Insulin-like peptide 3 (INSL3)3 is a member of the insulin superfamily of hormones, which also includes insulin-like growth factors I and II (IGF-I and -II), relaxin-1, -2, and -3, and INSL4, -5, and -6. INSL3 is a 6-kDa peptide consisting of two chains (A and B) linked by two intermolecular disulfide bonds, with a third intermolecular disulfide bond in the A-chain (Fig. 1) (1). It is expressed primarily in the Leydig cells of fetal and adult testes and in the thecal cells of the ovary (1). The receptor for INSL3 has recently been identified and shown to belong to the family of leucine-rich repeat-containing G-protein-coupled receptors (LGRs) (2). INSL3 binds with high affinity to LGR8 and has very low affinity for the paralagous receptor LGR7, which is the receptor for relaxin (3). It is now well accepted that INSL3 is a member of the relaxin peptide family (4), and LGR7 and LGR8 have been classified as relaxin family peptide receptors, RXFP1 and RXFP2, respectively (5). Importantly, relaxin can bind to and activate LGR8 in some species, although with lesser affinity and potency than INSL3 (6,7).Although the precise physiological functions of INSL3 have not been fully determined, it has been shown to play a pivotal role in testicular descent and ovarian function. In male mice, deletion of either the ...

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Section: Methodsmentioning

confidence: 99%

Section: Design and Synthesis Of Cyclic Disulfide-constrained B-chainmentioning

confidence: 99%

Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo

Borgo

Hughes

Bathgate

et al. 2006

Journal of Biological Chemistry

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“…Turn formation has been proposed for short peptides in lipophilic environments, as well as “neo-glycosylated” enkephalin analogues in F 3 CCH 2 OH . There was more variation in the CD curves (and presumably in the conformations) measured in H 2 O than in the presence of micelles.…”

Section: Introductionmentioning

confidence: 88%

Glycopeptide-Membrane Interactions: Glycosyl Enkephalin Analogues Adopt Turn Conformations by NMR and CD in Amphipathic Media

et al. 2003

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Four enkephalin analogues (Tyr-D-Thr-Gly-Phe-Leu-Ser-CONH(2), 1, and the related O-linked glycopeptides bearing the monosaccharide beta-glucose, 2, the disaccharide beta-maltose, 3, and the trisaccharide beta-maltotriose, 4) were synthesized, purified by HPLC, and biophysical studies were conducted to examine their interactions with membrane model systems. Glycopeptide 2 has been previously reported to penetrate the blood-brain barrier (BBB), and produce potent analgesia superior to morphine in mice (J. Med. Chem.2000, 43, 2586-90 and J. Pharm. Exp. Ther. 2001, 299, 967-972). The parent peptide and its three glycopeptide derivatives were studied in aqueous solution and in the presence of micelles using 2-D NMR, CD, and molecular mechanics (Monte Carlo studies). Consistent with previous conformational studies on cyclic opioid agonist glycopeptides, it was seen that glycosylation did not significantly perturb the peptide backbone in aqueous solution, but all four compounds strongly associated with 5-30 mM SDS or DPC micelles, and underwent profound membrane-induced conformational changes. Interaction was also observed with POPC:POPE:cholesterol lipid vesicles (LUV) in equilibrium dialysis experiments. Although the peptide backbones of 1-4 possessed random coil structures in water, in the presence of the lipid phase they each formed a nearly identical pair of structures, all with a stable beta-turn motif at the C-terminus. Use of spin labels (Mn(2+) and 5-DOXYL-stearic acid) allowed for the determination of the position and orientation of the compounds relative to the surface of the micelle.

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“…Mean residue ellipticity (θ MR ) is expressed in degrees × cm 2 /dmole. CD spectra evaluations were based on comparison with known peptide conformations [18] and the amount of α-helicity was calculated according to the algorithm of Greenfield and Fasman [19].…”

Section: Circular Dichroism (Cd) Spectroscopymentioning

confidence: 99%

Conformationally constrained single-chain peptide mimics of relaxin B-chain secondary structure

2005

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Relaxin is a member of the insulin superfamily and has many biological actions including angiogenesis and collagen degradation. It is a 6 kDa peptide hormone consisting of two peptide chains (A and B) tethered by two disulphide bonds. Past structure-function relationship studies have shown the key receptor binding site of relaxin to be principally situated within the B-chain alpha-helix. Molecular dynamic simulations were performed to aid the design of conformationally constrained relaxin B-chain analogues that possess alpha-helical structure and relaxin-like activity. Restraints included disulphide bonds, both single and double, and lactam bonds. Each peptide was prepared by solid phase synthesis and, following purification, subjected to detailed conformational analysis by circular dichroism spectroscopy. Of 15 prepared relaxin B-chain mimetics, one was able to mimic the secondary structure of the native ligand as indicated by biomolecular recognition/interaction analysis using surface enhanced laser desorption ionization mass spectroscopy together with a relaxin antibody. However, none of the mimetics possess characteristic relaxin-like biological activity which strongly indicates that the pharmacophore comprises additional structural elements other than the relaxin B-chain alpha-helix. These findings will assist in the design and preparation of novel relaxin agonists and antagonists.

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Circular dichroism study of the carbohydrate-modified opioid peptides

Cited by 7 publications

References 28 publications

Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo

Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo

Glycopeptide-Membrane Interactions: Glycosyl Enkephalin Analogues Adopt Turn Conformations by NMR and CD in Amphipathic Media

Conformationally constrained single-chain peptide mimics of relaxin B-chain secondary structure

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