Gluthatione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine (GluCysGly), in which the glutamic acid is connected via its gamma carboxy group. It is an essential physiological antioxidant that detoxifies reactive oxygen species (ROS), free radicals, peroxides, lipid peroxides, heavy metals, and reactive metabolites of xenobiotics and thus, it prevents damage to cells. Oxidative stress depletes cellular GSH stores and may cause damage to biomolecules, organelles, and cells.

Chemical structure of Glutathione (GSH).

Reactive metabolites (RMs) play a crucial role in drug discovery and need to be evaluated during lead optimization and toxicologic profiling. They may be formed during the enzymatic breakdown of medications, e.g., by enzymes of the cytochrome P family (CYP), leading to toxicity and adverse drug effects.

Fluorescent – more precisely Dansyl-labeled – GSH (HAA7945), with its soft nucleophilic thiol, is a standard trapping reagent (TR) for soft electrophilic reactive metabolites (RMs), which are otherwise themselves instable and thus difficult to analyze. The fluorescent dansyl moiety allows for the facile detection of labeled reaction products ( λ_Ex ~340 nm; λ_Em ~ 525 nm).

However, for hard electrophilic reactive metabolites, only few fluorescent trapping reagents exist, which mainly lead to the formation of unstable imine derivatives, which cannot be detected.

In this regard, we added CysGluDan (LS-4665) to our portfolio, which captures both “soft” and “hard” reactive metabolites upon formation of a stable thiazolidine ring structure.

Chemical structure of Dansyl-GSH (HAA7945) and CysGluDan (LS-4665). The Dansyl-moiety is highlighted in green.

As an example, the Parkinson drug Pargyline forms propargyl aldehyde upon CYP oxidation. Propargyl aldehyde carries a hard and soft nucleophilic site and can react with two molecules of CysGluDan leading to the formation of a detectable conjugate.

Proposed reaction of propargyl aldehyde with two molecules of CysGluDan.

References:

Development of fluorescent-labeled trapping reagents based on cysteine to detect soft and hard electrophilic reactive metabolites; C.Shibazaki, T. Ohe, K. Takahashi, S. Nakamura, T. Mashino; Drug Metab Pharmacokinet. 2021; 39: 100386. https://doi.org/10.1016/j.dmpk.2021.100386

Development of a fluorescent-labeled trapping reagent to detect reactive acyl glucuronides; C. Shibazaki, O. Mashita, K. Takahashi, S. Nakamura, T. Mashino, T. Ohe; Chem Res Toxicol. 2021. 34(11): 2343-2352. https://doi.org/10.1021/acs.chemrestox.1c00236

Development of a fluorescent-labeled trapping reagent to evaluate the risk posed by acyl-CoA conjugates; C. Shibazaki, T. Mashino, T. Ohe; Drug Metab Pharmacokinet. 2023; 52: 100509. https://doi.org/10.1016/j.dmpk.2023.100509

Application of the hard and soft, acids and bases (HSAB) theory to toxicant-target interactions; R. M. LoPachin, T. Gavin, A. DeCaprio, D. S. Barber; Chem Res Toxicol. 2011; 25(2): 239-251. https://doi.org/10.1021/tx2003257

A review of CYP-mediated drug Interactions: Mechanisms and in vitro drug-drug interaction assessment; J. Lee, J. L. Beers, R. M. Geffert, K. D. Jackson; Biomolecules 2024; 14(1): 99. https://doi.org/10.3390/biom14010099

Role of cytochrome P450s in the generation and metabolism of reactive oxygen species; A. Veith, B. Moorthy; Curr. Opin. Toxicol. 2018; 7: 44-51. https://doi.org/10.1016/j.cotox.2017.10.003

Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib; G. M. Amaya, R. Durandis, D. S. Bourgeois, J. A. Perkins, A. A. Abouda, K. J. Wines, M. Mohamud, S. A. Starks, R. N. Daniels, K. D. Jackson; Chem. Res. Toxicol. 2018; 31(7): 570-581. https://doi.org/10.1021/acs.chemrestox.8b00005