A carnosine analog mitigates metabolic disorders of obesity by reducing carbonyl stress.

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward α,ß-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine.

Reactivity, Selectivity, and Reaction Mechanisms of Aminoguanidine, Hydralazine, Pyridoxamine, and Carnosine as Sequestering Agents of Reactive Carbonyl Species: A Comparative Study.

Reactive carbonyl species (RCS) are endogenous or exogenous byproducts involved in the pathogenic mechanisms of different oxidative-based disorders. Detoxification of RCS by carbonyl quenchers is a promising therapeutic strategy. Among the most studied quenchers are aminoguanidine, hydralazine, pyridoxamine, and carnosine; their quenching activity towards four RCS (4-hydroxy-trans-2-nonenal, methylglyoxal, glyoxal, and malondialdehyde) was herein analyzed and compared. Their ability to prevent protein carbonylation was evaluated in vitro by using an innovative method based on high-resolution mass spectrometry (HRMS). The reactivity of the compounds was RCS dependent: carnosine efficiently quenched 4-hydroxy-trans-2-nonenal, pyridoxamine was particularly active towards malondialdehyde, aminoguanidine was active towards methylglyoxal and glyoxal, and hydralazine efficiently quenched all RCS. Reaction products were generated in vitro and were characterized by HRMS. Molecular modeling studies revealed that the reactivity was controlled by specific stereoelectronic parameters that could be used for the rational design of improved carbonyl quenchers.

A novel high resolution MS approach for the screening of 4-hydroxy-trans-2-nonenal sequestering agents.

An in vitro high resolution mass spectrometry (MS) method was set-up to test the ability of compounds, mixtures and extracts to inhibit protein carbonylation induced by reactive carbonyl species (RCS). The method consists of incubating the protein target (ubiquitin) with 4-hydroxy-trans-2-nonenal (HNE) in the presence and absence of the tested compound. After 24h of incubation, the reaction is stopped and the protein is analyzed by high-resolution MS. The extent of protein carbonylation is determined by measuring the area of the +11 multicharged peak of the HNE adduct in respect to the native form. The method was validated by measuring the effect of well-known RCS sequestering agents, namely aminoguanidine, pyridoxamine, hydralazine and carnosine, yielding a good reproducibility and the possibility to be automatable. All the compounds were found to dose-dependently inhibit the protein carbonylation with the following order of potency carnosine≈hydralazine≫aminoguanidine>pyridoxamine, as determined by calculating the UC50 values, that is the concentration required to inhibit ubiquitin carbonylation by 50%. A good correlation was found with the results obtained by measuring HNE consumption using an HPLC method optimized by a mobile phase set at pH 7.4, in order to stabilize the eluted adducts. The MS approach was then applied to test the effect of two selected natural extracts on protein carbonylation, i.e. green coffee bean extract and procyanidins from Vitis vinifera. In summary, this paper reports a validated and highly reproducible MS method to test the ability of pure compounds as well as natural extracts to act as protein carbonylation inhibitors.

Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents.

The study reports a set of forty proteinogenic histidine-containing dipeptides as potential carbonyl quenchers. The peptides were chosen to cover as exhaustively as possible the accessible chemical space, and their quenching activities toward 4-hydroxy-2-nonenal (HNE) and pyridoxal were evaluated by HPLC analyses. The peptides were capped at the C-terminus as methyl esters or amides to favor their resistance to proteolysis and diastereoisomeric pairs were considered to reveal the influence of configuration on quenching. On average, the examined dipeptides are less active than the parent compound carnosine (ßAla + His) thus emphasizing the unfavorable effect of the shortening of the ßAla residue as confirmed by the control dipeptide Gly-His. Nevertheless, some peptides show promising activities toward HNE combined with a remarkable selectivity. The results emphasize the beneficial role of aromatic and positively charged residues, while negatively charged and H-bonding side chains show a detrimental effect on quenching. As a trend, ester derivatives are slightly more active than amides while heterochiral peptides are more active than their homochiral diastereoisomer. Overall, the results reveal that quenching activity strongly depends on conformational effects and vicinal residues (as evidenced by the reported QSAR analysis), offering insightful clues for the design of improved carbonyl quenchers and to rationalize the specific reactivity of histidine residues within proteins.