Carnosine derivatives as novel RCS sequestering agents in preventing protein carbonylation and related cellular dysfunction

Several evidences strongly support a pathogenic role for Reactive Carbonyl Species (RCS), such as in the case of diabetic-related diseases, age-dependent tissue dysfunction, and metabolic distress syndrome. Hence, RCS can be considered a potential biological target for drug discovery. The most promising pharmacological strategy to neutralize/reduce RCS is based on nucleophilic compounds capable to form covalent and unreactive adducts with RCS (RCS sequestering agents) such as pyridoxamine (PYR), hydralazine (HY), dihydralazine (di-HY), and aminoguanidine (AG). However these compounds are characterized by a lack of selectivity since they also react with physiological aldehydes, such as pyridoxal. We recently found that the endogenous dipeptide carnosine (beta-alanyl-L-histidine, CAR) is a specific quencher of alfa,beta-unsaturated aldehydes due to its peculiar mechanism involving the Schiff base formation between the beta-alanine amino group and the RCS aldehyde followed by the Michael adduction between the C3 of the aldehyde and the Ntau of the histidine group. We also found that CAR exogenously given to Zucker obese rats (30 mg/Kg die for 24 weeks) greatly reduces dyslipidemia, hypertension, albuminuria and protein carbonylation. However, the therapeutic use of CAR is limited since it is unstable in human plasma due to serum carnosinase. Hence, our interest was to derive carnosine analogues characterized by (i) carnosinase stability and (ii) a grater 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-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 favour 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 three fold HNE-quenching ability increase compared to D-CAR.