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.

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.

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.

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.

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.

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.

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.

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.

4-Amino-1,2-dithiolane-4-carboxylic acid (Adt) as cysteine conformationally restricted analogue. Synthetic protocol for Adt containing peptides.

An efficient and versatile protocol to incorporate the achiral and C(alpha,alpha)-tetrasubstituted 4-amino-1,2-dithiolane-4-carboxylic acid Adt (1) residue into peptides is described. The 2,2-bis[(benzylthio)methyl]glycine N-carboxy anhydride (5) was found to be the key reactive intermediate from which both Boc-Adt-OMe (8) and the glutathione analogue H-Glu(-Adt-Gly-OH)-OH (12) can be obtained.

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.

Novel glutathione analogues containing the dithiol and disulfide form of the Cys-Cys dyad.

The glutathione analogue gamma-(H-Glu-OH)-Cys-Cys-OH (5), containing the 8-membered disulfide ring -Cys-Cys replacing the native-Cys-Gly fragment, has been synthesized and characterized together with its reduced dithiol form gamma-(H-Glu-OH)-Cys-Cys-OH (6).

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.

Hypotaurine disproportionation reaction: identification of products.

Hypotaurine, concentrated under reduced pressure in HCl solution, partially (30-40%) degrades into taurine (about 30%), 2-aminoethyl-2-aminoethanethiolsulfonate (about 10%) and ethanolamine. The degradation products were identified using LC/APCI-MS, NMR, amino acid analysis and various chromatographies. The identities were confirmed by comparing the HPLS, MS and NMR characteristics of authentic compounds. One of the degradation processes during concentration of HCl solution of hypotaurine is therefore a disproportionation reaction which can interfere with the experimental results, when studying hypotaurine in biological systems.

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.

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).

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

The backbone-modified glutathione analogue gamma-(L-gamma-azaglutamyl)-L-cysteinyl-glycine 7, characterized by the presence of a NHCONH urea linkage deriving from the replacement of the native Glu gamma-CH2 with the aza (NH) group, was synthesized and fully characterized by FAB-MS, 1H- and 13C-NMR. Potential of 7 and its oxidized form 6 as gamma-glutamyltransferase inhibitors was investigated. Both compounds 7 and 6 were found to be competitive inhibitors of hog kidney gamma-glutamyltransferase (EC 2.3.2.2.) by binding at the donor site: the reduced analogue is a more efficient inhibitor than glutathione of the gamma-glutamyl transfer reaction. Inhibition at the acceptor site, which is also present, appears to be more complex. In particular, un-competitive inhibition is observed for compound 7. The results indicate that gamma-azapeptides of type 7 may represent interesting targets in the search for stable inhibitors of gamma-glutamyltransferases.

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.

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.