Molecular mechanism underlying the cerebral effect of Gly-Pro-Glu tripeptide bound to L-dopa in a Parkinson's animal model.

Oxidative stress is a critical contributing factor to neurodegenerative disorders. Therefore, the inhibition of ROS formation, responsible for chronic detrimental neuroinflammation, is an important strategy for preventing the neurodegenerative disease and for neuroprotective therapy. Gly-Pro-Glu (GPE) is the N-terminal tripeptide of insulin-like growth factor-I, which is naturally cleaved in the plasma and brain tissues. GPE has neuroprotective effects since it crosses the blood-CSF and the functional CSF-brain barriers and binds to glial cells. It has been shown that GPE improves motor behaviour in rats after 6-OHDA lesion, although it does not rescue dopaminergic neurons. Thus, we hypothesized that the GPE therapeutic efficacy in a Parkinson model might be improved by combining GPE to L: -dopa. Here, we used an animal model that represents a progressive chronic Parkinson's disease (PD) model, characterized by high levels of oxidative stress and inflammation. We showed that the co-drug, in which L: -dopa is covalently linked to the GPE tripeptide, by down-regulating the expression of inflammatory genes, decreases the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced inflammatory response and, by up-regulating tyrosine hydroxylase, reduces MPTP-induced neurotoxicity. Furthermore, by determining the nuclear translocation/activation of Nrf2 and NF-κB, we showed that systemic administration of the co-drug activates Nrf2-induced antioxidant response while suppressing NF-κB inflammatory pathway. Data suggest that the binding of L: -dopa to GPE tripeptide might represent a promising strategy to supply L: -dopa to parkinsonian patients.

Detection of levodopa, dopamine and its metabolites in rat striatum dialysates following peripheral administration of L-DOPA prodrugs by mean of HPLC-EC.

A high performance liquid chromatography (HPLC) method was developed to detected simultaneously L-dihydroxyphenylalanine (L-DOPA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum dilaysates following oral administration of L-DOPA or its prodrugs. The chromatographic system uses a reversed-phase C18 column with electrochemical detection at +0.30 V. Mobile phase consisted of 0.05 M citric acid, sodium EDTA 50 microM, sodium octylsulphonate 0.4 nM at pH of 2.9 and 8% methanol (v/v) at a flow rate of 1 ml/min. The calibration curves were linear over the concentration range of 10nm to 100 microM and the lower limits of detections were 125 fmol for L-DOPA, 50 fmol for DOPAC, 250 fmol for DA and 150 fmol for HVA at signal noise to ratio of 3. The repeatability (or intra-day precision), expressed by the relative standard deviation, were better than 4%. The construction of microdialysis probes has been described. The in vitro relative recoveries of each microdialysis probe were evaluated and the results show that they are similar and reproducible for all the analytes with CVs from 1 to 4%. The HPLC-EC method was applied to detect the extracellular levels of L-DOPA, DA, DOPAC and HVA in the striatum dialysates of freely moving rats after oral administration of six new potential L-DOPA prodrugs.

Design and synthesis of glutathione analogues.

This review reports recent structural modifications (since 1989) performed on the glutathione molecular both in the oxidized and reduced form. Relevant chemical aspects, biochemical consequences and therapeutical implications are illustrated. Natural thiols related to glutathione are also considered.

Inhibitors of Zn-dependent metallopeptidases: synthesis and activity of N-(2-furoyl)-(Z)-alpha, beta-didehydroleucyl-L-tryptophan.

In order to gain information on structure activity relationships of peptidic inhibitors of Zn-dependent metalloproteinases "hemorrhagins", the conformationally restricted model N-(2-furoyl)-(Z)-alpha,beta-didehydroleucyl-L-tryptophan 2 was synthesized and its activity compared to that of related previously studied substrates. The new model 2 exhibits an inhibitory activity on proteinase II from Crotalus Adamanteus snake venom, sensibly lower than that of related substrates. This result indicates that the reduction of the conformational space due to the presence of the alpha,beta-didehydro-amino acid residue in 2 does not favour the fitting and binding at the enzyme active site.

Synthesis and activity of novel glutathione analogues containing an urethane backbone linkage.

The new GSH analogues H-Glo(-Ser-Gly-OH)-OH (5), its O-benzyl derivative 4, and H-Glo(-Asp-Gly-OH)-OH (9), characterized by the replacement of central cysteine with either serine or aspartic acid, and containing an urethanic fragment as isosteric substitution of the scissile gamma-glutamylic junction, have been synthesized and characterized. Their ability to inhibit human GST P1-1 (hGST P1-1) in comparison with H-Glu(-Ser-Gly-OH)-OH and H-Glu(-Asp-Gly-OH)-OH, which are potent competitive inhibitors of rat GST 3-3 and 4-4, has been evaluated. In order to further investigate the effect of the isosteric substitution on the binding abilities of the new GSH analogues 4, 5 and 9, the previously reported cysteinyl-containing analogue H-Glo(-Cys-Gly-OH)-OH has been also evaluated as a co-substrate for hGSTP1-1.

psi(SO2NH) transition state isosteres of peptides. Synthesis and bioactivity of sulfonamido pseudopeptides related to carnosine.

This paper reports the synthesis of tauryl dipeptides related to carnosine. In particular H-Tau-His-OH (5), H-Tau-His(pi-Me)-OH (6) and H-Tau-His(tau-Me)-OH (9) are described. The enzyme carnosinase has been isolated from pig kidney and after purification has been used to test the stability and the inhibitory activity of the three new analogues. H-Tau-His-OH (5) and H-Tau-His(tau-Me)-OH (9) were found to possess weak inhibitory properties towards carnosinase, while H-Tau-His(pi-Me)-OH (6) proved to be devoid of any significant activity. All the three sulfonamido pseudopeptides 5, 6 and 9 show stability to carnosinase activity.

Synthesis and biological evaluation of a novel pyroglutamyl-modified TRH analogue.

The TRH analogue 3, incorporating the (S)-isothiazolidine-1,1-dioxide-3-carboxylic acid (1) moiety in place of the native L-pyroglutamic acid (pGlu) residue, has been synthesized and fully characterized by 1H and 13C NMR. The effects of replacing pGlu with its sulphonamido counterpart on biological activity have been investigated. This peptide, which is significantly stabilized towards hydrolysis by pyroglutamyl peptidase type I (PP I, EC 3.4.19.3), has shown to maintain in vitro prolactin-releasing activity.

GPE and GPE analogues as promising neuroprotective agents.

The tripeptide glycine-proline-glutamate (GPE) is the naturally cleaved N-terminal tripeptide of insulin-like growth factor-1 (IGF-1) in brain tissues by an acid protease. Although GPE does not bind to IGF-1 receptors and its mode of action is not clear, in vitro studies have demonstrated its ability to stimulate acetylcholine and dopamine release, as well as to protect neurones from diverse induced brain injures. More importantly, GPE has been shown to have potent neuroprotective effects in numerous animal models of hypoxic-ischemic brain injury and neurodegenerative diseases such as Parkinson's, Alzheimer's and Huntington's diseases. As a consequence, GPE was suggested to be a potential target for the rational design of neuroprotective agents. Unfortunately, the use of GPE as a therapeutic agent is limited because of its unfavorable biochemical and pharmacokinetic properties. This review will focus on structural modifications performed on the GPE molecule in order to obtain bioactive analogues with increased pharmacokinetic profile useful for the treatment of central nervous system (CNS) injures and neurodegenerative disorders.

2,5-diketopiperazines as neuroprotective agents.

2,5-diketopiperazines are the simplest cyclic peptides found in nature, commonly biosynthesized from amino acids by different organisms, and represent a promising class of biologically active natural products. Their peculiar heterocyclic structure confers high stability against the proteolysis and constitutes a structural requirement for the active intestinal absorption. Furthermore, the diketopiperazine-based motif is considered as a novel brain shuttle for the delivery of drugs with limited ability to cross the blood-brain barrier (BBB) and can be proposed as an ideal candidate for the rational development of new therapeutic agents. Although these cyclic peptides have been known since the beginning of the 20th century, only recently have they attracted substantial interest with respect to the wide spectrum of their biological properties, including antitumor, antiviral, antifungal, antibacterial and antihyperglycemic activities. In addition to these, the most challenging function of the diketopiperazine derivatives is related with their remarkable neuroprotective and nootropic activity. The aim of the present paper is to provide an overview of the two major classes of diketopiperazines, the TRH-related and the unsaturated derivatives both characterized by a significant ability to protect against neurotoxicity in several experimental models. The neuroprotective profile of these compounds suggests that they may have a future utility in the therapy of neuronal degeneration in vivo, potentially through several different mechanisms.

Biochemical properties of new synthetic carnosine analogues containing the residue of 2,3-diaminopropionic acid: the effect of N-acetylation.

Three novel carnosine analogues 7-9 containing the residue of L(+)2,3-diaminopropionic acid with different degree of N-acetylation instead of beta-alanine have been synthesized and characterized. Comparative analysis of hydrolysis by carnosinase revealed that the mono- and bis-acetylated compounds 8 and 9 are resistant to enzymatic hydrolysis and act as competitive inhibitors of this enzyme. The hydroxyl radical scavenging potential of the three analogues was evaluated by their ability to inhibit iron/H(2)O(2)-induced degradation of deoxyribose. The second-order rate constants of the reaction of compounds 7-9 with hydroxyl radical were almost identical to that of carnosine. These compounds were also found to act as protective agents against peroxynitrite-dependent damage as assessed by their ability to prevent nitration of free tyrosine induced by this species.

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate.

Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-gamma-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC50=0.11+/-0.01 microM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-gamma-(gamma-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the gamma-glutamyl-cysteine peptide bond, has been developed as gamma-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the gamma-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.

Cyclization under mild conditions of anthraniloyl- and N-methyl-anthraniloyl dipeptides.

Anthraniloyl- and N-methyl-anthraniloyl dipeptides containing C-terminal L and D proline residues have been synthesized and subjected to cyclization reaction under mild conditions. The influence of substituents and of the chirality of the residues on the output of the reaction is discussed.

Identification of an oxidation product of aminoethylcysteine ketimine dimer.

In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine dimer by oxygen reactive species, we give here data for the identification of the alpha, beta unsaturated sulfoxide as the main product of interaction of the dimer with H2O2. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography.

Identification of new products of S-aminoethylcysteine ketimine autoxidation.

In continuation of a previous work (Pecci et al., 1993), dedicated to the detection of the autoxidation products of S-aminoethylcysteine ketimine (AECK), we give here data for the identification of 2,3,6,7-tetrahydro-4H-[1,4]thiazino[2,3-b]thiazine, thiomorpholine-3-one and 5,5', 6,6'-tetrahydro-2,2'-dihydroxy-3,3'-bi-2H-thiazine among the products of AECK autoxidation. Identification has been done on the basis of mass spectrometry and NMR spectral analyses of the isolated products.

Serine-containing 10-membered cyclodepsipeptides. Synthesis and molecular structure of PhCH2CO-DSer-Pro-Pro- and PhCH2CO-DSer-Pro-DPro-.

As a part of a research program aimed at studying synthesis and conformation of small ring peptides, the cyclization of diastereoisomeric N-phenylacetyl-seryl-propyl-proline tripeptides has been examined. Two 10-membered peptide lactones, PhCH2CO-DSer-Pro-Pro- 5a and PhCH2CO-DSer-Pro-DPro- 5b, have been isolated by treating the corresponding linear p-nitrophenyl esters with DBU in dry benzene. In these two compounds the serine lactone fragment (a common structural feature of several bioactive cyclodepsipeptides) is inserted into a highly strained small ring system. The conformation in the crystal of 5a and 5b has been studied by X-ray analysis. Both the 10-membered rings of 5a and 5b adopt an overall cis-cis-trans conformation in which the lactone junction is trans. The deviations from planarity of the peptide units vary from delta omega = 30 degrees for the DSer-Pro bond in 5b to delta omega = 5-6 degrees for the DSer-Pro bond in 5a and Pro-DPro bond in 5b. The skeletal atoms of 5b, containing the Pro-DPro sequence, are related by a pseudo-symmetry mirror plane passing through the Pro carbonyl and the opposite DSer C beta H2 group. In both the molecules the exocyclic amide bond adopts an extended conformation with respect to the DSer-Pro ring junction; this arrangement gives rise to a C5-type ring structure which is well evidenced in the case of 5a.

Prevention of peroxynitrite-dependent damage by carnosine and related sulphonamido pseudodipeptides.

The naturally occurring dipeptides carnosine and anserine have been proposed to act as antioxidants in vivo. We investigated whether these compounds can act as protective agents able to counteract peroxynitrite-dependent reactions. The results showed that the dipeptides efficiently protect tyrosine against nitration, alpha1-antiproteinase against inactivation and human low-density lipoprotein against modification by peroxynitrite. Carnosine exerts its protective effect at concentrations similar to those found in human tissues. In addition, some synthetic pseudodipeptides, stucturally related to carnosine but stable to hydrolytic enzymes, possess protective properties against peroxynitrite-dependent damage similar to the natural dipeptides. These pseudodipeptides may represent stable mimics of the biologically active carnosine suitable for pharmacological applications.

Synthesis and activity of the glutathione analogue gamma-(L-gamma-oxaglutamyl)-L-cysteinyl-glycine.

An efficient synthesis of the backbone modified glutathione analogue gamma-(L-gamma-oxaglutamyl)-L-cysteinyl-glycine (7), characterized by the presence of an urethane O-CO-NH linkage replacing the gamma-glutamylic CH2CO-NH fragment is described. The new analogue has been fully characterized by 1H- and 13C-NMR, and FAB-MS. Compound 7 was tested for inhibition of gamma-glutamyl-transferase activity and was found to be a non-competitive inhibitor of hog kidney gamma-glutamyltransferase (EC 2.3.2.2).

Cyclization under mild conditions of salicyloyl-dipeptides.

Carboxy-activated linear peptides 6(a-c) of general formula Sal-Xaa-Pro-ONp (Xaa = Phe: Gly; Aib) were synthesized and treated at room temperature with 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) in benzene solution. The tetrahedral adducts (oxa-cyclols) 7, 11 and 12, tautomeric forms of the corresponding 10-membered cyclodepsitripeptides, have been isolated in each of the three models examined. These adducts, which contain the hydroxylated carbon atom located at the junction between two 6-membered rings, do not show a tendency to isomerize into the corresponding macrocyclic lactones, regardless of the nature of the substituents on the C alpha carbon atom of the central residue. Partially cyclized dimeric products 8 and 13, identified as N-(Sal-Xaa-Pro)-dioxopiperazines (Xaa = Phe;Aib), have been also isolated from the cyclization reactions.

Ten-membered cyclotripeptides. III. Synthesis and conformation of cyclo(-Me beta Ala-Phe-Pro-) and cyclo(-Me beta Ala-Phe-DPro-).

The 10-membered cyclotripeptide cyclo(-Me beta Ala-Phe-Pro) 3 and its diastereoisomer cyclo(-Me beta Ala-Phe-DPro-) 4 have been synthesized under mild cyclization conditions starting from linear precursors containing C-terminal proline. The crystal and molecular structure of the two models has been determined by X-ray crystallography. Analysis of the NMR spectra supported by NOE data clearly indicates that the conformations found in the crystals are retained in solution. Both cyclotripeptides exhibit a cis-cis-trans backbone conformation. The two tertiary peptide bonds, at the proline and Me beta Ala nitrogen atoms, adopt a cis conformation whereas the CO-NH junctions are trans in both the models. The deviations from planarity of the peptide units vary from delta omega values of ca. 18 degrees for the Pro-Me beta Ala and DPro-Me beta Ala bonds to ca. 7 degrees for Phe-Pro and Phe-DPro bonds. Relevant conformational details of 3 and 4, as revealed by X-ray and NMR analysis, are reported. Crystals of 3 are monoclinic: P2(1), a = 5.317(2), b = 17.059(6), c = 9.514(3) A, beta = 99.18(3), Z = 2. The final R and Rw are 0.054 and 0.071 respectively. Crystals of 4 are orthorhombic: P2(1)2(1)2(1), a = 8.797(2), b = 19.440(9), c = 21.605(10) A, Z = 8. The final R and Rw are 0.069 and 0.104 respectively.