Among the molecular mechanisms responsible of oxidative damage involved in the pathogenesis and/or progression of several degenerative disorders including aging, inflammation, diabetes, cardiovascular and neurodegenerative diseases, structure modification of protein, lipids and nucleic acids induced by reactive oxygen species (ROS) and reactive carbonyl species (RCS) derived from lipid oxidation plays a key role. RCS, characterized by a keto/aldehydic function, are important cytotoxic mediators, since by inducing irreversible structural modifications to biomolecules, lead to alteration of the cellular function [1-4]. Most of the biological effects of RCS generated by oxidation of omega-6 polyunsaturated fatty acids, such as dialdehydes (malondiladehyde, glyoxal) and alpha,beta-unsaturated aldehydes, 4-hydroxy-trans-2-nonenal (HNE) and acrolein, are attributed to their strong electrophilic nature: these compounds can react, through a Michael addition, with the nucleophilic sites of proteins/peptides (lysine, cysteine and histidine residues), and with nucleic acids (deoxyguanosine) to form covalently modified biomolecules which can alter/disrupt important cellular functions and induce mutations . The causal involvement of unsaturated aldehydes in the lipid peroxidation-mediated pathologies has been only recently demonstrated by the use of mono- and poli-clonal antibodies raised against HNE or acrolein . HNE or acrolein adducts with proteins have been immunohistochemically detected in bioptic and autoptic specimens of subjects affected by diabetes, atherosclerosis, muscolar distrophy, rheumatoid arthtritis, actinic elastosis, cerebral ischemia, and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases [5-9]. Hence, RCS can be considered an important target for the development of a new class of biologically active compounds with carbonyl-quencher properties, nucleophilic compounds able to deactivate the reactive carbonyl function through the formation of Michael adducts or Schiff bases, as demonstrated for aminoguanidine, hydralazine and pyridoxamine [8, 10]. However, the low selectivity towards lipoxidation-derived aldehydes represents the main limiting factor to the pharmacological use of these compounds: hydralazine and aminoguanidine, being characterized by a strong nucleophilic center, react not only with RCS, but also with physiologically relevant aldehydes (pyridoxal phosphate, Schiff base formation), inducing their depletion. In addition, the promiscuous activity of some RCS quenchers, as aminoguanidine (inhibitory action on inducible NO-synthase) or hydralazine (vasodilating and antihypertensive effect) greatly limits their clinical use as carbonyl trapping agents . On the basis of these considerations, we have undertaken in the last years a research program with the aim to identify endogenous peptides able to quench RCS, and in particular the most toxic alfa,beta-unsaturated aldehydes. The strategy was firstly based on a peptidomic approach focused to identify endogenous compounds involved in the detoxification of RCS in those tissues (skeletal muscle) highly susceptible of oxidative attack . This approach allowed identifying, besides GSH, a series of histidine-containing peptides such as carnosine (β-alanyl-L-histidine), anserine and homocarnosine (gamma-aminobutiryl-L-histidine), dipeptides present in high concentrations in human skeletal muscle and brain, whose carbonyl quenching ability was previously proposed [13,14]. Further in vitro studies performed in our laboratory [15-17] confirmed the potent quenching activity of carnosine and analogues towards HNE and acrolein. By ESI-MS/MS and NMR analyses it was possible to characterize in homogeneous solution the conjugated products of carnosine with HNE and acrolein, and to elucidate the mechanisms of interaction; by LC-MS/MS analysis (ESI interface) their formation has been confirmed in complex biological matrices (spontaneously oxidized rat skeletal muscle). These results have outstanding biological relevance since they demonstrate the existence in this tissue, highly susceptible to peroxidative attack, of a histidine-dependent detoxification pathway against cytotoxic HNE alternative/concomitant to that involving thiol-containing peptides. In addition, it has been demonstrated for the first time the protective effect of carnosine in a cellular model (human keratinocyte cell line NCT2544) that mimics the skin damage induced by UV radiation (sequential exposure to UVB and HNE) [18-20]. More recently, in order to gain a better understanding of the biological role of histidine-containing peptides in the oxidative-mediated physio-pathological processes, highly specific and sensitive LC-MS/MS methods have been developed for the determination of the endogenous levels of carnosine and homocarmnosine in biological matrices (to study their hematic and tissue distribution in the rat) , and for determination of the corresponding Michael adducts with HNE in complex biological matrices (rat skeletal muscle) . In this study it was possibile to demonstrate that the carnosine/HNE adduct can be considered as an early, specific and stable marker of lipid peroxidation in those biological districts where it is specifically located, and unequivocally confirm its endogenous role as an aldehyde sequestering agent. Notwithstanding the high reactivity, specificity and safety , carnosine is rapidly hydrolyzed in plasma and kidney by carnosinases (specific hydrolases that catalyze the cleavage of the dipeptide with formation of b-alanine and histidine, totally devoid of quenching ability). This greatly limits its potential pharmacological application as detoxifying agent for RCS to prevent RCS-induced damage. Hence, L-carnosine was used in the first drug discovery approach as a model for developing novel long-lasting drug candidates which, maintaining the trapping activity and safety of the parent compound, will result more resistant to the enzymatic hydrolysis. To do this, an in silico model for carnosinase has been developed using the experimental structures of homologous dipeptidyl hydrolases (the enzyme structure by X-rays is not yet available) which allows to screen by molecular docking new carnosine derivatives, highly resistant to carnosinase . By the application of this in silico model, it was possible to identify D-carnosine (β-alanyl-D-histidine) as target compound. D-carnosine is in fact characterized by a carbonyl quenching activity comparable to that of L-carnosine (as determined in vitro according to the protocol already applied to L-carnosine), but it is totally resistant to the carnosinase hydrolytic action, as experimentally demonstrated using human serum as enzyme source and HPLC-ESI-MS/MS analysis to monitor its stability in serum. On the basis of these premises, the first step of the incoming project will focus as a first objective, on the identification of a series of D-carnosine analogues with the final aim to optimize the following parameters: absorption, reactivity towards α,β-unsaturated aldehydes and dicarbonyls (1,2- 1,3- and 1,4-dicarbonyls), selectivity, metabolic stability and pharmacokinetics, target tissue tropism and distribution. In detail, the research program will consist of the following phases: 1. Design and synthesis of D-carnosine analogues. The aim is to develop D-carnosine analogues characterized by a greater carbonyl quenching activity. On the basis of the previously demonstrated mechanism of HNE/carnosine interaction , this approach will involve suitable chemical modifications in order to stabilize the imine intermediate and then to favour the intramolecular Michael adduction (in collaboration with other academic and industrial partners). 2. Evaluation of the carbonyl-quenching activity in vitro. The results of this phase will allow identifying not only the more potent and selective carbonyl quenchers, but also those with the lower toxicological profile (lower reactivity towards physiologically relevant aldehydes), thus to eliminate aspecific and potentially toxic compounds in the first steps of drug development. In detail, through the development and the application of medium-high throughput screening by mass spectrometry, for each selected candidate the following parameters will be evaluated: reactivity towards alpha,beta-unsaturated aldehydes (HNE and acrolein) as prototypes of lipid peroxidation products; reactivity towards dialdehydes (glyoxal and methylglyoxal) as prototypes of glucose oxidation; pyridoxal phosphate as a prototype of physiological non cytotoxic aldehydes. The determination of the rate constants for reaction will enable to identify the compound/s to be screened in the successive phases of the project. The mass spectrometric approach will also allow to characterize the reaction products and to elucidate the mechanisms of reaction, useful information in the design phase. In addition, the determination of the carbonyl-quenching activity will be performed using carbonylated proteins (albumin, actin), to demonstrate the ability of the selected molecule/s to prevent and/or to rigenerate the native protein through a de-carbonylation reaction. This approach involves the preparation of carbonylated proteins, according to already developed methodologies [25,26] and the subsequent mass spectrometric analysis (direct infusion experiments under ESI conditions). 3. Metabolic stability and metabolic pre-screening It is well known that metabolic screening and structural characterization of metabolites, as one of major efforts to reduce attrition rates in drug development, has increasingly become an integral part of the ADMET-guided lead optimization process in drug discovery. Hence, the research program involves the evaluation of the metabolic stability in the early phase of drug development, with the final aim to identify and to optimize the lead compound/s on the basis of the metabolic parameters. This screening phase will be carried out by evaluating: a) the stability to the carnosinase action, by applying the in silico model of the enzyme already developed ; b) the metabolic stability in rat and human plasma and the in vitro metabolic profile (rat and human liver subcellular fractions) through the application of tandem mass spectrometric techniques integrated with automated systems and data analysis (high-throughput metabolic screening). 4. Design and synthesis of lipophilic derivatives of D-carnosine Because the highly hydrophilic character of D-carnosine and derivatives (unlike L-carnosine, D-carnosine itself is not recognized by the active transport system PEPT1), this phase involves the design and synthesis (in collaboration with other academic and industrial partners) of lipophilic derivatives (logP 2-3) of the compound/s selected from the above reported steps, with the final aim to optimize the absorption profile. 5. ADME studies In the last phase of the project, an ADME (absorption, distribution, metabolism, excretion) pilot study of the lead compound will be performed in the animal following oral administration. This, through the determination of plasma and tissue levels by the already developed LC-MS/MS methodology , suitably modified. 1. Halliwell B, et al. Free Radicals in Biology and Medicine (2001) Oxford Science Pubblications, 3° Edition. 2. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 1991;11:81. 3. Uchida K. Role of reactive aldehyde in cardiovascular diseases. Free Radic. Biol. Med. 2000;28:1685. 4. Poli G, Schaur RJ. 4-Hydroxynonenal in the pathomechanisms of oxidative stress. IUBMB Life 2000;50:315. 5. Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res. 2003;42:318. 6. Zarkovic N. 4-hydroxynonenal as a bioactive marker of pathophysiological processes. Mol. Aspects Med. 2003;24:281. 7. Zarkovic N. 4-hydroxynonenal and neurodegenerative diseases. Mol. Aspects Med. 2003; 24:293. 8. Carini M, Aldini G, Maffei Facino R. Sequestering agents of intermediate reactive aldehydes as inhibitors of advanced lipoxidation end-products (ALEs). In “Redox Proteomics: from Protein Modifications to Cellular Dysfunction and Diseases” (Ed. I. Dalle-Donne, A. Scaloni, and A. Butterfield); Hoboken: John Wiley & Sons Inc., pp. 887-929 (2006). 9. Carini M, Aldini G, Maffei Facino R. Mass spectrometry for detection of 4-hydroxy-trans-2-nonenal (HNE) adducts with peptides and proteins. Mass Spectrom. Rev. 2004;23:285. 10. Dalle-Donne I, Aldini G, Carini M, Colombo R, Rossi R, Milzani A. Protein carbonylation, cellular dysfunction, and disease progression J. Cell. Mol. Med. 2006 10(2):389-406. 11. Aldini G. Dalle-Donne I, Maffei Facino R, Milzani A, Carini M. Intervention strategies to inhibit protein carbonylation by lipoxidation-derived reactive carbonyls. Med Res Rev., 2006 in press (published online 16 october 2006 in Wiley InterScience www.interscience.wiley.com) 12. Aldini G, Dalle-Donne I, Milzani A, Colombo R, Maffei Facino R, Carini M. Advanced lipoxidation end-products as potential drug targets in preventing protein carbonylation and related cellular dysfunction. ChemMedChem, 2006 1(10):1045-58. 13. Hipkiss AR, Preston JE, Himsworth DT, Worthington VC, Keown M, Michaelis J, Lawrence J, Mateen A, Allende L, Eagles PA, Abbott NJ. Pluripotent protective effects of carnosine, a naturally occurring dipeptide. Ann N Y Acad Sci. 1998 20;854:37-53. 14. Hipkiss AR. Carnosine and protein carbonyl groups: a possible relationship. Biochemistry (Mosc). 2000 ;65(7):771-8. Review. 15. Aldini G, Granata P, Carini M. Detoxification of cytotoxic alpha,beta-unsaturated aldehydes by carnosine: characterization of conjugated adducts by electrospray ionization tandem mass spectrometry and detection by LC/MS in rat skeletal muscle. J. Mass Spectrom. 2002;37:1219. 16. Aldini G, Carini M, Beretta G, Bradamante S, Maffei Facino R. Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction? Biochem. Biophys. Res. Commun. 2002;298:699. 17. Carini M, Aldini G, Beretta G, Arlandini E, Maffei Facino R. Acrolein-sequestering ability of endogenous dipeptides: characterization of carnosine and homocarnosine/acrolein adducts by electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 2003;38:996. 18. Carini M, Aldini G, et al. Detoxification of cytotoxic unsaturated aldehydes in keratinocytes is a GSH-dependent pathway: inhibition by UVB and protective effect of histidine and cysteine-containing peptides. Preprints of the 22th IFSCC Congress, 2002 Edinburgh, pp.129-143. 19. Aldini G, Granata P, Orioli M, Santaniello E, Carini M. Detoxification of 4-hydroxynonenal (HNE) in keratinocytes: characterization of conjugated metabolites by liquid chromatography/electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 2003;38:1160. 20. Aldini G. Granata P., Beretta G., Marinello C., Carini M., Maffei Facino R. Effects of UVB radiation on 4-hydroxy-2-trans-nonenal metabolism and toxicity in human keratinocytes. Chem Res Toxicol, 2007 accepted 21. Aldini G, Orioli M., Carini M, Maffei Facino R. Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 2004;39:1417. 22. Orioli M, Aldini G, Beretta G, Maffei Facino R, Carini M. LC-ESI-MS/MS determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and histidine-containing peptides as early markers of oxidative stress in excitable tissues. J. Chromatogr. B 2005;827:109. 23. Aldini G, Maffei Facino R, Beretta G, Carini M. Carnosine and related dipeptides as quencher of reactive carbonyl species: from structural studies to therapeutic intervention. Biofactors 2005;24:77. 24. Vistoli G, Perdetti A, Cattaneo M, Aldini G, Testa B. Homology modeling of human serum carnosinase, a potential medicinal target, and MD simulations of its allosteric activation by citrate, J.Med.Chem. 2006; 49:3269-77. 25. Aldini G, Dalle-Donne I, Vistoli G, Maffei Facino R, Carini M. 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|Titolo:||ANALYTICAL APPROACHES IN THE DISCOVERY OF INHIBITORS OF DEGENERATIVE DISORDERS INDUCED BY CARBONYL STRESS|
|Supervisori e coordinatori interni:||DE MICHELI, CARLO|
|Data di pubblicazione:||2009|
|Settore Scientifico Disciplinare:||Settore CHIM/08 - Chimica Farmaceutica|
|Citazione:||ANALYTICAL APPROACHES IN THE DISCOVERY OF INHIBITORS OF DEGENERATIVE DISORDERS INDUCED BY CARBONYL STRESS ; tutor: Marina Carini ; coordinatore Carlo de Micheli. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE FARMACEUTICHE "PIETRO PRATESI", 2009. ((22. ciclo, Anno Accademico 2006/2007.|
|Appare nelle tipologie:||13 - Tesi di dottorato discussa entro ottobre 2010|