Penindolone Derivatives Glucuronidation Assay

Penindolone and its derivatives HDYL-GQQ-2399, QL-Vir-09 and lbh3-78-2 (all HPLC purity > 95%, Figure S1), were synthesized by Laboratory of Natural Product Chemistry (School of Medicine, Ocean University of China). Mephenytoin, diclofenac sodium, chlorzoxazone, 6-hydroxychlorzoxazone, 4-acetaminophen were purchased from J&K Scientific (Beijing, China). Dextromethorphan, midazolam, 4-hydroxymephenytoin, 1-hydroxymidazolam, chenodeoxycholic acid 24-acyl-β-D-glucuronide, serotonin-β-D-glucuronide and 3′-azido-3′-deoxythymidine-β-D-glucuronide were acquired from Toronto Research Chemicals (Toronto, Canada). Glucose-6-phosphate (G-6-P), β-nicotinamide adenine dinucleotide phosphate (β-NADP), glucose-6-phosphate dehydrogenase (G-6-P-DH), phenacetin, 4-hydroxydiclofenac sodium, ticlopidine, β-glucuronidase (Type HP-2, aqueous solution, ≥100,000 units/mL) and trifluoperazine N-glucuronide were purchased from Sigma-Aldrich (Shanghai, China). Paeonol, estradiol, chenodeoxycholic acid, trifluoperazine, serotonin, propofol, zidovudine, bilirubin, lithocholic acid, hecogenin, troglitazone, niflumic acid, fluconazole, alamethicin and formic acid (FA) were acquired from Aladdin Biochemical (Shanghai, China). β-Estradiol-3-glucuronide was obtained from APExBIO (Shanghai, China). Propofol-β-D-glucuronide was purchased from GlpBio (Shanghai, China). Ibrutinib, UDP-D-glucuronide trisodium salt (UDPGA) and theophylline were obtained from Macklin Biochemical (Shanghai, China). PEG 300 was purchased from Sinopharm Chemical Reagent. (Shanghai, China). Dimethyl sulfoxide (DMSO) was obtained from Sangon Biotech (Shanghai, China). Isopropanol, methanol, acetonitrile and water were purchased from Merck (Darmstadt, Germany).
Mouse and rat liver microsomes (LMs) were prepared following previously established methods [16 (link)]. Mouse liver microsomes (MLM) were prepared with 10 Kunming males (6–8 weeks), and rat liver microsomes (RLMs) were prepared with 6 Sprague-Dawley males (6–8 weeks). Male Mongolia human microsomes (HLM), prepared from 30 male donors (19–78 years old), were obtained from RILD Liver Disease Research Co., Ltd. (Shanghai, China). LMs were activated by preincubation with alamethicin (50 μg/mg protein) on ice for 30 min before their use in UGTs incubations [17 (link)].