Targeted Quantitative Proteomics for the Analysis of 14 UGT1As and -2Bs in Human Liver Using NanoUPLC–MS/MS with Selected Reaction Monitoring

Targeted quantitative proteomics using heavy isotope dilution techniques is increasingly being utilized to quantify proteins, including UGT enzymes, in biological matrices. Here we present a multiplexed method using nanoLC–MS/MS and multiple reaction monitoring (MRM) to quantify 14 UGT1As and UGT2Bs in liver matrices. Where feasible, we employ two or more proteotypic peptides per protein, with only four proteins quantified with only one proteotypic peptide. We apply the method to analysis of a library of 60 human liver microsome (HLM) and matching S9 samples. Ten of the UGT isoforms could be detected in liver, and the expression of each was consistent with mRNA expression reported in the literature. UGT2B17 was unusual in that ∼30% of liver microsomes had no or little (<0.5 pmol/mg protein) content, consistent with a known common polymorphism. Liver S9 UGT concentrations were approximately 10–15% those of microsomes. The method was robust, precise, and reproducible and provides novel UGT expression data in human liver that will benefit rational approaches to evaluate metabolism in drug development

DataSheet1_Impact of pregnancy related hormones on drug metabolizing enzyme and transport protein concentrations in human hepatocytes.PDF

Pregnancy alters the disposition and exposure to multiple drugs indicated for pregnancy-related complications. Previous in vitro studies have shown that pregnancy-related hormones (PRHs) alter the expression and function of certain cytochrome P450s (CYPs) in human hepatocytes. However, the impact of PRHs on hepatic concentrations of non-CYP drug-metabolizing enzymes (DMEs) and transport proteins remain largely unknown. In this study, sandwich-cultured human hepatocytes (SCHH) from five female donors were exposed to vehicle or PRHs (estrone, estradiol, estriol, progesterone, cortisol, and placental growth hormone), administered individually or in combination, across a range of physiologically relevant PRH concentrations for 72 h. Absolute concentrations of 33 hepatic non-CYP DMEs and transport proteins were quantified in SCHH membrane fractions using a quantitative targeted absolute proteomics (QTAP) isotope dilution nanoLC-MS/MS method. The data revealed that PRHs altered the absolute protein concentration of various DMEs and transporters in a concentration-, isoform-, and hepatocyte donor-dependent manner. Overall, eight of 33 (24%) proteins exhibited a significant PRH-evoked net change in absolute protein concentration relative to vehicle control (ANOVA p < 0.05) across hepatocyte donors: 1/11 UGTs (9%; UGT1A4), 4/6 other DMEs (67%; CES1, CES2, FMO5, POR), and 3/16 transport proteins (19%; OAT2, OCT3, P-GP). An additional 8 (24%) proteins (UGT1A1, UGT2B4, UGT2B10, FMO3, OCT1, MRP2, MRP3, ENT1) exhibited significant PRH alterations in absolute protein concentration within at least two individual hepatocyte donors. In contrast, 17 (52%) proteins exhibited no discernable impact by PRHs either within or across hepatocyte donors. Collectively, these results provide the first comprehensive quantitative proteomic evaluation of PRH effects on non-CYP DMEs and transport proteins in SCHH and offer mechanistic insight into the altered disposition of drug substrates cleared by these pathways during pregnancy.</p

Bevirimat Inhibits p24 Release from the Capsid p25 Precursor in Human Thymocytes.

<p>Thymocytes dispersed from NL4-3- and SP1/A1V-infected SCID-hu Thy/Liv implants were cultured in the presence of the indicated range of concentrations of bevirimat for 2 days. Purified virions was lysed and individual viral proteins were detected with HIV-1 Ig. Note the target-specific and dose-dependent reduction of CAp25 cleavage in the presence of bevirimat. The presence of the drug does not affect the stoichiometry of the precursor Pr55Gag polyprotein nor does it inhibit cleavage of the matrix protein, MAp17 (lower panel, longer exposure).</p

Postexposure Dosing of Bevirimat also Reduces Viral Load.

<p>Mice were treated by twice-daily oral gavage with bevirimat at 100 mg/kg per day beginning 1 day before (−1), 1 day after (+1), or 3 days after (+3) virus inoculation of Thy/Liv implants with HIV-1 NL4-3 and JD. Dosing was continued until implant collection 14 days (JD) and 21 days (NL4-3) after inoculation. Antiviral efficacy was assessed by determining cell-associated HIV-1 RNA and p24. There was no reduction in viral load in bevirimat-treated mice inoculated with bevirimat-resistant SP1/A1V, whereas 3TC treatment (30 mg/kg per day by twice-daily oral gavage) was highly effective. Data are expressed as means±SEM; *<i>p</i>≤0.05 for treated mice versus untreated mice by the Mann-Whitney U test for 6–8 mice per group.</p

In Vivo Competitions Reveal No Impairment of SP1/A1V Replication.

<p>Sequencing of CA-SP1 RNA from three SCID-hu Thy/Liv mice coinfected with equivalent infectious units of NL4-3 and SP1/A1V reveal no impairment of SP1/A1V replication 28 days after implant inoculation. The A-to-V substitution in SP1 is conferred by a C-to-T mutation at nucleotide 1880.</p

Mice have High Concentrations of Bevirimat in Plasma 0.5–1 h after Oral Administration.

<p>Serial plasma samples were collected after the final dose from SCID-hu Thy/Liv mice that had been treated with 100 mg/kg per day for 21 days. Mean±SEM for four mice.</p

In Vivo Competition Results for NL4-3 and Bevirimat-Resistant SP1/A1V.

a<p>Viral species identified by sequencing amplicons generated after RT-PCR of CA RNA as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001251#pone-0001251-g006" target="_blank">Figure 6</a>.</p

The A1V Mutation in SP1 Does Not Affect Kinetics of Viral Replication or Thymocyte Depletion.

<p>Bevirimat-resistant SP1/A1V replicates and depletes thymocytes with kinetics comparable to wild-type NL4-3 in SCID-hu Thy/Liv mice. Viral replication was assessed by determining implant viral load (A), and thymocyte depletion was assessed by total implant cellularity, thymocyte viability, and CD4/CD8 ratio (B) for NL4-3-infected versus SP1/AIV-infected mice for 6 mice per group. Data are expressed as means±SEM; there were no statistically significant differences in viral load or thymocyte depletion at any of the three time points. The number in each graph is the proportion of SP1/A1V to NL4-3 in area under the curve.</p

Protease Inhibitor-Resistant HIV-1 has Impaired CA-SP1 Cleavage.

<p>Wild-type NL4-3 and protease mutant NL4-3 PR_I54V+V82A virions were collected from transfected 293T cells grown in the presence (20 µM) or absence of bevirimat and analyzed for particle maturation by Western blot using a p24 monoclonal antibody. The protease mutant is deficient in CA-SP1 cleavage and is comparable to bevirimat-treated wild-type virus, however, the drug has a more dramatic effect on CA-SP1 cleavage in HIV-1 PR_I54V+V82A.</p