ABSTRACT
1. A protocol has been developed and validated for the high-throughput screening of eight major human cytochrome P450 (CYP) isozymes inhibition (CYP 1A2, 2C9, 2C19, 2D6, 3A4, 2B6, 2C8 and 2E1) using an in vitro probe cocktail containing eight substrates by overcoming the unfavorable effect of assay conditions on CYP2E1 inhibition data. 2. The cocktail consisting of selective probe substrates like tacrine (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4), bupropion (CYP2B6), paclitaxel (CYP2C8) and chlorzoxazone (CYP2E1) was incubated with human liver microsomes. 3. The method was investigated by incubating well-known CYP inhibitors {alphanaphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), N-3-benzylnirvanol (CYP2C19), quinidine (CYP2D6), ketoconazole (CYP3A4), ticlopidine (CYP2B6), quercetin (CYP2C8) and 4-methylpyrazole (CYP2E1)} with the substrate cocktail. A fast gradient liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for this study. 4. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those in the literature. DMSO has significant effect and itself inhibits CYP2E1. DMSO should not exceed 0.1% for the determination of reliable CYP2E1 inhibition profile. This cocktail assay offers an efficient and robust method to determine the CYP450 isoforms inhibition profiles of large numbers of compounds in a quick turnaround time.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P450 Family 2/metabolism , Drug Evaluation, Preclinical/methods , Chromatography, Liquid , Cytochrome P450 Family 2/antagonists & inhibitors , Cytochrome P450 Family 3/antagonists & inhibitors , Cytochrome P450 Family 3/metabolism , Dimethyl Sulfoxide/pharmacology , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Reproducibility of Results , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
KEY MESSAGE: A U-box E3 ubiquitin ligase GhPUB17 is inhibited by GhCyP3 with antifungal activity and acts as a negative regulator involved in cotton resistance to Verticillium dahliae. E3 ubiquitin ligases, the key component enzymes of the ubiquitin-proteasome system, which contains the most diverse structural and functional members involved in the determination of target specificity and the regulation of metabolism, have been well documented in previous studies. Here, we identify GhPUB17, a U-box E3 ligase in cotton that has ubiquitination activity and is involved in the cotton immune response to Verticillium dahliae. The expression level of GhPUB17 is downregulated in the ssn mutant with a constitutively activated immune response (Sun et al., Nat Commun 5:5372, 2014). Infection with V. dahliae or exogenous hormone treatment, including jasmonic acid and salicylic acid, significantly upregulated GhPUB17 in cotton roots, which suggested a possible role for this E3 ligase in the plant immune response to pathogens. Moreover, GhPUB17-knockdown cotton plants are more resistant to V. dahliae, whereas GhPUB17-overexpressing plants are more susceptible to the pathogen, which indicated that GhPUB17 is a negative regulator of cotton resistance to V. dahliae. A yeast two-hybrid (Y2H) assay identified GhCyP3 as a protein that interacts with GhPUB17, and this finding was confirmed by further protein interaction assays. The downregulation of GhCyP3 in cotton seedlings attenuated the plants' resistance to V. dahliae. In addition, GhCyP3 showed antifungal activity against V. dahliae, and the E3 ligase activity of GhPUB17 was repressed by GhCyP3 in vitro. These results suggest that GhPUB17 negatively regulates cotton immunity to V. dahliae and that the antifungal protein GhCyP3 likely interacts with and inhibits the ligase activity of GhPUB17 and plays an important role in the cotton-Verticillium interaction.
