Introduction Reactive carbonyl species (RCS) are important cytotoxic mediators generated by lipid oxidation of PUFAs, leading to alteration of the cellular function and inducing irreversible structural modifications to biomolecules. RCS belong to different chemical classes such as alfa,beta-unsaturated aldehydes [4-hydroxy-trans-2-nonenal (HNE), acrolein (ACR)], dialdehydes [malondialdehyde (MDA), glyoxal (GO)], levuglandines and prostaglandin member of J2 series. RCS are electrophilic and reactive compounds capable to form covalent adducts with nucleophilic molecules in particular proteins and nucleic acids. RCS and the corresponding adducts with proteins (carbonylated proteins) are widely used as biomarkers of lipid peroxidation and, in general, of oxidative stress. Moreover, there are several convincing evidences supporting a pathogenic role for RCS, such as in the case of diabetic-related diseases, age-dependent tissue dysfunction, and metabolic distress syndrome. Consequently, RCS, in addition of being a predictive biomarker, also represents a biological target for drug discovery (1). Aim of the work The present work is devoted to better understand the molecular and cellular effects of RCS and to design novel compounds able to inhibit the RCS-derived cellular dysfunction, the RCS sequestering agents (RCS-SA). To reach these goals, the following steps have been conducted: (i) to study the effects of RCS on protein and cellular function; (ii) to design specific and efficient sequestering agents of RCS; (iii) to set-up an analytical method aimed to screen reactivity, selectivity and to elucidate the reaction mechanisms of RCS-SA; (iv) to identify a biomarker of protein carbonylation to monitor the efficacy of RCS-SA in in vivo studies. RCS effect on protein and cellular function The cause or effect role of RCS has been evaluated in cellular models by using 15d-PGJ2, a neurotoxic prostaglandin member of J2 series, as model of RCS. By using biotinylated 15d-PGJ2, it was found that actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change and consequently disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization (2). We further studied the effect of acrolein and HNE on actin function and the sites and mechanism of adduction fully characterized by MS analyses (3). Discovery of specific and efficient sequestering agents of RCS Taking RCS and carbonylation damage as drug-targets, different molecular strategies have been up to now considered in order to neutralize/reduce these pathogenetic factors and the most promising is that based on nucleophilic compounds capable to form covalent and unreactive adducts with RCS. Among the most studied RCS sequestering agent, the vitamer pyridoxamine (PYR), hydralazine (HY), and aminoguanidine (AG). Most of the RCS-SA above listed are characterized by a high nucleophilic amino group accompanied by a low basicity favoring the unprotonated form. This feature, if from one side permits a high reactivity towards a broad range of RCS, from another side greatly limits the specificity of the RCS sequestering agents, since it makes the compounds reactive toward biogenic and physiological aldehydes, such as pyridoxal phosphate (4). We recently found that carnosine (beta-alanyl-L-histidine, CAR), an endogenous dipeptide present in mM concentrations in some tissues such as skeletal muscle, is a specific quencher of alfa,beta-unsaturated aldehydes (5). The reaction mechanism involves both the Schiff base formation between the beta-alanine amino group and the aldehydic function, which then catalyses the Michael adduction between the C3 of the aldehyde and the Ntau of the histidine group. This peculiar mechanism of reaction makes CAR highly specific towards alfa,beta-unsaturated. The carbonyl quenching ability of CAR has been confirmed by LC–MS/MS analysis (ESI interface) in complex biological matrices, by detecting the conjugated adducts in spontaneously oxidized rat skeletal muscle. This indicates that histidine-containing dipeptides behave as detoxifying agents for cytotoxic aldehydes, by reacting with HNE in biological systems through a “sacrificial” mechanism that mimics the preferred HNE addition sites in proteins; so CAR can be considered a molecule active against cytotoxic carbonyls. Although the important features of CAR as RCS sequestering agent, a possible therapeutic use of CAR is limited since it is unstable in human plasma due to the presence of carnosinase, a specific dipeptidase which catalyzes the hydrolytic cleavage of the dipeptide (T1/2 of carnosine in human serum < 5 min). Moreover the reactivity of CAR towards RCS is significant lower in respect to that of AG, HY and PYR. Hence, a rational drug design approach was taken with the aim to develop a CAR derivative characterized by the following features: (i) high selectivity towards cytotoxic RCS; (ii) resistant to carnosinase; (iii) more reactive in respect to CAR towards alfa,beta-unsaturated aldehydes. We found that the isomerization of L to D-histidine aminoacid leads to a derivative [(beta-alanyl-D-histidine, D-carnosine (D-CAR)] which is not recognized by the catalytic site of carnosinase as evaluated by in silico analysis. The stability of D-CAR to carnosinase was then confirmed in human serum (no significant loss of D-CAR up to 24 h); moreover, D-CAR was found to posses the same quenching activity towards HNE and ACR, in respect to CAR. The second issue to be solved regards the increase of reactivity towards RCS in respect to the parent compound. Since it is well established that the formation of an immine group at physiological conditions strongly depends on the basicity of the amino group, that influences the fraction of amine present in the unprotonated form, a simple approach to increase the carnosine reactivity is to design derivatives characterized by a lower basicity. Unfortunately, such an approach is not largely exploitable since the introduced modifications would mine the specificity of obtained derivatives hampering the specific Michael adduction. Hence, we focused our attention to such specific Michael reaction, by modulating the conformational profile of the Schiff base intermediate in order to favor a close conformation in which the imidazole ring approaches enough the C3 of the Schiff base to form the corresponding Michael adduct. A series of D-carnosine derivatives was then designed by in silico approaches to find out those characterized by a favorable folded conformational profile. The analogues were then synthesized and the quenching ability, stability in human plasma, basicity and the folded conformation evaluated. By this way a set of phenyl derivatives was identified, characterized by high stability in human plasma (no significant loss of the parent compound after 60 min of incubation), selectivity (no cross reactivity towards pyridoxal) and by a quenching activity towards HNE significantly increased in respect to CAR (the most reactive compound is characterized by a quenching reactivity increased of three folds in respect to carnosine) (manuscript in preparation). A Direct Infusion Mass Spectrometric Method to Study Reactivity, Selectivity and Reaction Mechanisms of Carbonyl Sequestering Agents One of the limiting step in the discovery of novel RCS-SA is the lack of analytical methods capable to screen the reactivity and selectivity. Hence we developed a novel mass spectrometric approach capable to simultaneously provide information on reactivity, selectivity and to elucidate the reaction mechanism of the carbonyl quenchers tested. The method involves: a) incubation of the target cytotoxic (HNE, GO, MGO) or physiological (pyridoxal) aldehyde with the compounds to be tested; b) sample spiking with the internal standard and direct infusion experiments (triple quadrupole, equipped with ESI and APCI sources); c) determination of reactivity and selectivity, by measuring the relative consumption of the tested compound; d) identification of the reaction products in full scan mode and structure characterization by tandem MS experiments. The method was firstly validated using well-known quenchers such as CAR, PYR, AG and HY and the corresponding reaction products fully characterized. The reactivity towards HNE was HY>AG>PYR>CAR and all the compounds, except CAR, were able to react with pyridoxal thus to demonstrate a lack of selectivity. The method has been then successfully applied to screen the CAR phenyl derivatives as above described (MS in preparation). Identification of biomarker of protein carbonylation to monitor the efficacy of RCS-SA in vivo studies Aim of this step was to identify a biomarker of protein carbonylation to be used in in vivo studies. By using a proteomic approach we found that albumin (HSA) is the main serum protein to be modified by RCS, such as HNE. The high reactivity of HNE towards HSA was then studied in in vitro models. By using a direct infusion ESI-MS approach we found that HNE rapidly reacts with HSA because of the covalent adduction to the different accessible nucleophilic residues of the protein. An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving several Lys, His and Cys nucleophilic sites. The most reactive HNE-adduction site was found to be Cys34 (MA), a suitable tag of HNE-adducted albumin that can be used as an useful biomarkers of oxidative and carbonylation damage in humans (6,7). Conclusion In the present work we confirmed that RCS, through the formation of Michael and Schiff base adducts, are involved in protein and cellular derangement. We then discovered a novel class of RCS-SA, the phenyl derivatives of D-CAR, reactive and selective towards RCS, as well as stable in human serum. Future studies will deal with the effect of these compounds in reducing RCS-dependent diseases and protein carbonylation, in animal models, such as the obese Zucker rats. References (1) G. Aldini, I. Dalle-Donne, R. Maffei Facino, A. Milzani, M. Carini. Med Res Rev. 2007;27(6):817-68.. (2) G. Aldini, M. Carini, G. Vistoli, T. Shibata, Y. Kusano, L. Gamberoni, I. Dalle-Donne, A.Milzani, K. Uchida. Biochemistry 46 (2007) 2707-18. (3) I. Dalle-Donne, M. Carini, G. Vistoli, L. Gamberoni, D. Giustarini, R. Colombo, R. Maffei Facino, R. Rossi, A. Milzani, G. Aldini. Free Radic Biol Med. 42 (2007) 583-98. (4) G. Aldini, I. Dalle-Donne, R. Colombo, R. Maffei Facino, A. Milzani, M. Carini. ChemMedChem. 1 (2006) 1045-58. (5) G. Aldini, M. Carini, G. Beretta, S. Bradamante, R. Maffei Facino. Biochem Biophys Res Commun. (298) 2002 699-706. (6) G. Aldini, L. Gamberoni, M. Orioli, G. Beretta, L. Regazzoni, R. Maffei Facino, M. Carini. J Mass Spectrom. 41 (2006) 1149-61. (7) G. Aldini, G. Vistoli, L. Regazzoni, L. Gamberoni, R. Maffei Facino, S. Yamaguchi, K Uchida and M. Carini [inviato]

Discovery of inhibitors of carbonyl related degenerative disease : peptides and derivatives as detoxifying agents of cytotoxic aldehydes / L. Gamberoni ; Tutor: G. Aldini. - : . DIPARTIMENTO DI SCIENZE FARMACEUTICHE "PIETRO PRATESI", 2007 Feb 01. ((20. ciclo, Anno Accademico 2004/2005.

Discovery of inhibitors of carbonyl related degenerative disease : peptides and derivatives as detoxifying agents of cytotoxic aldehydes

L. Gamberoni
2007-02-01

Abstract

Introduction Reactive carbonyl species (RCS) are important cytotoxic mediators generated by lipid oxidation of PUFAs, leading to alteration of the cellular function and inducing irreversible structural modifications to biomolecules. RCS belong to different chemical classes such as alfa,beta-unsaturated aldehydes [4-hydroxy-trans-2-nonenal (HNE), acrolein (ACR)], dialdehydes [malondialdehyde (MDA), glyoxal (GO)], levuglandines and prostaglandin member of J2 series. RCS are electrophilic and reactive compounds capable to form covalent adducts with nucleophilic molecules in particular proteins and nucleic acids. RCS and the corresponding adducts with proteins (carbonylated proteins) are widely used as biomarkers of lipid peroxidation and, in general, of oxidative stress. Moreover, there are several convincing evidences supporting a pathogenic role for RCS, such as in the case of diabetic-related diseases, age-dependent tissue dysfunction, and metabolic distress syndrome. Consequently, RCS, in addition of being a predictive biomarker, also represents a biological target for drug discovery (1). Aim of the work The present work is devoted to better understand the molecular and cellular effects of RCS and to design novel compounds able to inhibit the RCS-derived cellular dysfunction, the RCS sequestering agents (RCS-SA). To reach these goals, the following steps have been conducted: (i) to study the effects of RCS on protein and cellular function; (ii) to design specific and efficient sequestering agents of RCS; (iii) to set-up an analytical method aimed to screen reactivity, selectivity and to elucidate the reaction mechanisms of RCS-SA; (iv) to identify a biomarker of protein carbonylation to monitor the efficacy of RCS-SA in in vivo studies. RCS effect on protein and cellular function The cause or effect role of RCS has been evaluated in cellular models by using 15d-PGJ2, a neurotoxic prostaglandin member of J2 series, as model of RCS. By using biotinylated 15d-PGJ2, it was found that actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change and consequently disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization (2). We further studied the effect of acrolein and HNE on actin function and the sites and mechanism of adduction fully characterized by MS analyses (3). Discovery of specific and efficient sequestering agents of RCS Taking RCS and carbonylation damage as drug-targets, different molecular strategies have been up to now considered in order to neutralize/reduce these pathogenetic factors and the most promising is that based on nucleophilic compounds capable to form covalent and unreactive adducts with RCS. Among the most studied RCS sequestering agent, the vitamer pyridoxamine (PYR), hydralazine (HY), and aminoguanidine (AG). Most of the RCS-SA above listed are characterized by a high nucleophilic amino group accompanied by a low basicity favoring the unprotonated form. This feature, if from one side permits a high reactivity towards a broad range of RCS, from another side greatly limits the specificity of the RCS sequestering agents, since it makes the compounds reactive toward biogenic and physiological aldehydes, such as pyridoxal phosphate (4). We recently found that carnosine (beta-alanyl-L-histidine, CAR), an endogenous dipeptide present in mM concentrations in some tissues such as skeletal muscle, is a specific quencher of alfa,beta-unsaturated aldehydes (5). The reaction mechanism involves both the Schiff base formation between the beta-alanine amino group and the aldehydic function, which then catalyses the Michael adduction between the C3 of the aldehyde and the Ntau of the histidine group. This peculiar mechanism of reaction makes CAR highly specific towards alfa,beta-unsaturated. The carbonyl quenching ability of CAR has been confirmed by LC–MS/MS analysis (ESI interface) in complex biological matrices, by detecting the conjugated adducts in spontaneously oxidized rat skeletal muscle. This indicates that histidine-containing dipeptides behave as detoxifying agents for cytotoxic aldehydes, by reacting with HNE in biological systems through a “sacrificial” mechanism that mimics the preferred HNE addition sites in proteins; so CAR can be considered a molecule active against cytotoxic carbonyls. Although the important features of CAR as RCS sequestering agent, a possible therapeutic use of CAR is limited since it is unstable in human plasma due to the presence of carnosinase, a specific dipeptidase which catalyzes the hydrolytic cleavage of the dipeptide (T1/2 of carnosine in human serum < 5 min). Moreover the reactivity of CAR towards RCS is significant lower in respect to that of AG, HY and PYR. Hence, a rational drug design approach was taken with the aim to develop a CAR derivative characterized by the following features: (i) high selectivity towards cytotoxic RCS; (ii) resistant to carnosinase; (iii) more reactive in respect to CAR towards alfa,beta-unsaturated aldehydes. We found that the isomerization of L to D-histidine aminoacid leads to a derivative [(beta-alanyl-D-histidine, D-carnosine (D-CAR)] which is not recognized by the catalytic site of carnosinase as evaluated by in silico analysis. The stability of D-CAR to carnosinase was then confirmed in human serum (no significant loss of D-CAR up to 24 h); moreover, D-CAR was found to posses the same quenching activity towards HNE and ACR, in respect to CAR. The second issue to be solved regards the increase of reactivity towards RCS in respect to the parent compound. Since it is well established that the formation of an immine group at physiological conditions strongly depends on the basicity of the amino group, that influences the fraction of amine present in the unprotonated form, a simple approach to increase the carnosine reactivity is to design derivatives characterized by a lower basicity. Unfortunately, such an approach is not largely exploitable since the introduced modifications would mine the specificity of obtained derivatives hampering the specific Michael adduction. Hence, we focused our attention to such specific Michael reaction, by modulating the conformational profile of the Schiff base intermediate in order to favor a close conformation in which the imidazole ring approaches enough the C3 of the Schiff base to form the corresponding Michael adduct. A series of D-carnosine derivatives was then designed by in silico approaches to find out those characterized by a favorable folded conformational profile. The analogues were then synthesized and the quenching ability, stability in human plasma, basicity and the folded conformation evaluated. By this way a set of phenyl derivatives was identified, characterized by high stability in human plasma (no significant loss of the parent compound after 60 min of incubation), selectivity (no cross reactivity towards pyridoxal) and by a quenching activity towards HNE significantly increased in respect to CAR (the most reactive compound is characterized by a quenching reactivity increased of three folds in respect to carnosine) (manuscript in preparation). A Direct Infusion Mass Spectrometric Method to Study Reactivity, Selectivity and Reaction Mechanisms of Carbonyl Sequestering Agents One of the limiting step in the discovery of novel RCS-SA is the lack of analytical methods capable to screen the reactivity and selectivity. Hence we developed a novel mass spectrometric approach capable to simultaneously provide information on reactivity, selectivity and to elucidate the reaction mechanism of the carbonyl quenchers tested. The method involves: a) incubation of the target cytotoxic (HNE, GO, MGO) or physiological (pyridoxal) aldehyde with the compounds to be tested; b) sample spiking with the internal standard and direct infusion experiments (triple quadrupole, equipped with ESI and APCI sources); c) determination of reactivity and selectivity, by measuring the relative consumption of the tested compound; d) identification of the reaction products in full scan mode and structure characterization by tandem MS experiments. The method was firstly validated using well-known quenchers such as CAR, PYR, AG and HY and the corresponding reaction products fully characterized. The reactivity towards HNE was HY>AG>PYR>CAR and all the compounds, except CAR, were able to react with pyridoxal thus to demonstrate a lack of selectivity. The method has been then successfully applied to screen the CAR phenyl derivatives as above described (MS in preparation). Identification of biomarker of protein carbonylation to monitor the efficacy of RCS-SA in vivo studies Aim of this step was to identify a biomarker of protein carbonylation to be used in in vivo studies. By using a proteomic approach we found that albumin (HSA) is the main serum protein to be modified by RCS, such as HNE. The high reactivity of HNE towards HSA was then studied in in vitro models. By using a direct infusion ESI-MS approach we found that HNE rapidly reacts with HSA because of the covalent adduction to the different accessible nucleophilic residues of the protein. An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving several Lys, His and Cys nucleophilic sites. The most reactive HNE-adduction site was found to be Cys34 (MA), a suitable tag of HNE-adducted albumin that can be used as an useful biomarkers of oxidative and carbonylation damage in humans (6,7). Conclusion In the present work we confirmed that RCS, through the formation of Michael and Schiff base adducts, are involved in protein and cellular derangement. We then discovered a novel class of RCS-SA, the phenyl derivatives of D-CAR, reactive and selective towards RCS, as well as stable in human serum. Future studies will deal with the effect of these compounds in reducing RCS-dependent diseases and protein carbonylation, in animal models, such as the obese Zucker rats. References (1) G. Aldini, I. Dalle-Donne, R. Maffei Facino, A. Milzani, M. Carini. Med Res Rev. 2007;27(6):817-68.. (2) G. Aldini, M. Carini, G. Vistoli, T. Shibata, Y. Kusano, L. Gamberoni, I. Dalle-Donne, A.Milzani, K. Uchida. Biochemistry 46 (2007) 2707-18. (3) I. Dalle-Donne, M. Carini, G. Vistoli, L. Gamberoni, D. Giustarini, R. Colombo, R. Maffei Facino, R. Rossi, A. Milzani, G. Aldini. Free Radic Biol Med. 42 (2007) 583-98. (4) G. Aldini, I. Dalle-Donne, R. Colombo, R. Maffei Facino, A. Milzani, M. Carini. ChemMedChem. 1 (2006) 1045-58. (5) G. Aldini, M. Carini, G. Beretta, S. Bradamante, R. Maffei Facino. Biochem Biophys Res Commun. (298) 2002 699-706. (6) G. Aldini, L. Gamberoni, M. Orioli, G. Beretta, L. Regazzoni, R. Maffei Facino, M. Carini. J Mass Spectrom. 41 (2006) 1149-61. (7) G. Aldini, G. Vistoli, L. Regazzoni, L. Gamberoni, R. Maffei Facino, S. Yamaguchi, K Uchida and M. Carini [inviato]
ALDINI, GIANCARLO
Settore CHIM/08 - Chimica Farmaceutica
Discovery of inhibitors of carbonyl related degenerative disease : peptides and derivatives as detoxifying agents of cytotoxic aldehydes / L. Gamberoni ; Tutor: G. Aldini. - : . DIPARTIMENTO DI SCIENZE FARMACEUTICHE "PIETRO PRATESI", 2007 Feb 01. ((20. ciclo, Anno Accademico 2004/2005.
Doctoral Thesis
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/229972
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact