Peakview version 2

Rat liver microsomes (final concentration = 2 mg/mL) were incubated with SCH 58261 (5 µg/mL) in a reaction mixture that consisted of 2 mM NADPH, 5 mM UDPGA, and 0.5mM GSH at 37 °C. The reaction was initiated by adding the cofactor solutions containing NADPH, UDPGA, and GSH to the rat liver microsome suspension with a 3-min pre-incubation. The microsomes, mixed-cofactor, and SCH 58261 were mixed and incubated for 0 and 60 min at 37 °C. The reaction was stopped by adding ACN. After vortexing for 1 min and centrifuging at 8000× g for 10 min, the supernatant was transferred to another Eppendorf tube. The supernatants were evaporated to dryness under vacuum in a rotary evaporator with a cold trap (Eyela CVE-3110 & UT-1000, Tokyo, Japan). The dried residue was re-constituted to 210 μL of DW/MeOH (2:1), vortexed, centrifuged at 10,000× g for 5 min, and the supernatant was transferred to an LC vial for analysis. PeakView^® Version 2.2 (Sciex, Redwood City, CA, USA) and MetabolitePilot^™ Version 2.0.2 (Sciex, Redwood City, CA, USA) were used for the structural elucidation of SCH 58261 metabolites.

Data acquisition and a LC-qTOF-MS operation were conducted using Analyst^® TF Version 1.6 (Sciex, Foster City, CA, USA). α-Amanitin was quantified by MultiQuant^® Version 2.1.1 (Sciex, Foster City, CA, USA) using peak integration. The PK parameters of α-amanitin were calculated by WinNonlin^® version 8.1.0 (Certara, Princeton, NJ, USA) in a non-compartment analysis. For MetID analysis, PeakView^® Version 2.2 (Sciex, Foster City, CA, USA) and MetabolitePilot™ Version 2.0.2 (Sciex, Foster City, CA, USA) were used for the structural elucidation of α-amanitin metabolites. Excel 2016 spreadsheet (Microsoft^®) was also used to process the statistical analysis of results.

Data acquisition and LC-MS/MS operation were conducted using Analyst® TF Version 1.6 (Sciex, Redwood City, CA, USA). MultiQuant® Version 2.1.1 (Sciex, Redwood City, CA, USA) was used for peak integration for MMAF quantification. The descriptive statistics for the qualification studies were calculated with Excel 2015 (Microsoft, Seoul, Korea). Pharmacokinetic parameters were calculated in a non-compartmental analysis using WinNonlin® version 8.0.0 (Certara, Princeton, NJ, USA). PeakView® Version 2.2 (Sciex, Redwood City, CA, USA) and MetabolitePilot^TM Version 2.0.2 (Sciex, Redwood City, CA, USA) were used for the structural elucidation of metabolites. MedChem Designer (Simulations Plus, Inc, Lancaster, CA, USA) was used for in silico metabolite prediction.

Mass spectrometry measurements were performed with an electrospray Q-TOF mass spectrometer (Waters) equipped with the Nanomate device (Advion, Inc.). The HD_A_384 chip (5 μm I.D. nozzle chip, flow rate range 100−500 nL/min) was calibrated before use. For ESI − MS measurements, the Q-TOF instrument was operated in RF quadrupole mode with the TOF data being collected between m/z 400 and 2990. Collision energy was set to 10 eV and argon was used as the collision gas. PsKAI2 proteins (50 µM) in 50 mM ammonium acetate (pH 6.8) in presence or without (-)-GR24 (500 µM) were incubated for 10 min at room temperature before denaturation in 50% acetonitrile and 1% formic acid. The solutions were directly injected for Mass spectra acquisition or digested before LC-MS/MS analyses. Mass Lynx version 4.1 (Waters) and Peakview version 2.2 (Sciex) software were used for acquisition and data processing, respectively. Deconvolution of multiply charged ions was performed by applying the MaxEnt algorithm (Sciex). The average protein masses were annotated in the spectra and the estimated mass accuracy was ± 2 Da. External calibration was performed with NaI clusters (2 μg/μL, isopropanol/H₂O 50/50, Waters) in the acquisition m/z mass range.

Data acquisition and LC-qTOF-MS operation was conducted using Analyst^® TF Version 1.6 (Sciex, Foster City, CA, USA). MultiQuant^® Version 2.1.1 (Sciex, Foster City, CA, USA) was used for the peak integration for Daporinad quantification. PeakView^® Version 2.2 (Sciex, Foster City, CA, USA) and MetabolitePilot™ Version 2.0.2 (Sciex, Foster City, CA, USA) were used for the structural elucidation of Daporinad metabolites. The descriptive statistics for the qualification studies were calculated with Excel 2015 (Microsoft). Pharmacokinetic parameters were calculated in a non-compartmental analysis using WinNonlin^® version 8.0.0 (Certara, Princeton, NJ, USA).