Barbara Scolaro

Glycodermorphins: opioid peptides with potent and prolonged analgesic activity and enhanced blood-brain barrier penetration

1 In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O‐βglucosylated analogs ([Ser7‐O‐βGlc]dermorphin and [Ser7‐O‐βGlc(Ac)4]‐dermorphin) and C‐αgalactosylated analogs ([Ala7‐C‐αGal]dermorphin and [Ala7‐C‐αGal(Ac)4]‐dermorphin). 2 O‐ and C‐glycosylation of dermorphin halved the peptide affinity for brain μ‐opioid receptors and the biological potency in guinea‐pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8–40 pmol) and subcutaneously (s.c., 0.5–3 μmol kg−1) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5–10 times both biological activity on GPI and μ‐receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two‐three times lower (s.c.) than dermorphin potency. 3 Blood‐Brain Barrier Permeability Index (BBB‐PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O‐β‐linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT‐1 of the endothelial barrier. However the calculated BBB‐PI for the C‐αgalactoside was about two times higher than that of the O‐βglucoside, excluding the implication of GLUT‐1 that is known to be selective for O‐β‐links and preferring for the exose glucose. 4 The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.

Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins

Sequential glycopeptides [Thr(beta-D-galactose)-Ala-Ala]n, with n ranging from 2 to 7, as models of natural antifreeze glycoproteins were synthesized by the continuous flow, solid phase procedure. The conformational properties of these materials in solution were investigated by c.d. and 1H-n.m.r. spectroscopy. In aqueous solution the c.d. pattern is practically independent of chain length and is very similar to that of natural antifreeze glycoproteins. The results are interpreted in terms of random coil structure. The absence of ordered structures is further confirmed by n.m.r. data. A small amount of ordered conformation can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. The c.d. pattern of all glycopeptides in water at temperatures higher than 50 degrees C are compatible with the presence of a small amount of alpha-helix or 3(10) helix. Since the glyco-hexapeptide is too short to form an alpha-helix, the hypothesis is made that in the glycopeptides in water at high temperature a small amount of 3(10) helix is formed. The same is observed for the 21-residue glycopeptide in presence of 85% (v/v) TFE. In this medium, the c.d. data on the glyco-hexapeptide are more compatible with the presence of a small amount of beta-structure.

Semisynthetic glycoproteins: preparation of glycosylated ribonuclease S′ analogues

Abstract Glycopeptide analogues of ribonuclease S-peptide, des 16–20 [(Gal β)Thr3]-S-peptide and des 16–20 [(Gal β)Thr6]-S-peptide, were synthesized by the solid-phase procedure based on the Fmoc chemistry. Reassociation of the synthetic glycopeptides with S-protein yielded enzymatically active, glycosylated RNase S′ variants. The conformational properties of the S-peptide analogues in aqueous buffer and in the presence of trifluoroethanol were investigated by circular dichroism spectroscopy. The activation curves of the S-protein with varying amounts of the synthetic analogues, using C > p as the substrate, were determined and the dissociation constants (Kd), the free energies of binding (−ΔG), and the kinetic parameters (Km and k2) for the RNase S′ adducts were calculated and compared with the corresponding values obtained for the S-peptide. The properties of the semisynthetic, glycosylated enzymes agree with the results of previous investigations on the conformational and catalytic features of S-peptide analogues and related RNase S′.

Improved solid-phase synthesis of bombesin by continuous flow procedure using fmoc amino acids

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.

Linear and cyclic Thr6-Bradykinin analogues containing N-benzylglycine as a replacement for residues Phe5 and Phe8

N-Benzylglycine (Nphe) is an achiral structural isomer of phenylalanine that may be useful as an amino acid replacement in SAR studies. The first incorporation of Nphe into a biologically active peptide was reported by using bradykinin (BK) [1]. In continuation of our investigations on the structure-activity relationship of BK and BK analogues [2] we focused our attention on Thr6-BK (H-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH), a native kinin discovered in the venom of a solitary wasp and present also in ant venom and in frog skin. Thr6-BK proved to be about 10 times more potent than BK in the insect central nervous system [3] as well as in stimulating smooth muscle contraction [4]. The structural analysis of Thr6-BK and its analogues is made particularly interesting by the fact that despite the high sequence homology with native BK (only one conservative substitution: Ser6/Thr6) there is a marked and significant difference in the biological profile of the two peptides. The conformation of the native Thr6-BK has been already investigated in DMSO by NMR spectroscopy and computer simulations [5]. We report in this communication the synthesis and some preliminary pharmacological experiments and structural investigations of new linear and cyclic Thr6-BK analogues in which either one or both the Phe residues in the peptide sequence have been substituted by N-benzylglycine.

Studies on the Involvement of a His Residue of mt1 GPCR TM Helix V in the Interaction with Melatonin

Melatonin (N-acetyl-5-methoxytryptamine, MLT) is a hormone produced mainly by the pineal gland in most vertebrate species, including humans. The physiological role of MLT is related to both chronobiology and modulation of the body’s hormonal milieu. MLT appears to exert these effects through specific high affinity receptors. MLT has also been shown to have powerful antioxidant properties acting as an efficient free radicals scavenger.