Tenofovir Activation Is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor prescribed for the treatment and prevention of human immunodeficiency virus infection and the treatment of chronic hepatitis B virus infection. Here, we demonstrate that creatine kinase brain-type (CKB) can form tenofovir-diphosphate (TFV-DP), the pharmacologically active metabolite, in vitro and identify nine missense mutations (C74S, R96P, S128R, R132H, R172P, R236Q, C283S, R292Q, and H296R) that diminish this activity. Additional characterization of these mutations reveals that five (R96P, R132H, R236Q, C283S, and R292Q) have ATP dephosphorylation catalytic efficiencies less than 20% of those of the wild type (WT), and seven (C74S, R96P, R132H, R172P, R236Q, C283S, and H296P) induce thermal instabilities. To determine the extent CKB contributes to TFV activation in vivo, we generated a CKB knockout mouse strain, Ckbtm1Nnb. Using an in vitro assay, we show that brain lysates of Ckbtm1Nnb male and female mice form 70.5 and 77.4% less TFV-DP than wild-type brain lysates of the same sex, respectively. Additionally, we observe that Ckbtm1Nnb male mice treated with tenofovir disoproxil fumarate for 14 days exhibit a 22.8% reduction in TFV activation in the liver compared to wild-type male mice. Lastly, we utilize mass spectrometry-based proteomics to elucidate the impact of the knockout on the abundance of nucleotide and small molecule kinases in the brain and liver, adding to our understanding of how the loss of CKB may be impacting tenofovir activation in these tissues. Together, our data suggest that disruptions in CKB may lower levels of active drugs in the brain and liver.

Tenofovir Douche as HIV Preexposure Prophylaxis for Receptive Anal Intercourse: Safety, Acceptability, Pharmacokinetics, and Pharmacodynamics (DREAM 01).

BACKGROUND: Despite highly effective HIV preexposure prophylaxis (PrEP) options, no options provide on-demand, nonsystemic, behaviorally congruent PrEP that many desire. A tenofovir-medicated rectal douche before receptive anal intercourse may provide this option. METHODS: Three tenofovir rectal douches-220 mg iso-osmolar product A, 660 mg iso-osmolar product B, and 660 mg hypo-osmolar product C-were studied in 21 HIV-negative men who have sex with men. We sampled blood and colorectal tissue to assess safety, acceptability, pharmacokinetics, and pharmacodynamics. RESULTS: The douches had high acceptability without toxicity. Median plasma tenofovir peak concentrations for all products were several-fold below trough concentrations associated with oral tenofovir disoproxil fumarate (TDF). Median colon tissue mucosal mononuclear cell (MMC) tenofovir-diphosphate concentrations exceeded target concentrations from 1 hour through 3 to 7 days after dosing. For 6-7 days after a single product C dose, MMC tenofovir-diphosphate exceeded concentrations expected with steady-state oral TDF 300 mg on-demand 2-1-1 dosing. Compared to predrug baseline, HIV replication after ex vivo colon tissue HIV challenge demonstrated a concentration-response relationship with 1.9 log10 maximal effect. CONCLUSIONS: All 3 tenofovir douches achieved tissue tenofovir-diphosphate concentrations and colorectal antiviral effect exceeding oral TDF and with lower systemic tenofovir. Tenofovir douches may provide a single-dose, on-demand, behaviorally congruent PrEP option, and warrant continued development. Clinical Trials Registration . NCT02750540.

Tenofovir Activation is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor prescribed for the treatment and prevention of human immunodeficiency virus infection, and the treatment of chronic hepatitis B virus infection. Here, we demonstrate that creatine kinase brain-type (CKB) can form tenofovir-diphosphate (TFV-DP), the pharmacologically active metabolite, in vitro, and identify nine missense mutations (C74S, R96P, S128R, R132H, R172P, R236Q, C283S, R292Q, and H296R) that diminish this activity. Additional characterization of these mutations reveal that five (R96P, R132H, R236Q, C283S, and R292Q) have ATP dephosphorylation catalytic efficiencies less than 20% of wild-type (WT), and seven (C74S, R96P, R132H, R172P, R236Q, C283S, and H296P) induce thermal instabilities. To determine the extent CKB contributes to TFV activation in vivo, we generated a CKB knockout mouse strain, Ckbtm1Nnb. Using an in vitro assay, we show that brain lysates of Ckbtm1Nnb male and female mice form 70.5% and 77.4% less TFV-DP than wild-type brain lysates of the same sex, respectively. Additionally, we observe that Ckbtm1Nnb male mice treated with tenofovir disoproxil fumarate for 14 days exhibit a 22.8% reduction in TFV activation in liver compared to wild-type male mice. Lastly, we utilize mass spectrometry-based proteomics to elucidate the impact of the knockout on the abundance of nucleotide and small molecule kinases in the brain and liver, adding to our understanding of how loss of CKB may be impacting tenofovir activation in these tissues. Together, our data suggest that disruptions in CKB may lower levels of active drug in brain and liver.

The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.

Biotransformation of Efavirenz and Proteomic Analysis of Cytochrome P450s and UDP-Glucuronosyltransferases in Mouse, Macaque, and Human Brain-Derived In Vitro Systems.

Antiretroviral drugs such as efavirenz (EFV) are essential to combat human immunodeficiency virus (HIV) infection in the brain, but little is known about how these drugs are metabolized locally. In this study, the cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT)-dependent metabolism of EFV was probed in brain microsomes from mice, cynomolgus macaques, and humans as well as primary neural cells from C57BL/6N mice. Utilizing ultra high performance liquid chromatography high-resolution mass spectrometry (uHPLC-HRMS), the formation of 8-hydroxyefavirenz (8-OHEFV) from EFV and the glucuronidation of P450-dependent metabolites 8-OHEFV and 8,14-dihydroxyefavirenz (8,14-diOHEFV) were observed in brain microsomes from all three species. The direct glucuronidation of EFV, however, was only detected in cynomolgus macaque brain microsomes. In primary neural cells treated with EFV, microglia were the only cell type to exhibit metabolism, forming 8-OHEFV only. In cells treated with the P450-dependent metabolites of EFV, glucuronidation was detected only in cortical neurons and astrocytes, revealing that certain aspects of EFV metabolism are cell type specific. Untargeted and targeted proteomics experiments were used to identify the P450s and UGTs present in brain microsomes. Eleven P450s and 11 UGTs were detected in human brain microsomes, whereas seven P450s and 14 UGTs were identified in mouse brain microsomes and 15 P450s and four UGTs, respectively, were observed in macaque brain microsomes. This was the first time many of these enzymes have been noted in brain microsomes at the protein level. This study indicates the potential for brain metabolism to contribute to pharmacological and toxicological outcomes of EFV in the brain. SIGNIFICANCE STATEMENT: Metabolism in the brain is understudied, and the persistence of human immunodeficiency virus (HIV) infection in the brain warrants the evaluation of how antiretroviral drugs such as efavirenz are metabolized in the brain. Using brain microsomes, the metabolism of efavirenz by both cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) is established. Additionally, proteomics of brain microsomes characterizes P450s and UGTs in the brain, many of which have not yet been noted in the literature at the protein level.

Atp7b-dependent choroid plexus dysfunction causes transient copper deficit and metabolic changes in the developing mouse brain.

Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-ß-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.

Achieving a Deeper Understanding of Drug Metabolism and Responses Using Single-Cell Technologies.

Recent advancements in single-cell technologies have enabled detection of RNA, proteins, metabolites, and xenobiotics in individual cells, and the application of these technologies has the potential to transform pharmacological research. Single-cell data has already resulted in the development of human and model species cell atlases, identifying different cell types within a tissue, further facilitating the characterization of tumor heterogeneity, and providing insight into treatment resistance. Research discussed in this review demonstrates that distinct cell populations express drug metabolizing enzymes to different extents, indicating there may be variability in drug metabolism not only between organs, but within tissue types. Additionally, we put forth the concept that single-cell analyses can be used to expose underlying variability in cellular response to drugs, providing a unique examination of drug efficacy, toxicity, and metabolism. We will outline several of these techniques: single-cell RNA-sequencing and mass cytometry to characterize and distinguish different cell types, single-cell proteomics to quantify drug metabolizing enzymes and characterize cellular responses to drug, capillary electrophoresis-ultrasensitive laser-induced fluorescence detection and single-probe single-cell mass spectrometry for detection of drugs, and others. Emerging single-cell technologies such as these can comprehensively characterize heterogeneity in both cell-type-specific drug metabolism and response to treatment, enhancing progress toward personalized and precision medicine. SIGNIFICANCE STATEMENT: Recent technological advances have enabled the analysis of gene expression and protein levels in single cells. These types of analyses are important to investigating mechanisms that cannot be elucidated on a bulk level, primarily due to the variability of cell populations within biological systems. Here, we summarize cell-type-specific drug metabolism and how pharmacologists can utilize single-cell approaches to obtain a comprehensive understanding of drug metabolism and cellular heterogeneity in response to drugs.

Interaction of Depot Medroxyprogesterone Acetate and Tenofovir Disoproxil Fumarate/Emtricitabine on Peripheral Blood Mononuclear Cells and Cervical Tissue Susceptibility to HIV Infection and Pharmacokinetics.

BACKGROUND: Depot medroxyprogesterone acetate (DMPA) is a widely used contraceptive method. HIV pre-exposure prophylaxis with emtricitabine and tenofovir disoproxil fumarate (F/TDF) is highly effective in reducing HIV acquisition in women. We sought to determine the impact of DMPA on F/TDF pharmacokinetics and pharmacodynamics. METHODS: Twelve healthy premenopausal cisgender women were enrolled and each completed 4 sequential conditions: (1) baseline, (2) steady-state F/TDF alone, (3) steady-state F/TDF + DMPA, and (4) DMPA alone. Assessments included clinical, pharmacokinetic, viral infectivity (ex vivo challenge of peripheral blood mononuclear cells by X4- and R5-tropic green fluorescent protein pseudoviruses and cervical tissue by HIV BaL ), endocrine, immune cell phenotyping, and renal function. RESULTS: Compared with baseline, F/TDF (± DMPA) significantly decreased both %R5- and X4-infected CD4 T cells and F/TDF + DMPA decreased cervical explant p24 (all P < 0.05). The %R5- and X4-infected CD4 T cells were higher during DMPA alone than during F/TDF periods and lower than baseline (not statistically significant). Cervical explant p24 fell between baseline and F/TDF values (not statistically significant). There were neither statistically significant differences in F/TDF pharmacokinetics, including total or renal clearance of either antiviral drug, nor changes in glomerular filtration rate with the addition of DMPA. There were few immune cell phenotypic differences across conditions. CONCLUSIONS: F/TDF decreased HIV infection in both challenge assays, whereas DMPA alone did not enhance HIV infection in either challenge assay. DMPA did not alter F/TDF pharmacokinetics or renal function.

Insights into protein post-translational modification landscapes of individual human cells by trapped ion mobility time-of-flight mass spectrometry.

Single cell proteomics is a powerful tool with potential for markedly enhancing understanding of cellular processes. Here we report the development and application of multiplexed single cell proteomics using trapped ion mobility time-of-flight mass spectrometry. When employing a carrier channel to improve peptide signal, this method allows over 40,000 tandem mass spectra to be acquired in 30 min. Using a KRASG12C model human-derived cell line, we demonstrate the quantification of over 1200 proteins per cell with high relative sequence coverage permitting the detection of multiple classes of post-translational modifications in single cells. When cells were treated with a KRASG12C covalent inhibitor, this approach revealed cell-to-cell variability in the impact of the drug, providing insight missed by traditional proteomics. We provide multiple resources necessary for the application of single cell proteomics to drug treatment studies including tools to reduce cell cycle linked proteomic effects from masking pharmacological phenotypes.