Quantitative Evaluation of Reactivity and Toxicity of Acyl Glucuronides by [³⁵S]Cysteine Trapping

Abstract

Acyl glucuronides (AGs) are reactive metabolites of carboxylic acid-containing drugs, which are associated with idiosyncratic toxicity (IDT) such as anaphylaxis, drug-induced liver injury, and so on. In this study, we developed a new in vitro approach for the quantitative assessment of the reactivity of AGs and their toxicity risk. Thirteen test drugs were incubated with human liver microsomes and uridine 5′-diphospho-glucuronic acid in the presence of cysteine (Cys) as a trapping agent. Both acylation and glycation Cys adducts formed from the AGs of the test drugs and were analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. Acylation Cys adduct formation can closely reflect the reactivity of AGs to predict their IDT risk. Subsequently, we performed a quantitative trapping assay using radiometric analysis, with [35S]-labeled Cys ([35S]­Cys) as the trapping agent, and the results showed that the test drugs associated with IDT resulted in a high product formation of [35S]­Cys adducts. In conclusion, this approach can be used for the easy and quantitative evaluation of the reactivity of AGs without the need for authentic AG standards and to screen the potential IDT of new chemical entities during the early drug discovery phase.

Bioactivation of α,β-Unsaturated Carboxylic Acids Through Acyl Glucuronidation

“…This incredible stability indicates that while M6 is converted to a great electrophile for protein conjugation, it is not activated to undergo degredation or other metabolic processes. Bioactivation by glucuronidation has only been previously described to result in transacylation or glycation via Amadori rearrangement (Skonberg et al, 2008;Miyashita et al, 2014;Ding et al, 1993;Harada et al, 2019). No GSH addition to the glucuronic acid moiety was observed, even though acyl migration isomers were detected in these in vitro incubations.…”

“…There was no glucuronic acid replacement product by GSH or cysteine from these incubations. Direct replacement of acyl glucuronide by cysteine thiol, followed by S-to N-acyl rearrangement to form a more stable conjugate, has previously been reported (Harada et al, 2019). This produces an isomer with the same molecular ion in mass spectral analysis.…”

Development of a Fluorescent-Labeled Trapping Reagent to Detect Reactive Acyl Glucuronides

“…From these aspects, trapping reagents must possess two amino groups in their neighborhood to form stable glycation adducts. Although cysteine also has two nucleophilic groups, it does not form glycation adducts in the preaddition method . It is likely that the adducts formed after acyl migration have a thioester, which is still unstable.…”

“…In the trapping assay with 17 medicines containing carboxylic acid, the trapping ability of Dap-Dan was examined to determine whether it could distinguish between acyl glucuronides derived from withdrawn/warning class and safe-class compounds. These drugs were previously evaluated by their half-lives or the detection of adducts in trapping assays with dKF or cysteine. ,, Although the half-life evaluation in potassium phosphate buffer showed a good correlation with drug toxicity, no adducts were detected in preaddition conditions with both trapping reagents. As shown in Table , Dap-Dan formed acylation adducts with some medicines classified as withdrawn and warning, whereas no acylation adducts were detected with safe-class drugs.…”

“…Additionally, false-positive and false-negative results are frequently observed with dKF , which implies a need for improvement. Cysteine and N -acetyl cysteine have also been employed as trapping reagents. − However, despite having a free amino group, cysteine does not form glycation adducts under physiological conditions . Since it is inferred that the glycation of proteins is implicated in toxicity more than acylation, a trapping reagent that efficiently forms glycation adducts is preferable.…”