Cytotoxic reactive carbonyl species (RCS) : from metabolic studies to the discovery of novel RCS sequestering agents

Reactive carbonyl species (RCS), such as α,β-unsaturated aldehydes [4-hydroxy-trans-2-nonenal (HNE), acrolein (ACR)], dialdehydes [malondialdehyde (MDA), glyoxal (GO)] and levuglandines are important cytotoxic mediators leading to alteration of the cellular function and involved in several physio-pathological conditions including diabetic-related diseases, age-dependent tissue dysfunction, and metabolic distress syndrome. The main detoxification pathways of RCS involve oxidation–reduction of the carbonyl function, and glutathione (GSH) conjugation by GSH-S-transferases. More recently, by using a peptidomic mass spectrometric approach, we identified endogenous histidine dipeptides, such as carnosine (β-alanyl-L-histidine, CAR), as additional nucleophilic agents able to detoxify the α,β-unsaturated aldehydes through a Michael adduction [1]. This novel metabolic pattern has been then confirmed in vivo in the Zucker obese rats, a well established animal model of carbonyl damage, by detecting in urine the Michael adduct CAR-HNE. When RCS are massively generated, or when the detoxification systems are hampered, RCS covalently react with several macromolecules such as DNA and proteins, leading to cell and tissue damages. Hence, RCS are a potential drug target and there is great interest in searching novel bioactive molecules effective in detoxifying these compounds through a direct mechanism (RCS sequestering effect), or by restoring/ameliorating the metabolic efficiency [2]. Although CAR is a selective and endogenous quencher of RCS, its pharmacological use is limited since it is rapidly hydrolized by a specific serum dipeptidase (carnosinase). Hence, we were aimed to derive CAR analogues characterized by (i) carnosinase stability and (ii) a greater reactivity towards RCS, even maintaining the same selectivity. The stability was reached by the isomerization of L- to D-histidine, leading to β-alanyl-D-histidine (D-carnosine, D-CAR), which is not recognized by carnosinase, but maintains the same quenching activity of L-CAR. The increase of reactivity was reached by modulating the conformational profile of the Schiff’s base intermediate, in order to favor a close conformation in which the imidazole ring approaches enough the C3 of the Schiff’s base to form the corresponding Michael adduct. A series of D-CAR derivatives was analyzed by in silico approaches to find out those characterized by a favorable folded conformational profile. The most promising were synthesized and the stability and quenching ability evaluated. By this way a set of phenyl derivatives was identified, characterized by high stability in human plasma, and by a nearly doubled HNE-quenching ability compared to D-CAR. [1] Aldini, G et al. Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction? Biochem Biophys Res Commun. 2002;298(5):699-706. [2] Aldini, G et al. Lipoxidation-derived reactive carbonyl species as potential drug targets in preventing protein carbonylation and related cellular dysfunction. ChemMedChem. 2006;1(10):1045-58.