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

Acyl glucuronides are common metabolites of carboxylic acid-containing compounds. Since acyl glucuronides sometimes show high reactivity, they are considered to be involved in drug toxicity. Therefore, it is important to evaluate the risk posed by acyl glucuronides in the development of safe drugs; however, there are no suitable evaluation methods for the early stages of drug discovery. We aimed to develop a trapping reagent that detects reactive acyl glucuronides to assess their risk. We designed a diamine-structured trapping reagent, Dap-Dan, and compared its trapping ability with the reported one that has an amino group, and results showed that Dap-Dan showed higher accuracy. In the trapping assay with 17 medicines containing a carboxylic acid, Dap-Dan trapped acyl glucuronides that had a higher risk of toxicity. In conclusion, Dap-Dan can be useful for evaluating the risk of reactive acyl glucuronides.

Development of a fluorescent-labeled trapping reagent to evaluate the risk posed by acyl-CoA conjugates.

Although acyl-CoA conjugates are known to have higher reactivity than acyl glucuronides, few studies have been conducted to evaluate the risk of the conjugates. In the present study, we aimed to develop a trapping assay for acyl-CoA conjugates using trapping reagents we have developed previously. It was revealed that Cys-Dan, which has both a thiol and an amino group, was the most effective in forming stable adducts containing an amide bond after intramolecular acyl migration. Additionally, we also developed a hepatocyte-based trapping assay in the present study to overcome the shortcomings of liver microsomes. Although liver microsomes are commonly used as enzyme sources in trapping assays, they lack some of the enzymes required for drug metabolism and detoxification systems. In human hepatocytes, our three trapping reagents, CysGlu-Dan, Dap-Dan and Cys-Dan, captured CYP-dependent reactive metabolites, reactive acyl glucuronides, and reactive acyl-CoA conjugates, respectively. The work suggests that the trapping assay with the reagents in hepatocytes is useful to evaluate the risk of reactive metabolites in drug discovery.

Synthesis and evaluation of tofacitinib analogs designed to mitigate metabolic activation.

Tofacitinib (TFT), a JAK inhibitor used for the treatment of rheumatoid arthritis and other diseases, is associated with severe liver injury that is believed to be caused by its reactive aldehyde or epoxide metabolites. In this study, we synthesized six tofacitinib analogs designed to avoid the formation of reactive metabolites and evaluated their JAK3 inhibitory activity, metabolic stability, CYP3A time-dependent inhibition, and cytotoxicity. Our data indicated that purine analog 3, which showed little inhibition of CYP3A and cytotoxicity and inhibited JAK3 in the nanomolar range, could be a safer drug candidate than TFT. In addition, the results of the bioactivation study using TFT and its analogs suggest that the epoxide metabolite might contribute to TFT-induced CYP3A4 mechanism-based inhibition and hepatic toxicity.

Development of fluorescent-labeled trapping reagents based on cysteine to detect soft and hard electrophilic reactive metabolites.

Trapping assays are conducted at lead optimization stages to detect reactive metabolites (RMs) that can contribute to drug toxicity. The commonly used dansyl glutathione (dGSH) provides a sensitive analysis owing to the fluorescent label, however, it captures only soft electrophilic RMs. TRs for hard electrophilic RMs, few of which are labeled fluorescently, can detect hard electrophilic aldehydes only by forming unstable imine derivatives. In this study, we aimed to develop novel fluorescently labeled TRs that detect both soft and hard electrophilic RMs and form stable ring structures with aldehydes. We designed four dansylated TRs based on cysteine, which has both soft and hard nucleophilic groups. To evaluate the reactivity of the TRs, we incubated them with several substrates and found that one of the TRs (CysGlu-Dan) detected all the soft and hard electrophilic RMs. We also examined the inhibition potential of each TR for seven major CYPs involved in drug metabolism and found that CysGlu-Dan showed an inhibitory profile similar to that of dGSH. In conclusion, CysGlu-Dan can be used to evaluate the risk of RMs in drug discovery.