A targeted covalent inhibitor of p97 with proteome-wide selectivity

A resurging interest in targeted covalent inhibitors (TCIs) focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target. p97 is an emerging protein target for cancer therapy, viral infections and neurodegenerative diseases. Extensive efforts were devoted to the development of p97 inhibitors. The most promising inhibitor of p97 was in phase 1 clinical trials, but failed due to the off-target-induced toxicity, suggesting the selective inhibitors of p97 are highly needed. We report herein a new type of TCIs (i.e., FL-18) that showed proteome-wide selectivity towards p97. Equipped with a Michael acceptor and a basic imidazole, FL-18 showed potent inhibition towards U87MG tumor cells, and in proteome-wide profiling, selectively modified endogenous p97 as confirmed by in situ fluorescence scanning, label-free quantitative proteomics and functional validations. FL-18 selectively modified cysteine residues located within the D2 ATP site of p97. This covalent labeling of cysteine residue in p97 was verified by LC‒MS/MS-based site-mapping and site-directed mutagenesis. Further structure-activity relationship (SAR) studies with FL-18 analogs were established. Collectively, FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity, thus providing a promising scaffold for cancer therapy.

Simultaneous Determination of Methadone, Venlafaxine and Their Metabolites in Rat Plasma by UPLC-MS/MS and Pharmacokinetics Study / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of methadone,venlafaxine and their metabolites in rat plasma,and to use it for the study of pharmacokinetic in rats. METHODS:UPLC-MS/MS method was adopted to determine plasma after precipitated with acetonitrile using diazepam as internal standard. The determination was performed on Waters Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile-0.1％ formic acid (gradient elution) at the flow rate of 0.4 mL/min. The column temperature was set at 40 ℃,and sample size was 2 μL. ESI was used for positive ion scanning by multiple reaction monitoring (MRM) mode. The ion pairs for quantitative analysis were m/z 310.4→265.4(methadone),m/z 278.2→234.1 (EDDP),m/z 278.1→57.8(venlafaxine),m/z 263.9→57.9(O-desvenlafaxine),m/z 285.1→193.1(internal standard). Eight SD rats were given methadone hydrochloride 6 mg/kg and venlafaxine hydrochloride 10 mg/kg intragastrically(4 rats for each drug);blood samples (0.3 mL) were collected from the tail vein before and 0.083,0.167,0.25,0.5,0.75,1,1.5,2,3,4,6,8,10,12, and 24 h after medication. Pharmacokinetic parameters were calculated by using DAS 3.0 software. RESULTS:The linear ranges of methadone,EDDP,venlafaxine and O-desvenlafaxine were 0.5-250 ng/mL(r=0.9997),0.5-250 ng/mL(r=0.9992),0.4-200 ng/mL(r=0.9999),0.4-200 ng/mL(r=0.9999),respectively. The limits of quantitation were 0.5,0.5,0.4,0.4 ng/mL. RSDs of intra-day and inter-day were all lower than 9.0％(n=6). The method recoveries were 94.20％-102.87％,90.93％- 102.94％, 92.95％-101.61％,90.33％-101.97％(RSD≤5.5％,n=6). The extraction recoveries were 85.90％-94.45％,85.97％- 91.66％, 87.97％ -93.58％ , 88.53％ -94.54％(RSD≤5.9％ ,n=6). The matrix effects were 95.96％ -97.78％ , 92.33％ -97.40％ , 95.28％-97.71％,95.33％-95.74％(RSD≤4.9％,n=3). The pharmacokinetic parameters included that t1/2 were (1.42 ± 1.02),(2.59 ± 0.76),(0.63 ± 0.08),(1.29 ± 0.14) h;cmax were (52.21 ± 5.42),(25.68 ± 3.45),(45.68 ± 2.29),(47.63 ± 13.09) μg/L;MRT0-24 h were (3.55 ± 0.21),(3.98 ± 0.41),(1.44 ± 0.21),(2.01 ± 0.17) h;AUC0-24 h were (201.95 ± 51.14),(86.092 ± 15.95),(75.38 ± 23.95),(82.90 ± 23.44)μg·h/L. CONCLUSIONS:The method is specific, absolute separated, rapid and sensitive, and can be used for the simultaneous determination of methadone,venlafaxine and their metabolites in plasma and pharmacokinetics research.