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 KRAS
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 KRAS
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.
For better drugs, diversify clinical trials Bumpus, Namandjé N
Science (American Association for the Advancement of Science),
02/2021, Letnik:
371, Številka:
6529
Journal Article
Recenzirano
Genetic variation in drug metabolism can affect drug outcomes
COVID-19 has highlighted the devastating disparities that exist for Black, Latinx, and Indigenous people in health care and medicine in ...the United States and other parts of the world. As a result, the importance of ethnic diversity in clinical trials has entered public discourse. This moral issue is enough to warrant elevation. But there are also scientific considerations because a lack of diversity in clinical trials hinders the ability to fully understand variation and predict drug outcomes across populations, including those most hard-hit by a disease.
Sterile alpha motif and HD domain protein 1 (SAMHD1) is a unique enzyme that plays important roles in nucleic acid metabolism, viral restriction, and the pathogenesis of autoimmune diseases and ...cancer. Although much attention has been focused on its dNTP triphosphohydrolase activity in viral restriction and disease, SAMHD1 also binds to single-stranded RNA and DNA. Here we utilize a UV cross-linking method using 5-bromodeoxyuridine-substituted oligonucleotides coupled with high-resolution mass spectrometry to identify the binding site for single-stranded nucleic acids (ssNAs) on SAMHD1. Mapping cross-linked amino acids on the surface of existing crystal structures demonstrated that the ssNA binding site lies largely along the dimer–dimer interface, sterically blocking the formation of the homotetramer required for dNTPase activity. Surprisingly, the disordered C-terminus of SAMHD1 (residues 583–626) was also implicated in ssNA binding. An interaction between this region and ssNA was confirmed in binding studies using the purified SAMHD1 583–626 peptide. Despite a recent report that SAMHD1 possesses polyribonucleotide phosphorylase activity, we did not detect any such activity in the presence of inorganic phosphate, indicating that nucleic acid binding is unrelated to this proposed activity. These data suggest an antagonistic regulatory mechanism in which the mutually exclusive oligomeric state requirements for ssNA binding and dNTP hydrolase activity modulate these two functions of SAMHD1 within the cell.
The well accepted "free drug hypothesis" for small-molecule drugs assumes that only the free (unbound) drug concentration at the therapeutic target can elicit a pharmacologic effect. Unbound (free) ...drug concentrations in plasma are readily measurable and are often used as surrogates for the drug concentrations at the site of pharmacologic action in pharmacokinetic-pharmacodynamic analysis and clinical dose projection in drug discovery. Furthermore, for permeable compounds at pharmacokinetic steady state, the free drug concentration in tissue is likely a close approximation of that in plasma; however, several factors can create and maintain disequilibrium between the free drug concentration in plasma and tissue, leading to free drug concentration asymmetry. These factors include drug uptake and extrusion mechanisms involving the uptake and efflux drug transporters, intracellular biotransformation of prodrugs, membrane receptor-mediated uptake of antibody-drug conjugates, pH gradients, unique distribution properties (covalent binders, nanoparticles), and local drug delivery (e.g., inhalation). The impact of these factors on the free drug concentrations in tissues can be represented by
, the ratio of free drug concentration between tissue and plasma at steady state. This review focuses on situations in which free drug concentrations in tissues may differ from those in plasma (e.g.,
> or <1) and discusses the limitations of the surrogate approach of using plasma-free drug concentration to predict free drug concentrations in tissue. This is an important consideration for novel therapeutic modalities since systemic exposure as a driver of pharmacologic effects may provide limited value in guiding compound optimization, selection, and advancement. Ultimately, a deeper understanding of the relationship between free drug concentrations in plasma and tissues is needed.
Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor that is administered as a prodrug for use in both HIV treatment and prevention. TFV requires two sequential phosphorylation steps to ...form the pharmacologically active metabolite, tenofovir‐diphosphate (TFV‐DP). Previous studies have shown that creatine kinase muscle‐type (CKM) can catalyze the final phosphorylation step in colon tissue. Thus, to discover other TFV activating enzymes, we used CKM as a starting point and identified creatine kinase brain‐type (CKB) as a candidate TFV activating enzyme, as it is 80% homologous to CKM. To test the hypothesis that CKB can phosphorylate TFV in a manner similar to CKM, we performed in vitro activity assays by incubating recombinantly expressed CKB or CKM with reaction substrates, phosphocreatine and tenofovir‐monophosphate. TFV metabolites were detected using ultra‐high performance liquid chromatography tandem mass spectrometry (uHPLC/MS). Analysis of TFV‐DP levels revealed no significant difference between CKB and CKM catalyzed reactions. These data indicate that CKB may contribute to TFV activation in tissues where it is expressed, thus genetic variation in CKB may play a role in inter‐individual variability observed in TFV efficacy. To investigate the potential impact of genetic variation, fifteen naturally occurring missense mutations were chosen and mutant CKB enzymes were recombinantly expressed in E. coli. The purified enzymes were used in our in vitro activity assay and TFV metabolites were subsequently detected by uHPLC/MS. Eight mutations (C74S, R96P, S128R, R132H, R172P, R236Q, R292Q, and H296R) demonstrated a statistically significant reduction in the formation of TFV‐DP compared to that of wild‐type CKB. To gain insight into how these mutations disrupt enzymatic function, we exploited the reverse canonical reaction (ATP dephosphorylation) in an enzyme coupled system, from which Michaelis‐Menten curves were constructed and resulting kinetic parameters were used to calculate catalytic efficiencies. Five mutations (R96P, R132H, R236Q, R292Q, and H296R) resulted in catalytic efficiencies less than 20% of wild‐type, which can be attributed to increases in Km. Lastly, thermal stability was examined using differential scanning fluorimetry and showed four mutations (C74S, R96P, R172P, and H296P) had significantly lower melting temperatures than wild‐type. Additionally, seven mutations (C74S, R96P, R132H, R172G, R172P, R236Q, and H296P) displayed melting curves with double peaks or shouldering, suggesting local domain melting or dimer instability induced by the mutation. Together these data support our hypothesis that CKB contributes to TFV activation and indicates that naturally occurring mutations can diminish TFV‐DP formation, catalytic efficiency, and thermal stability in vitro.
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-β-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.
Oral preexposure prophylaxis (PrEP) trials report disparate efficacy attributed to variable adherence. HPTN 066 was conducted to establish objective, quantitative benchmarks for discrete, regular ...levels of adherence using directly observed dosing of tenofovir (TFV) disoproxil fumarate (TDF)/emtricitabine (FTC). Healthy, HIV-uninfected men and women were randomized to one of four oral regimens of fixed-dose TDF 300 mg/FTC 200 mg tablet for 5 weeks with all doses observed: one tablet weekly (one/week), one tablet twice weekly (two/week), two tablets twice weekly (four/week), or one tablet daily (seven/week). Trough serum TFV and FTC, peripheral blood mononuclear cell (PBMC), and CD4(+) TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) concentrations were determined throughout dosing and 2 weeks after the last dose. Rectosigmoidal, semen, and cervicovaginal samples were collected for drug assessment at end of dosing and 2 weeks later in a subset of participants. The 49 enrolled participants tolerated the regimens well. All regimens achieved steady-state concentrations by the second dose for serum TFV/FTC and by 7 days for PBMC TFV-DP/FTC-TP. Steady-state median TFV-DP predose concentrations demonstrated dose proportionality: one/week 1.6 fmol/10(6) PBMCs, two/week 9.1, four/week 18.8, seven/week, 36.3. Further, TFV-DP was consistently quantifiable 2 weeks after the last dose for the ≥4/week regimens. Adherence benchmarks were identified using receiver operating characteristic curves, which had areas under the curve ≥0.93 for all analytes in serum and PBMCs. Intersubject and intrasubject coefficients of variation (%CV) ranged from 33% to 63% and 14% to 34%, respectively, for all analytes in serum and PBMCs. Steady-state PBMC TFV-DP was established earlier and at lower concentrations than predicted and was the only analyte demonstrating predose concentration dose proportionality. Steady-state daily dosing serum TFV and PBMC TFV-DP was consistent with highly effective PrEP clinical trials. HPTN 066 provides adherence benchmarks for oral TFV/FTC regimens to assist interpreting study outcomes.
Cytochrome P450-dependent metabolism of the anti-HIV drug nevirapine (NVP) to 12-hydroxy-NVP (12-OHNVP) has been implicated in NVP toxicities. We investigated the impact of twelfth-position ...trideuteration (12-D3NVP) on the hepatic metabolism of and response to NVP. Formation of 12-OHNVP decreased in human (10.6-fold) and mouse (4.6-fold) hepatocytes incubated with 10 μM 12-D3NVP vs NVP. An observed kinetic isotope effect of 10.1 was measured in human liver microsomes. During mouse hepatocyte treatment (400 μM) with NVP or 12-D3NVP, cell death was reduced 30% with 12-D3NVP vs NVP, while glucuronidated and glutathione-conjugated metabolites increased with 12-D3NVP vs NVP. Using mass spectrometry proteomics, changes in hepatocyte protein expression, including an increase in stress marker insulin-like growth factor-binding protein 1 (IGFBP-1), were observed with 12-D3NVP vs NVP. These results demonstrate that while deuteration can reduce P450 metabolite formation, impacts on phase II metabolism and hepatocyte protein expression should be considered when employing deuteration to reduce P450 metabolite-related hepatotoxicity.
This study was designed to assess the dose-response relationship between tissue, blood, vaginal and rectal compartment concentrations of tenofovir (TFV) and tenofovir diphosphate (TFVdp) and ex vivo ...rectal HIV suppression following oral tenofovir disoproxil fumarate (TDF) and rectal administration of TFV 1% vaginally-formulated gel.
Phase 1, randomized, two-site (US), double-blind, placebo-controlled study of sexually-abstinent males and females.
Eighteen participants received a single 300 mg exposure of oral TDF and were then randomized 2∶1 to receive a single then seven-daily rectal exposures of TFV 1% gel (40 mg TFV per 4 ml gel application) or hydroxyethyl-cellulose (HEC) placebo gel. Blood and rectal biopsies were collected for pharmacokinetic TDF and TFVdp analyses and ex vivo HIV-1 challenge.
There was a significant fit for the TFVdp dose-response model for rectal tissue (p = 0.0004), CD4+MMC (p<0.0001), CD4-MMC (p<0.0001), and TotalMMC (p<0.0001) compartments with r2 ranging 0.36-0.64. Higher concentrations of TFVdp corresponded with lower p24, consistent with drug-mediated virus suppression. The single oral treatment failed to provide adequate compartment drug exposure to reach the EC50 of rectal tissue TFVdp predicted to be necessary to suppress HIV in rectal tissue. The EC50 for CD4+MMC was within the single topical treatment range, providing evidence that a 1% topical, vaginally-formulated TFV gel provided in-vivo doses predicted to provide for 50% efficacy in the ex vivo assay. The 7-daily topical TFV gel treatment provided TFVdp concentrations that reached EC90 biopsy efficacy for CD4-MMC, CD4+MMC and TotalMMC compartments.
The TFVdp MMC compartment (CD4+, CD4- and Total) provided the best surrogate for biopsy infectibility and the 7-daily topical TFV gel treatment provided the strongest PK profile for HIV suppression. ClinicalTrials.gov NCT00984971.
Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been ...previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.
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