Covalent inhibition of carboxylesterase-2 by sofosbuvir and its effect on the hydrolytic activation of tenofovir disoproxil  Yuanjun Shen, Bingfang Yan  Journal of Hepatology  Volume 66, Issue 3, Pages 660-661 (March 2017) DOI: 10.1016/j.jhep.2016.11.025 Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Inhibition of CES2 by sofosbuvir. (A) Inhibition of microsomal hydrolysis by sofosbuvir microsomes for human liver (0.25μg/per well) or kidney (1μg/per well) were incubated with sofosbuvir for 120min at 0–50μM in a total volume of 90μl and then 10μl of p-nitrophenylacetate (PNPA) was added at a final concentration of 1mM. The hydrolysis of PNPA was monitored with a microplate-reader from an increase in the absorbance at 400nm. (B) Native-gel electrophoresis stained for hydrolytic activity microsomes (0.25μg) were incubated with sofosbuvir at 0–10μM and subjected to native gel electrophoresis and stained for esterase activity with 4-methylumbelliferylacetate. (C) Inhibited hydrolysis of tenofovir disoproxil fumarate lysates from CES2 transfected cells were pre-incubated with sofosbuvir at 20μM or the reaction buffer at 37°C for 120min, followed by addition of tenofovir disoproxil fumarate at a final concentration of 20μM. The incubations lasted for an additional 30min and were then mixed with acetonitrile at a final concentration of 66%. The reactions were centrifuged to remove the proteins and the supernatants were analyzed by high-performance liquid chromatography (Hitachi-300). (This figure appears in colour on the web.) Journal of Hepatology 2017 66, 660-661DOI: (10.1016/j.jhep.2016.11.025) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions