Free radicals generated in biological systems, particularly alkyl peroxy (RCOO), which occurs in lipid peroxidation and superoxide ion (O2-), which is formed during normal metabolism, have been implicated in aging and in age-related degenerative diseases 1,4. There is evidence that lipid peroxidation, the presence of iron and the depletion of natural antioxidants are common epiphenomenona of some pathologies in the central nervous system (CNS). The role of iron in catalysing oxygen-derived free radicals, and consequently its role in disease processes, is well known. Currently a tremendous effort is under way to study the properties of naturally occurring antioxidants (vitamin E, ascorbic acid, lipoic acid, flavonoids containing catechol groups, stilbenes related to resveratrol, etc.)1. Even though vitamin E has shown some promise in the treatment of AD, it is unsuitable for acute situations because of its slow CNS uptake. There is therefore a necessity for new synthetic antioxidants to optimise antioxidant activity. However, none of the currently available antioxidants have proven efficacious in a large-scale controlled study. The essential considerations are the design of the antioxidant drug and the appropriate targeting to the affected site within the timeframe of maximal tissue damage and with minimal side effects1,4. EURODESY will provide training in the synthesis of series of antioxidants, combining in one molecular scaffold structural features responsible for the radical scavenging activity of vitamin E or lipoic acid connected through spacers with catechol moieties, as well as hydroxy stilbenoid dendrimers and iron chelators. The synthetic methodologies will be based on our own experience5 and on literature procedures.
Over the past decade, it has become clear that the brain is a steroidogenic organ. The steroids synthesised by the brain and nervous system, given the name neurosteroids, have potent and specialised activity at both central and peripheral neuronal targets. Both in vitro and in vivo studies strongly suggest therapeutic potential for such steroids in a variety of developmental, stress-related or age-related neurological and psychiatric disorders2. In humans, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEAS) are the most abundant circulating steroids during development, and their levels decline with aging. As both age and stress are associated with neuronal vulnerability to degeneration, several investigators have hypothesised that DHEA and DHEAS may be active in protecting the brain from damage due to neurodegeneration and/or ischemic events caused by stroke or trauma to the central nervous system. Recent studies using in vivo and in vitro models, as well as epidemiological data, suggest that estrogens provide neuroprotection of CNS cells implicated in the etiology of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases6. EURODESY will offer training in the synthesis of DHEA analogues with modifications at position 3 and allopregnanolone analogues with modifications at positions 3, 17, 20 or 21 with the aim of inhibiting metabolic transformation to androgens or estrogens and achieving optimum neuroprotective activity. Based on our previous experience we have designed new molecules to be synthesised in the context of the proposed project.
Cannabinoids have a long history of consumption for recreational and medical reasons. The primary active constituent of the hemp plant, Cannabis sativa, is Ä 9-tetrahydrocannabinol (Ä 9-THC). Two subtypes of cannabinoid receptors have been identified to date, the CB1 receptor, essentially located in the CNS, but also in peripheral tissues, and the CB2 receptor, found only at the periphery. The identification of Ä 9-tetrahydrocannabinol (Ä 9-THC) as the major active component of marijuana (Cannabis sativa), the recent emergence of potent synthetic ligands and the identification of anandamide and sn-2 arachidonylglycerol as putative endogenous ligands for cannabinoid receptors in the brain, have contributed to advancing cannabinoid pharmacology and approaching the neurobiological mechanisms involved in physiological and behavioral effects of cannabinoids. Emerging research indicates that cannabinoids possess neuroprotective properties6,7. Researchers at the National Institutes for Mental Health (NIMH) demonstrated that the cannabinoids THC and cannabidiol (CBD) are potent anti-oxidants in animals8. NIMH scientists found that CBD protected rat brain cells that had been exposed to toxic levels of glutamate better than standard anti-oxidants like vitamins C and E. Moreover, it was recently observed that some cannabinoids are potent antioxidants that can protect neurons from death even without cannabinoid receptor activation6. It seems that cannabinoids could delay or even stop progressive degeneration of brain dopaminergic systems, a process for which there is presently no prevention. In combination with currently used drugs, cannabinoids might represent, qualitatively, a new approach to the treatment of Parkinson's disease PD, making it more effective. In EURODESY novel cannabinoids will be synthesised with modifications at 1' and or 7' position of the cannabinoid side chain (cyclic substituents and different functional groups, respectively) in order to explore the effect of these substitutions on the ability of the novel analogues to recognise the two known cannabinoid receptors.
GP and PhK inhibitors
Inhibitors of GP have been proposed as a therapeutic strategy for improving glycaemic control in T2D mellitus and various studies have shown the efficacy of such compounds at lowering blood glucose or inhibiting liver glycogenolysis in vivo9. Inhibitor design for GP targets the AMP activatory and the glucose-6-P inhibitory allosteric sites, the catalytic site that binds glycogen and glucose-1-P, the inhibitor site located 12 A from the catalytic site and which binds caffeine and heterocyclic compounds such as flavopiridol, and a novel allosteric inhibitor site, shown to bind a number of indole-2-carboxamide inhibitors10. In this project we wish to extend our knowledge on molecular recognition of small molecules by the catalytic site of GP and to develop new potent inhibitors by using as scaffolds the inhibitor N-acetyl-â-D-glucopyranosylamine and oxadiazole derivatives of glucopyranose11. Furthermore, guided by the crystal structure of the ã truncated subunit of PhK and by employing the structure-based ligand design method, we wish to initiate the design, synthesis and assessment of potent inhibitors of ã truncated subunit of PhK12 by screening libraries of indole-imidazole hybrids connected at position C-3 of indole and 4(5) of imidazole through a variety of spacers. Identified lead compounds will be used as scaffold for the synthesis of 2nd generation of inhibitors according to the results obtained by 3D structural analysis. EURODESY will thus adopt a multidisciplinary approach to go from a protein crystal structure to the development of high affinity inhibitors.
Computational and biophysical studies
In parallel with the synthesis of the target compounds, molecular modelling and conformational analysis will also be performed. These techniques aid the design of bioactive compounds and support experimental phases. The high cost in the development of novel drugs has led nowadays to rational drug design, which combines the recent advancements of computational chemistry (QSAR, docking, conformational search methods etc.) as well as the experience and talent of the medicinal chemist. Ab-initio and semi-empirical techniques will be used as well as conformational search methods in order to examine the energy barriers of their flexible segments13. The interaction and binding of the ligand to the target site depends on factors such as the electronic distribution and the polarisabilities of the interacting groups and the expertise which the Biophysical and Computational Analysis Group has in the study of molecular structure and electronic properties will be employed for the development and application of these methods14. In addition, these methods may give plausible explanations for the experimental results and highlight new avenues for novel potential drugs. Conformational analysis involves the use of a combination of 2D NMR spectroscopy and conformational search methods. These methods coupled with distance geometry will be used to complement the experimental data. For the design and study of novel antioxidants, we shall develop methods to compute the bond dissociation enthalpy (BDE) of large molecules containing O-H, C-H, N-H, etc, since it has been shown that BDE may be used to predict antioxidant activity.
Oxidative cell death in neurodegenerative diseases
Oxidative stress has been causatively linked to the onset and progression of Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis and to neuronal damage ensuing cerebral ischemia. Hippocampal neurons are particularly sensitive to oxidative cell death. The HT22 mouse hippocampal cell line is devoid of ionotropic glutamate receptors. Nevertheless, glutamate inhibits cystine transport into HT22 cells, thus causing glutathione levels to drop, ROS production to burst and cell death to come about by some unknown mechanism15. Antioxidants (e.g Vitamin E), gonadal steroids (e.g. estrogens), neurosteroids (e.g. DHEA) and cannabinoids (e.g. cannabinol), are known to rescue HT22 cells from glutamate-induced cell death by non-receptor mechanisms16. However, while genetically engineered expression of CB1 receptor failed to sensitize HT22 cells to cannabinoids, expression of estrogen receptor alpha (ERá) and beta (ERâ) was pronouncedly effective in this respect, suggesting direct involvement of ER in neuroprotection17. Given that both ER types are widely expressed in the CNS; that ERá but not ERâ has been reported to mediate the neuroprotective effect of estradiol following brain injury; and that both ER types are known to bind a large selection of phenolic compounds with neuroptotective activity (xenoestrogens and phytoestrogens as well as various gonadal, adrenal and neuro steroids)18, in addition to estradiol, receptor involvement in neuroprotection warrants further investigation.
The antioxidant neuroprotective potential of the new compounds will be assessed using HT22 hippocampal neurons and clones thereof, stably transfected with ERá, ERâ or the empty expression vector to elucidate receptor involvement in oxidative stress and neurodegeneration. HEK239 cells genetically engineered to express an ER-regulated luciferase reporter as well as ERá or ERâ will be used to determine the ER transactivation potential of the new compounds and compared it to their neuroprotective activity and chemical antioxidant properties. Áll new compounds tested positive in oxidative cell death prevention will be assayed for their affinity for ERá and ERâ. Highly neuroprotective compounds, in particular, will be further selected for not increasing the proliferation of prostate (LNCaP), breast (MCF-7) and endometrial adenocarcinoma (Ishikawa) cells, as a means to minimize their cancer risk. Effort will be put into modifying the structure of the highly neuroprotective compounds so as to minimize their stimulation of cell proliferation while maintaining their neuroprotective activity to the highest possible level. Stable MCF-7 cells transfectants bearing highly sensitive estrogen-responsive luciferase reporters will be also employed to verify that the hormonal activity of the lead compounds is weak if not marginal19.
The major original results EURODESY is expected to produce is a series of novel anti-oxidants, neurosteroids, canabinoids and GP and PhK inhibitors, with better activity than the currently available derivatives. The innovative approach through which these compounds will be produced involves organic, organometallic, and pharmaceutical chemistry, structural biology, biophysical methods and molecular modelling a well as methods of evaluation of the biological activity of the new potentially therapeutical agents.
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